Patent application title: Protein Kinase Stress-Related Polypeptides and Methods of Use in Plants
Inventors:
Nocha Van Thielen (Chapel Hill, NC, US)
Oswaldo Da Costa E Silva (Neustadt, DE)
Ruoying Chen (Apex, NC, US)
Assignees:
BASF Plant Science GmbH
IPC8 Class: AA01H100FI
USPC Class:
800278
Class name: Multicellular living organisms and unmodified parts thereof and related processes method of introducing a polynucleotide molecule into or rearrangement of genetic material within a plant or plant part
Publication date: 2010-01-14
Patent application number: 20100011465
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Patent application title: Protein Kinase Stress-Related Polypeptides and Methods of Use in Plants
Inventors:
Ruoying Chen
Oswaldo da Costa e Silva
Nocha Van Thielen
Agents:
BASF CORPORATION
Assignees:
BASF Plant Science GmbH
Origin: LUDWIGSHAFEN, DE
IPC8 Class: AA01H100FI
USPC Class:
800278
Patent application number: 20100011465
Abstract:
A transgenic plant transformed by a Protein Kinase Stress-Related
Polypeptide (PKSRP) coding nucleic acid, wherein expression of the
nucleic acid sequence in the plant results in increased tolerance to
environmental stress as compared to a wild type variety of the plant.
Also provided are agricultural products, including seeds, produced by the
transgenic plants. Also provided are isolated PKSRPs, and isolated
nucleic acid coding PKSRPs, and vectors and host cells containing the
latter.Claims:
1. A transgenic plant cell transformed with an isolated polynucleotide
selected from the group consisting of:a) a polynucleotide sequence
comprising nucleotides 1 to 2400 of SEQ ID NO:19; andb) a polynucleotide
sequence encoding a polypeptide comprising amino acids 1 to 716 of SEQ ID
NO:20.
2. The plant cell of claim 1, wherein the polynucleotide has the sequence as set forth in SEQ ID NO:19.
3. The plant cell of claim 1, wherein the polynucleotide encodes the polypeptide having the sequence as set forth in SEQ ID NO:20.
4. A transgenic plant transformed with an isolated polynucleotide selected from the group consisting of:a) a polynucleotide sequence comprising nucleotides 1 to 2400 of SEQ ID NO:19; andb) a polynucleotide sequence encoding a polypeptide comprising amino acids 1 to 716 of SEQ ID NO:20.
5. The transgenic plant of claim 4, wherein the polynucleotide has the sequence as set forth in SEQ ID NO:19.
6. The transgenic plant of claim 4, wherein polynucleotide encodes the polypeptide having the sequence as set forth in SEQ ID NO:20.
7. The transgenic plant of claim 4, further defined as a monocot.
8. The plant of claim 4, further defined as a dicot.
9. The transgenic plant of claim 4, wherein the plant is selected from the group consisting of maize, wheat, rye, oat, triticale, rice, barley, soybean, peanut, cotton, rapeseed, canola, manihot, pepper, sunflower, tagetes, potato, tobacco, eggplant, tomato, Vicia species, pea, alfalfa, coffee, cacao, tea, Salix species, oil palm, coconut, perennial grasses, and a forage crop plant.
10. The transgenic plant of claim 9, which is maize.
11. The transgenic plant of claim 9, which is soybean.
12. The transgenic plant of claim 9, which is cotton.
13. The transgenic plant of claim 9, which is rapeseed or canola.
14. A seed which is true breeding for a transgene comprising a polynucleotide selected from the group consisting of:a) a polynucleotide sequence comprising nucleotides 1 to 2400 of SEQ ID NO:19; andb) a polynucleotide sequence encoding a polypeptide comprising amino acids 1 to 716 of SEQ ID NO:20.
15. The seed of claim 14, wherein the polynucleotide has the sequence as set forth in SEQ ID NO:19.
16. The seed of claim 14, wherein the polynucleotide encodes the polypeptide having the sequence as set forth in SEQ ID NO:20.
17. An isolated nucleic acid comprising a polynucleotide selected from the group consisting of:a) a polynucleotide sequence comprising nucleotides 1 to 2400 of SEQ ID NO:19; andb) a polynucleotide sequence encoding a polypeptide comprising amino acids 1 to 716 of SEQ ID NO:20.
18. The isolated nucleic acid of claim 17, wherein the polynucleotide has the sequence as set forth in SEQ ID NO:19.
19. The isolated nucleic acid of claim 17, wherein the polynucleotide encodes the polypeptide having the sequence as set forth in SEQ ID NO:20.
20. A method of producing a drought-tolerant transgenic plant, the method comprising the steps of:a) transforming a plant cell with an expression vector comprising a polynucleotide sequence encoding a polypeptide comprising amino acids 1 to 716 of SEQ ID NO:20;b) growing the transformed plant cell to generate transgenic plants; andc) screening the transgenic plants generated in step b) to identify a transgenic plant that expresses the polypeptide and exhibits increased tolerance to drought stress as compared to a wild type variety of the plant.
21. The method of claim 20, wherein the polynucleotide sequence comprises nucleotides 1 to 2400 of SEQ ID NO:19.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001]This application is a divisional of U.S. patent application Ser. No. 10/292,408, filed Nov. 12, 2002 and now U.S. Pat. No. 7,176,026, which claims the priority benefit of U.S. Provisional Patent Application Ser. No. 60/346,096, filed Nov. 9, 2001. This application is with allowed U.S. patent application Ser. No. 12/169,777, filed Jul. 9, 2008, which is with U.S. patent application Ser. No. 11/925,020, filed Oct. 26, 2007 and now U.S. Pat. No. 7,427,698, which is with U.S. patent application Ser. No. 11/609,353, filed Dec. 12, 2006 and now U.S. Pat. No. 7,303,919, which is also a divisional of U.S. patent application Ser. No. 10/292,408. The entire contents of each priority application identified above are hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0002]1. Field of the Invention
[0003]This invention relates generally to nucleic acid sequences encoding polypeptides that are associated with abiotic stress responses and abiotic stress tolerance in plants. In particular, this invention relates to nucleic acid sequences encoding polypeptides that confer drought, cold, and/or salt tolerance to plants.
[0004]2. Background Art
[0005]Abiotic environmental stresses, such as drought stress, salinity stress, heat stress, and cold stress, are major limiting factors of plant growth and productivity. Crop losses and crop yield losses of major crops such as soybean, rice, maize (corn), cotton, and wheat caused by these stresses represent a significant economic and political factor and contribute to food shortages in many underdeveloped countries.
[0006]Plants are typically exposed during their life cycle to conditions of reduced environmental water content. Most plants have evolved strategies to protect themselves against these conditions of desiccation. However, if the severity and duration of the drought conditions are too great, the effects on development, growth, and yield of most crop plants are profound. Continuous exposure to drought conditions causes major alterations in the plant metabolism which ultimately lead to cell death and consequently yield losses.
[0007]Developing stress-tolerant plants is a strategy that has the potential to solve or mediate at least some of these problems. However, traditional plant breeding strategies to develop new lines of plants that exhibit resistance (tolerance) to these types of stresses are relatively slow and require specific resistant lines for crossing with the desired line. Limited germplasm resources for stress tolerance and incompatibility in crosses between distantly related plant species represent significant problems encountered in conventional breeding, Additionally, the cellular processes leading to drought, cold, and salt tolerance in model drought- and/or salt-tolerant plants are complex in nature and involve multiple mechanisms of cellular adaptation and numerous metabolic pathways. This multi-component nature of stress tolerance has not only made breeding for tolerance largely unsuccessful, but has also limited the ability to genetically engineer stress tolerance plants using biotechnological methods.
[0008]Drought and cold stresses, as well as salt stresses, have a common theme important for plant growth, and that is water availability. Plants are exposed during their entire life cycle to conditions of reduced environmental water content, and most plants have evolved strategies to protect themselves against these conditions of desiccation. However, if the severity and duration of the drought conditions are too great, the effects on plant development, growth and yield of most crop plants are profound. Furthermore, most of the crop plants are very susceptible to higher salt concentrations in the soil. Because high salt content in some soils results in less water being available for cell intake, high salt concentration has an effect on plants similar to the effect of drought on plants. Additionally, under freezing temperatures, plant cells lose water as a result of ice formation that starts in the apoplast and withdraws water from the symplast. A plant's molecular response mechanisms to each of these stress conditions are common, and protein kinases play an essential role in these molecular mechanisms.
[0009]Protein kinases represent a superfamily, and the members of this superfamily catalyze the reversible transfer of a phosphate group of ATP to serine, threonine, and tyrosine amino acid side chains on target polypeptides. Protein kinases are primary elements in signaling processes in plants and have been reported to play crucial roles in perception and transduction of signals that allow a cell (and the plant) to respond to environmental stimuli. In particular, receptor protein kinases (RPKs) represent one group of protein kinases that activate a complex array of intracellular signaling pathways in response to the extracellular environment (Van der Gear et al., 1994, Annu. Rev. Cell Biol. 10:251-337). RPKs are single-pass transmembrane polypeptides that contain an amino-terminal signal sequence, extracellular domains unique to each receptor, and a cytoplasmic kinase domain. Ligand binding induces homo- or hetero-dimerization of RPKs, and the resultant close proximity of the cytoplasmic domains results in kinase activation by transphosphorylation. Although plants have many polypeptides similar to RPKs, no ligand has been identified for these receptor-like kinases (RLKs). The majority of plant RLKs that have been identified belong to the family of Serine/Threonine (Ser/Thr) kinases, and most have extracellular Leucine-rich repeats (Becraft, P W., 1998, Trends Plant Sci. 3:384-388).
[0010]Another type of protein kinase is the Ca+-dependent protein kinase (CDPK). This type of kinase has a calmodulin-like domain at the COOH terminus which allows response to Ca+ signals directly without calmodulin being present. Currently, CDPKs are the most prevalent Ser/Thr polypeptide kinases found in higher plants. Although their physiological roles remain unclear, they are induced by cold, drought, and abscisic acid (ABA) (Knight et al., 1991, Nature 352:524; Schroeder, J. I. and Thuleau, P., 1991, Plant Cell 3:555; Bush, D. S., 1995, Annu. Rev. Plant Phys. Plant Mol. Biol. 46:95; Urao, T. et al., 1994, Mol. Gen. Genet. 244:331).
[0011]Another type of signaling mechanism involves members of the conserved SNF1 Serine/Threonine polypeptide kinase family. These kinases play essential roles in eukaryotic glucose and stress signaling. Plant SNF1-like kinases participate in the control of key metabolic enzymes, including HMGR, nitrate reductase, sucrose synthase, and sucrose phosphate synthase (SPS). Genetic and biochemical data indicate that sugar-dependent regulation of SNF1 kinases involves several other sensory and signaling components in yeast, plants, and animals.
[0012]Additionally, members of the Mitogen-Activated Protein Kinase (MAPK) family have been implicated in the actions of numerous environmental stresses in animals, yeasts and plants. It has been demonstrated that both MAPK-like kinase activity and mRNA levels of the components of MAPK cascades increase in response to environmental stress and plant hormone signal transduction. MAP kinases are components of sequential kinase cascades, which are activated by phosphorylation of threonine and tyrosine residues by intermediate upstream MAP kinase kinases (MAPKKs). The MAPKKs are themselves activated by phosphorylation of serine and threonine residues by upstream kinases (MAPKKKs). A number of MAP Kinase genes have been reported in higher plants.
[0013]Another major type of environmental stress is lodging, which refers to the bending of shoots or stems in response to wind, rain, pests or disease. Two types of lodging occur in cereals: root-lodging and stem breakage. The most common type of lodging is root lodging, which occurs early in the season. Stem-breakage, by comparison, occurs later in the season as the stalk becomes more brittle due to crop maturation. Stem breakage has greater adverse consequences on crop yield, since the plants cannot recover as well as from the earlier root-lodging.
[0014]Lodging in cereal crops is influenced by morphological (structural) plant traits as well as environmental conditions. Lodging in cereals is often a result of the combined effects of inadequate standing power of the crop and adverse weather conditions, such as rain, wind, and/or hail. Lodging is also variety (cultivar) dependent. For example, a tall, weak-stemmed wheat cultivar has a greater tendency to lodge than a semi-dwarf cultivar with stiffer straw. In addition, the tendency of a crop to lodge depends on the resistance especially of the lower internodes. This is because the lower internodes have to resist the greatest movement of force. The weight of the higher internodes of the stems plus leaves and heads in relation to the stem (culm) will affect the resistance of a crop to lodging. The heavier the higher parts of the stem are and the greater the distance from their center of gravity to the base of the stem, the greater is the movement of the forces acting upon the lower internodes and the roots. Supporting this argument, it was found that the breaking strength of the lowest internode and shoot per root ratio were the most suitable indices of lodging. Furthermore, plant morphological (structural) characteristics such as plant height, wall thickness, and cell wall lignification can affect the ability of the plant to resist a lateral force.
[0015]Severe lodging is very costly due to its effects on grain formation and associated harvesting problems and losses, It takes about twice the time to harvest a lodged crop than a standing one. Secondary growth in combination with a flattened crop makes harvesting difficult and can subsequently lead to poor grain quality. Yield loss comes from poor grain filling, head loss, and bird damage. Yield losses are most severe when a crop lodges during the ten days following head emergence. Yield losses at this stage will range between 15% and 40%. Lodging that occurs after the plant matures will not affect the yield but it may reduce the amount of harvestable grain. For instance, when lodging occurs after the plant matures, neck breakage and the loss of the whole head can result; these often lead to severe harvest losses. In theses cases, farmers who straight combine their grain will likely incur higher losses than those who swath them, Accordingly, it is desirable to identify genes expressed in lodging resistant plants that have the capacity to confer lodging resistance to the host plant and to other plant species.
[0016]Although some genes that are involved in stress responses in plants have been characterized, the characterization and cloning of plant genes that confer stress tolerance remains largely incomplete and fragmented. For example, certain studies have indicated that drought and salt stress in some plants may be due to additive gene effects, in contrast to other research that indicates specific genes are transcriptionally activated in vegetative tissue of plants under osmotic stress conditions. Although it is generally assumed that stress-induced proteins have a role in tolerance, direct evidence is still lacking, and the functions of many stress-responsive genes are unknown.
[0017]There is a need, therefore, to identity genes expressed in stress tolerant plants that have the capacity to confer stress tolerance to its host plant and to other plant species. Newly generated stress tolerant plants will have many advantages, such as an increased range in which the crop plants can be cultivated, by for example, decreasing the water requirements of a plant species. Other desirable advantages include increased resistance to lodging, the bending of shoots or stems in response to wind, rain, pests, or disease.
SUMMARY OF THE INVENTION
[0018]This invention fulfills in part the need to identify new, unique protein kinases capable of conferring stress tolerance to plants upon over-expression. The present invention describes a novel genus of Protein Kinase Stress-Related Polypeptides (PKSRPs) and PKSRP coding nucleic acids that are important for modulating a plant's response to an environmental stress. More particularly, over-expression of these; PKSRP coding nucleic acids in a plant results in the plant's increased tolerance to an environmental stress.
[0019]The present invention includes an isolated plant cell comprising a PKSRP coding nucleic acid, wherein expression of the nucleic acid sequence in the plant cell results in increased tolerance to environmental stress as compared to a wild type variety of the plant cell. Namely, described herein are PK-3, PK-4, PK-10, and PK-11 from Physcomitrella patens; BnPK-1, BnPK-2, BnPK-3, and BnPK-4 from Brassica napus; GmPK-1, GmPK-2, GmPK-3, and GmPK-4, from Glycine max; and OsPK-1 from Oryza saliva.
[0020]The invention provides in some embodiments that the PKSRP and coding nucleic acid are those that are found in members of the genus Physcomitrella Brassica, Glycine, or Oryza. In another preferred embodiment, the nucleic acid and polypeptide are from a Physcomitrella patens plant, a Brassica napus plant, a Glycine max plant, or an Oryza saliva plant. The invention provides that the environmental stress can be increased salinity, drought, temperature, metal, chemical, pathogenic, and oxidative stresses, or combinations thereof. In preferred embodiments, the environmental stress can be drought or cold temperature.
[0021]The invention further provides a seed produced by a transgenic plant transformed by a PKSRP coding nucleic acid, wherein the plant is true breeding for increased tolerance to environmental stress as compared to a wild type variety of the plant. The invention further provides a seed produced by a transgenic plant expressing a PKSRP, wherein the plant is true breeding for increased tolerance to environmental stress as compared to a wild type variety of the plant.
[0022]The invention further provides an agricultural product produced by any of the below-described transgenic plants, plant parts or seeds. The invention further provides an isolated PKSRP as described below. The invention further provides an isolated PKSRP coding nucleic acid, wherein the PKSRP coding nucleic acid codes for a PKSRP as described below.
[0023]The invention further provides an isolated recombinant expression vector comprising a PKSRP coding nucleic acid as described below, wherein expression of the vector in a host cell results in increased tolerance to environmental stress as compared to a wild type variety of the host cell. The invention further provides a host cell containing the vector and a plant containing the host cell.
[0024]The invention further provides a method of producing a transgenic plant with a PKSRP coding nucleic acid, wherein expression of the nucleic acid in the plant results in increased tolerance to environmental stress as compared to a wild type variety of the plant comprising: (a) transforming a plant cell with an expression vector comprising a PKSRP coding nucleic acid, and (b) generating from the plant cell a transgenic plant with an increased tolerance to environmental stress as compared to a wild type variety of the plant. In preferred embodiments, the PKSRP and PKSRP coding nucleic acid are as described below.
[0025]The present invention further provides a method of identifying a novel PKSRP, comprising (a) raising a specific antibody response to a PKSRP, or fragment thereof, as described below; (b) screening putative PKSRP material with the antibody, wherein specific binding of the antibody to the material indicates the presence of a potentially novel PKSRP; and (c) identifying from the bound material a novel PKSRP in comparison to known PKSRP. Alternatively, hybridization with nucleic acid probes as described below can be used to identity novel PKSRP nucleic acids.
[0026]The present invention also provides methods of modifying stress tolerance of a plant comprising, modifying the expression of a PKSRP nucleic acid in the plant, wherein the PKSRP is as described below. The invention provides that this method can be performed such that the stress tolerance is either increased or decreased. Preferably, stress tolerance is increased in a plant via increasing expression of a PKSRP nucleic acid.
[0027]In another aspect, the invention provides methods of increasing a plant's resistance to lodging comprising, transforming a plant cell with an expression cassette comprising a PKSRP nucleic acid and generating a plant from the plant cell.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028]FIG. 1 shows a diagram of the plant expression vector pBPS-JH001 containing the super promoter driving the expression of the PKSRP coding nucleic acid ("Gene of Interest"). The components are: aacCI gentamycin resistance gene (Hajdukiewicz et al., 1994, Plant Molec. Biol. 25: 989-94), NOS promoter (Becker et al., 1992, Plant Molec. Biol. 20: 1195-97), g7T terminator (Becker et al., 1992), and NOSpA terminator (Jefferson et al., 1987, EMBO J. 6:3901-7).
[0029]FIG. 2 shows a diagram of the plant expression vector pBPS-SC022 containing the super promoter driving the expression of the PKSRP coding nucleic acid (Gene of Interest"). The components are: NPTII kanamycin resistance gene (Hajdukiewicz et al., 1994, Plant Molec. Biol. 25: 989-98), AtAct2-1 promoter (An et al., 1996, Plant J. 10: 107-21), and OCS3 terminator (Weigel et al., 2000, Plant Physiol 122:1003-13).
DETAILED DESCRIPTION OF THE INVENTION
[0030]The present invention may be understood more readily by reference to the following detailed description of the preferred embodiments of the invention and the Examples included herein. However, before the present compounds, compositions, and methods are disclosed and described, it is to be understood that this invention is not limited to specific nucleic acids, specific polypeptides, specific cell types, specific host cells, specific conditions, or specific methods, etc., as such may, of course, vary, and the numerous modifications and variations therein will be apparent to those skilled in the art. It is also to be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting. In particular, the designation of the amino acid sequences as "Protein Kinase Stress-Related Polypeptides" (PKSRPs), in no way limits the functionality of those sequences.
[0031]The present invention describes a novel genus of Protein Kinase Stress-Related Polypeptides (PKSRPs) and PKSRP coding nucleic acids that are important for modulating a plant's response to an environmental stress. More particularly, over-expression of these PKSRP coding nucleic acids in a plant results in the plant's increased tolerance to an environmental stress.
[0032]The present invention provides a transgenic plant cell transformed by a PKSRP coding nucleic acid, wherein expression of the nucleic acid sequence in the plant cell results in increased tolerance to environmental stress or increased resistance to lodging as compared to a wild type variety of the plant cell. The invention further provides transgenic plant parts and transgenic plants containing the plant cells described herein. In preferred embodiments, the transgenic plants and plant parts have increased tolerance to environmental stress or increased resistance to lodging as compared to a wild type variety of the plant. Plant parts include, but are not limited to, stems, roots, ovules, stamens, leaves, embryos, meristematic regions, callus tissue, gametophytes, sporophytes, pollen, microspores, and the like. In one embodiment the transgenic plant is male sterile. Also provided is a plant seed produced by a transgenic plant transformed by a PKSRP coding nucleic acid, wherein the seed contains the PKSRP coding nucleic acid, and wherein the plant is true breeding for increased tolerance to environmental stress as compared to a wild type variety of the plant. The invention further provides a seed produced by a transgenic plant expressing a PKSRP, wherein the seed contains the PKSRP, and wherein the plant is true breeding for increased tolerance to environmental stress as compared to a wild type variety of the plant. The invention also provides an agricultural product produced by any of the below-described transgenic plants, plant parts, and plant seeds. Agricultural products include, but are not limited to, plant extracts, proteins, amino acids, carbohydrates, fats, oils, polymers, vitamins, and the like.
[0033]As used herein, the term "variety" refers to a group of plants within a species that share constant characters that separate them from the typical form and from other possible varieties within that species. While possessing at least one distinctive trait, a variety is also characterized by some variation between individuals within the variety, based primarily on the Mendelian segregation of traits among the progeny of succeeding generations. A variety is considered "true breeding" for a particular trait if it is genetically homozygous for that trait to the extent that, when the true-breeding variety is self-pollinated, a significant amount of independent segregation of the trait among the progeny is not observed. In the present invention, the trait arises from the transgenic expression of one or more DNA sequences introduced into a plant variety.
[0034]The present invention describes for the first time that the Physcomitrella patens PKSRPs, PK-3, PK-4, PK-10, and PK-11; the Brassica napus PKSRPs, BnPK-1, BnPK-2, BnPK-3, and BnPK-4; the Glycine max PKSRPs, GmPK-1, GmPK-2, GmPK-3, and GmPK-4; and the Oryza saliva PKSRP OsPK-1 are useful for increasing a plant's tolerance to environmental stress. As used herein, the term polypeptide refers to a chain of at least four amino acids joined by peptide bonds. The chain may be linear, branched, circular or combinations thereof. Accordingly, the present invention provides isolated PKSRPs selected from the group consisting of PK-3, PK-4, PK-10, PK-11, BnPK-1, BnPK-2, BnPK-3, BnPK-4, GmPK-1, GmPK-2, GmPK-3, GmPK-4, and OsPK-1, and homologs thereof. In preferred embodiments, the PKSRP is selected from: 1) Physcomitrella patens Protein Kinase-3 (PK-3) polypeptide as defined in SEQ ID NO:3; 2) Physcomitrella patens Protein Kinase-4 (PK-4) polypeptide as defined in SEQ ID NO:6; 3) Physcomitrella patens Protein Kinase-10 (PK-10) polypeptide as defined in SEQ ID NO:9; 4) Physcomitrella patens Protein Kinase-1 (PK-11) polypeptide as defined in SEQ ID NO:12; 5) Brassica napus Protein Kinase-1 (BnPK-1) polypeptide as defined in SEQ ID NO:14; 6) Brassica napus Protein Kinase-2 (BnPK-2) polypeptide as defined in SEQ ID NO:16; 7) Brassica napus Protein Kinase-3 (BnPK-3) polypeptide as defined in SEQ ID NO:18; 8) Brassica napus Protein Kinase-4 (BnPK-4) polypeptide as defined in SEQ ID NO:20; 9) Glycine max Protein Kinase-1 (GmPK-1) polypeptide as defined in SEQ ID NO:22; 10) Glycine max Protein Kinase-2 (GmPK-2) polypeptide as defined in SEQ ID NO:24; 11) Glycine max Protein Kinase-3 (GmPK-3) polypeptide as defined in SEQ ID NO:26; 12) Glycine max Protein Kinase-4 (GmPK-4) polypeptide as defined in SEQ ID NO:28; 13) Oryza saliva Protein Kinase-1 (OsPK-1) polypeptide as defined in SEQ ID NO:30; and homologs and orthologs thereof. Homologs and orthologs of the amino acid sequences are defined below.
[0035]The PKSRPs of the present invention are preferably produced by recombinant DNA techniques. For example, a nucleic acid molecule encoding the polypeptide is cloned into an expression vector (as described below), the expression vector is introduced into a host cell (as described below) and the PKSRP is expressed in the host cell. The PKSRP can then be isolated from the cells by an appropriate purification scheme using standard polypeptide purification techniques. For the purposes of the invention, the term "recombinant polynucleotide" refers to a polynucleotide that has been altered, rearranged or modified by genetic engineering. Examples include any cloned polynucleotide, and polynucleotides that are linked or joined to heterologous sequences. The term "recombinant" does not refer to alterations to polynucleotides that result from naturally occurring events, such as spontaneous mutations. Alternative to recombinant expression, a PKSRP, or peptide can be synthesized chemically using standard peptide synthesis techniques. Moreover, native PKSRP can be isolated from cells (e.g., Physcomitrella patens, Brassica napus, Glycine max, or Oryza sativa), for example using an anti-PKSRP antibody, which can be produced by standard techniques utilizing a PKSRP or fragment thereof.
[0036]The invention further provides an isolated PKSRP coding nucleic acid. The present invention includes PKSRP coding nucleic acids that encode PKSRPs as described herein. In preferred embodiments, the PKSRP coding nucleic acid is selected from: 1) Physcomitrella patens Protein Kinase-3 (PK-3) nucleic acid as defined in SEQ ID NO:2; 2) Physcomitrella patens Protein Kinase-4 (PK-4) nucleic acid as defined in SEQ ID NO:5; 3) Physcomitrella patens Protein Kinase-10 (PK-10) nucleic acid as defined in SEQ ID NO:8; 4) Physcomitrella patens Protein Kinase-1 (PK-11) nucleic acid as defined in SEQ ID NO:1; 5) Brassica napus Protein Kinase-1 (BnPK-1) nucleic acid as defined in SEQ ID NO: 13; 6) Brassica napus Protein Kinase-2 (BnK-2) nucleic acid as defined in SEQ ID NO:15; 7) Brassica napus Protein Kinase-3 (BnPK-3) nucleic acid as defined in SEQ ID NO:17; 8) Brassica napus Protein Kinase-4 (BnPK-4) nucleic acid as defined in SEQ ID NO:19; 9) Glycine max Protein Kinase-1 (GmPK-1) nucleic acid as defined in SEQ ID NO:21; 10) Glycine max Protein Kinase-2 (GmPK-2) nucleic acid as defined in SEQ ID NO:23; 11) Glycine max Protein Kinase-3 (GmPK-3) nucleic acid as defined in SEQ ID NO:25; 12) Glycine max Protein Kinase-4 (GmPK-4) nucleic acid as defined in SEQ ID NO:27; 13) Oryza saliva Protein Kinase-1 (OsPK-1) nucleic acid as defined in SEQ ID NO:29; and homologs and orthologs thereof, Homologs and orthologs of the nucleotide sequences are defined below. In one preferred embodiment, the nucleic acid and polypeptide are isolated from the plant genus Physcomitrella, Brassica, Glycine, or Oryza. In another preferred embodiment, the nucleic acid and polypeptide are from a Physcomitrella patens (P. patens) plant, a Brassica napus plant, a Glycine max plant, or an Oryza sativa plant.
[0037]As used herein, the term "environmental stress" refers to any sub-optimal growing condition and includes, but is not limited to, sub-optimal conditions associated with salinity, drought, temperature, metal, chemical, pathogenic, and oxidative stresses, or combinations thereof. In preferred embodiments, the environmental stress can be selected from one or more of the group consisting of salinity, drought, or temperature, or combinations thereof, and in particular, can be selected from one or more of the group consisting of high salinity, low water content, or low temperature. Also included within the definition of "environmental stress" is lodging, or the bending of shoots or stems in response to elements such as wind, rain, pests, or disease. Accordingly, the present invention provides compositions and methods of increasing lodging resistance in a plant. It is also to be understood that as used in the specification and in the claims, "a" or "an" can mean one or more, depending upon the context in which it is used. Thus, for example, reference to "a cell" can mean that at least one cell can be utilized.
[0038]As also used herein, the term "nucleic acid" and "polynucleotide" refer to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term also encompasses RNA/DNA hybrids. These terms also encompass untranslated sequence located at both the 3' and 5' ends of the coding region of the gene: at least about 1000 nucleotides of sequence upstream from the 5' end of the coding region and at least about 200 nucleotides of sequence downstream from the 3' end of the coding region of the gene. Less common bases, such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others can also be used for antisense, dsRNA and ribozyme pairing. For example, polynucleotides that contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression. Other modifications, such as modification to the phosphodiester backbone, or the 2'-hydroxy in the ribose sugar group of the RNA can also be made. The antisense polynucleotides and ribozymes can consist entirely of ribonucleotides, or can contain mixed ribonucleotides and deoxyribonucleotides. The polynucleotides of the invention may be produced by any means, including genomic preparations, cDNA preparations, in vitro synthesis, RT-PCR, and in vitro or in vivo transcription.
[0039]An "isolated" nucleic acid molecule is one that is substantially separated from other nucleic acid molecules which are present in the natural source of the nucleic acid (i.e., sequences encoding other polypeptides). Preferably, an "isolated" nucleic acid is free of some of the sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in its naturally occurring replicon. For example, a cloned nucleic acid is considered isolated. In various embodiments, the isolated PKSRP nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 0.2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived (e.g., a Physcomitrella patens, a Brassica napus, a Glycine max, or an Oryza saliva cell). A nucleic acid is also considered isolated if it has been altered by human intervention, or placed in a locus or location that is not its natural site, or if it is introduced into a cell by agroinfection. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be free from some of the other cellular material with which it is naturally associated, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.
[0040]Specifically excluded from the definition of "isolated nucleic acids" are: naturally-occurring chromosomes (such as chromosome spreads), artificial chromosome libraries, genomic libraries, and cDNA libraries that exist either as an in vitro nucleic acid preparation or as a transfected/transformed host cell preparation, wherein the host cells are either an in vitro heterogeneous preparation or plated as a heterogeneous population of single colonies. Also specifically excluded are the above libraries wherein a specified nucleic acid makes up less than 5% of the number of nucleic acid inserts in the vector molecules, Further specifically excluded are whole cell genomic DNA or whole cell RNA preparations (including whole cell preparations that are mechanically sheared or enzymatically digested). Even further specifically excluded are the whole cell preparations found as either an in vitro preparation or as a heterogeneous mixture separated by electrophoresis wherein the nucleic acid of the invention has not further been separated from the heterologous nucleic acids in the electrophoresis medium (erg., further separating by excising a single band from a heterogeneous band population in an agarose gel or nylon blot).
[0041]A nucleic acid molecule of the present invention, e.g., a nucleic acid molecule having a nucleotide sequence of SEQ ID NO:2, SEQ ID NO:5 SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29; or a portion thereof can be isolated using standard molecular biology techniques and the sequence information provided herein. For example, a P. patens PKSRP cDNA can be isolated from a P. patens library using all or portion of one of the sequences of SEQ ID NO: 1 and SEQ ID NO:4. Moreover, a nucleic acid molecule encompassing all or a portion of one of the sequences of SEQ ID NO:2, SEQ ID NO:5 SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:29 can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon this sequence. For example, mRNA can be isolated from plant cells (e.g., by the guanidinium-thiocyanate extraction procedure of Chirgvin et al., 1979, Biochemistry 18:5294-5299) and cDNA can be prepared using reverse transcriptase (e.g., Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, Md.; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Petersburg, Fla.). Synthetic oligonucleotide primers for polymerase chain reaction amplification can be designed based upon one of the nucleotide sequences shown in SEQ ID NO:2, SEQ ID NO:5 SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:29. A nucleic acid molecule of the invention can be amplified using cDNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid molecule so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to a PKSRP nucleotide sequence can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
[0042]In a preferred embodiment, an isolated nucleic acid molecule of the invention comprises one of the nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO: 17 SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:29. These cDNAs may comprise sequences encoding the PKSRPs, (i.e., the "coding regions" of PK-3 and PK-4), as well as 5' untranslated sequences and 3, untranslated sequences. The coding region of PK-3 comprises nucleotides 138-1409 of SEQ ID NO:2 whereas the coding region of PK-4 comprises nucleotides 142-1395 of SEQ ID NO:5. It is to be understood that SEQ ID NO:2 and SEQ ID NO:5 comprise both coding regions and 5' and 3' untranslated regions. Alternatively, the nucleic acid molecules of the present invention can comprise only the coding region of any of the sequences in SEQ ID NO:2 and SEQ ID NO:5 or can contain whole genomic fragments isolated from genomic DNA. The present invention also includes PKSRP coding nucleic acids that encode PKSRPs as described herein. Preferred is a PKSRP coding nucleic acid that encodes a PKSRP selected from the group consisting of PK-3 as defined in SEQ ID NO:3, PK-4 as defined in SEQ ID NO:6, PK-10 as defined in SEQ ID NO:9, PK-11 as defined in SEQ ID NO:12, BnPK-1 as defined in SEQ ID NO:14, BnPK-2 as defined in SEQ ID NO:16, BnPK-3 as defined in SEQ ID NO:18, BnPK-4 as defined in SEQ ID NO:20, GmPK-1 as defined in SEQ ID NO:22, GmPK-2 as defined in SEQ ID NO:24, GmPK-3 as defined in SEQ ID NO:26, GmPK-4 as defined in SEQ ID NO:28, and OsPK-1 as defined in SEQ ID NO:30.
[0043]Moreover, the nucleic acid molecule of the invention can comprise a portion of the coding region of one of the sequences in SEQ ID NO:2, SEQ ID NO:5 SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29, for example, a fragment which can be used as a probe or primer or a fragment encoding a biologically active portion of a PKSRP. The nucleotide sequences determined from the cloning of the PKSRP genes from Physcomitrella patens, Brassica napus, Glycine max, and Oryza sativa allow for the generation of probes and primers designed for use in identifying and/or cloning PKSRP homologs in other cell types and organisms, as well as PKSRP homologs from other related species. The portion of the coding region can also encode a biologically active fragment of a PKSRP.
[0044]As used herein, the term "biologically active portion of" a PKSRP is intended to include a portion, e.g., a domain/motif, of a PKSRP that participates in modulation of stress tolerance in a plant, and more preferably, drought tolerance or salt tolerance. For the purposes of the present invention, modulation of stress tolerance refers to at least a 10% increase or decrease in the stress tolerance of a transgenic plant comprising a PKSRP expression cassette (or expression vector) as compared to the stress tolerance of a non-transgenic control plant. Methods for quantitating stress tolerance are provided at least in Example 7 below. In a preferred embodiment, the biologically active portion of a PKSRP increases a plant's tolerance to an environmental stress.
[0045]Biologically active portions of a PKSRP include peptides comprising amino acid sequences derived from the amino acid sequence of a PKSRP, e.g., an amino acid sequence of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30 or the amino acid sequence of a polypeptide identical to a PKSRP, which include fewer amino acids than a full length PKSRP or the full length polypeptide which is identical to a PKSRP, and exhibit at least one activity of a PKSRP. Typically, biologically active portions (e.g., peptides which are, for example, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids in length) comprise a domain or motif with at least one activity of a PKSRP. Moreover, other biologically active portions in which other regions of the polypeptide are deleted, can be prepared by recombinant techniques and evaluated for one or more of the activities described herein, Preferably, the biologically active portions of a PKSRP include one or more selected domains/motifs or portions thereof having biological activity such as a kinase domain. For example, the kinase domain of PK-3 spans amino acid residues 87-360 of SEQ ID NO:3, and the kinase domain of PK-4 spans amino acid residues 81-281 of SEQ ID NO:6. Accordingly, the present invention includes PKSRPs comprising amino acid residues 87-360 of SEQ ID NO:3 and amino acid residues 81-281 of SEQ ID NO:6.
[0046]The invention also provides PKSRP chimeric or fusion polypeptides. As used herein, a PKSRP "chimeric polypeptide" or "fusion polypeptide" comprises a PKSRP operatively linked to a non-PKSRP. A PKSRP refers to a polypeptide having an amino acid sequence corresponding to a PKSRP, whereas a non-PKSRP refers to a polypeptide having an amino acid sequence corresponding to a polypeptide which is not substantially identical to the PKSRP, e.g., a polypeptide that is different from the PKSRP and is derived from the same or a different organism. As used herein with respect to the fission polypeptide, the term "operatively linked" is intended to indicate that the PKSRP and the non-PKSRP are fused to each other so that both sequences fulfill the proposed function attributed to the sequence used. The non-PKSRP can be fused to the N-terminus or C-terminus of the PKSRP. For example, in one embodiment, the fusion polypeptide is a GST-PKSRP fusion polypeptide in which the PKSRP sequences are fused to the C-terminus of the GST sequences. Such fusion polypeptides can facilitate the purification of recombinant PKSRPs. In another embodiment, the fusion polypeptide is a PKSRP containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of a PKSRP can be increased through use of a heterologous signal sequence.
[0047]Preferably, a PKSRP chimeric or fusion polypeptide of the invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining and enzymatic ligation. In another embodiment, the fission gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (See, e.g., Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide), A PKSRP encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the PKSRP. In addition to fragments and fission polypeptides of the PKSRPs described herein, the present invention includes homologs and analogs of naturally occurring PKSRPs and PKSRP encoding nucleic acids in a plant. "Homologs" are defined herein as two nucleic acids or polypeptides that have similar, or substantially identical, nucleotide or amino acid sequences, respectively. Homologs include allelic variants, orthologs, paralogs, agonists and antagonists of PKSRPs as defined hereafter. The term "homolog" further encompasses nucleic acid molecules that differ from one of the nucleotide sequences shown in SEQ ID NO:2, SEQ ID NO:5 SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29 (and portions thereof) due to degeneracy of the genetic code and thus encode the same PKSRP as that encoded by the nucleotide sequences shown in SEQ ID NO:2, SEQ ID NO:5 SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29. As used herein a "naturally occurring" PKSRP refers to a PKSRP amino acid sequence that occurs in nature. Preferably, a naturally occurring PKSRP comprises an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:30.
[0048]An agonist of the PKSRP can retain substantially the same, or a subset, of the biological activities of the PKSRP. An antagonist of the PKSRP can inhibit one or more of the activities of the naturally occurring form of the PKSRP. For example, the PKSRP antagonist can competitively bind to a downstream or upstream member of the cell membrane component metabolic cascade that includes the PKSRP, or bind to a PKSRP that mediates transport of compounds across such membranes, thereby preventing translocation from taking place.
[0049]Nucleic acid molecules corresponding to natural allelic variants and analogs, orthologs and paralogs of a PKSRP cDNA can be isolated based on their identity to the Physcomitrella patens, Brassica napus, Glycine max, or Oryza sativa PKSRP nucleic acids described herein using PKSRP cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. In an alternative embodiment, homologs of the PKSRP can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the PKSRP for PKSRP agonist or antagonist activity. In one embodiment, a variegated library of PKSRP variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of PKSRP variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential PKSRP sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion polypeptides (e.g., for phage display) containing the set of PKSRP sequences therein. There are a variety of methods that can be used to produce libraries of potential PKSRP homologs from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene is then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential PKSRP sequences. Methods for synthesizing degenerate oligonucleotides are known in the art. See, e.g., Narang, S. A., 1983, Tetrahedron 39:3; Itakura et al., 1984, Annu. Rev. Biochem. 53:323; Itakura et al., 1984, Science 198:1056; Ike et al. 1983, Nucleic Acid Res. 11:477.
[0050]In addition, libraries of fragments of the PKSRP coding regions can be used to generate a variegated population of PKSRP fragments for screening and subsequent selection of homologs of a PKSRP. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a PKSRP coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA, which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal, C-terminal, and internal fragments of various sizes of the PKSRP.
[0051]Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of PKSRP homologs. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify PKSRP homologs (Arkin and Yourvan, 1992, PNAS 89:7811-7815; Delgrave et al., 1993, Polypeptide Engineering 6(3):327-331). In another embodiment, cell based assays can be exploited to analyze a variegated PKSRP library, using methods well known in the art. The present invention further provides a method of identifying a novel PKSRP, comprising (a) raising a specific antibody response to a PKSRP, or a fragment thereof, as described herein; (b) screening putative PKSRP material with the antibody, wherein specific binding of the antibody to the material indicates the presence of a potentially novel PKSRP; and (c) analyzing the bound material in comparison to known PKSRP, to determine its novelty.
[0052]As stated above, the present invention includes PKSRPs and homologs thereof. To determine the percent sequence identity of two amino acid sequences (e.g., one of the sequences of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30, and a mutant form thereof), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of one polypeptide for optimal alignment with the other polypeptide or nucleic acid). The amino acid residues at corresponding amino acid positions are then compared. When a position in one sequence (e.g., one of the sequences of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30) is occupied by the same amino acid residue as the corresponding position in the other sequence (e.g., a mutant form of the sequence selected from the polypeptide of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30), then the molecules are identical at that position. The same type of comparison can be made between two nucleic acid sequences.
[0053]The percent sequence identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent sequence identity=numbers of identical positions/total numbers of positions×100). Preferably, the isolated amino acid homologs included in the present invention are at least about 50-60%, preferably at least about 60-70%, and more preferably at least about 70-75%, 75-80%, 80-85%, 85-90% or 90-95%, and most preferably at least about 96%, 97%, 98%, 99% or more identical to an entire amino acid sequence shown in SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ TD NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30. In yet another embodiment, the isolated amino acid homologs included in the present invention are at least about 50-60%, preferably at least about 60-70%, and more preferably at least about 70-75%, 75-80%, 80-85%, 85-90% or 90-95%, and most preferably at least about 96%, 97%, 98%, 99% or more identical to an entire amino acid sequence encoded by a nucleic acid sequence shown in SEQ ID NO:2, SEQ ID NO:5 SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29. In other embodiments, the PKSRP amino acid homologs have sequence identity over at least 15 contiguous amino acid residues, more preferably at least 25 contiguous amino acid residues, and most preferably at least 35 contiguous amino acid residues of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30, In one embodiment of the present invention, the homolog has at least about 50-60%, preferably at least about 60-70%, more preferably at least about 70-75%, 75-80%, 80-85%, 85-90% or 90-95%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more sequence identity with the kinase domain of PK-3 (amino acids 87-360 of SEQ ID NO:3) or PK-4 (amino acids 81-281 of SEQ ID NO:6).
[0054]In another preferred embodiment, an isolated nucleic acid homolog of the invention comprises a nucleotide sequence which is at least about 50-60%, preferably at least about 60-70%, more preferably at least about 70-75%, 75-80%, 80-85%, 85-90% or 90-95%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more identical to a nucleotide sequence shown in SEQ ID NO:2, SEQ ID NO:5 SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29, or to a portion comprising at least 60 consecutive nucleotides thereof. The preferable length of sequence comparison for nucleic acids is at least 75 nucleotides, more preferably at least 100 nucleotides and most preferably the entire length of the coding region.
[0055]It is further preferred that the isolated nucleic acid homolog of the invention encodes a PKSRP, or portion thereof, that is at least 85% identical to an amino acid sequence of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16 SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID) NO:28, or SEQ ID NO:30 and that functions as a modulator of an environmental stress response in a plant. In a more preferred embodiment, overexpression of the nucleic acid homolog in a plant increases the tolerance of the plant to an environmental stress. In a further preferred embodiment, the nucleic acid homolog encodes a PKSRP that functions as a protein kinase.
[0056]For the purposes of the invention, the percent sequence identity between two nucleic acid or polypeptide sequences may be determined using the Vector NTI 6.0 (PC) software package (InforMax, 7600 Wisconsin Ave., Bethesda, Md. 20814). A gap opening penalty of 15 and a gap extension penalty of 6.66 are used for determining the percent identity of two nucleic acids. A gap opening penalty of 10 and a gap extension penalty of 0.1 are used for determining the percent identity of two polypeptides. All other parameters are set at the default settings. For purposes of a multiple alignment (Clustal W algorithm), the gap opening penalty is 10, and the gap extension penalty is 0.05 with blosum62 matrix. It is to be understood that for the purposes of determining sequence identity when comparing a DNA sequence to an RNA sequence, a thymidine nucleotide is equivalent to a uracil nucleotide.
[0057]In another aspect, the invention provides an isolated nucleic acid comprising a polynucleotide that hybridizes to the polynucleotide of SEQ ID NO:2, SEQ ID NO:5 SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29 under stringent conditions. More particularly, an isolated nucleic acid molecule of the invention is at least 15 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:2, SEQ ID NO:5 SEQ TD NO:8, SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29. In other embodiments, the nucleic acid is at least 30, 50, 100, 250 or more nucleotides in length. Preferably, an isolated nucleic acid homolog of the invention comprises a nucleotide sequence which hybridizes under highly stringent conditions to the nucleotide sequence shown in SEQ ID NO:2, SEQ ID NO:5 SEQ ID NO:8, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29, and functions as a modulator of stress tolerance in a plant. In a further preferred embodiment, overexpression of the isolated nucleic acid homolog in a plant increases a plant's tolerance to an environmental stress. In an even further preferred embodiment, the isolated nucleic acid homolog encodes a PKSRP that functions as a protein kinase.
[0058]As used herein with regard to hybridization for DNA to DNA blot, the term "stringent conditions" refers to hybridization overnight at 60° C. in 10× Denharts solution, 6×SSC, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA. Blots are washed sequentially at 62° C. for 30 minutes each time in 3×SSC/0.1% SDS, followed by 1×SSC/0.1% SDS and finally 0.1×SSC/0.1% SDS. As also used herein, "highly stringent conditions" refers to hybridization overnight at 65° C. in 10× Denharts solution, 6×SSC, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA. Blots are washed sequentially at 65° C. for 30 minutes each time in 3×SSC/0.1% SDS, followed by 1×SSC/0.1% SDS and finally 0.1×SSC/0.1% SDS. Methods for nucleic acid hybridizations are described in Meinkoth and Wahl, 1984, Anal. Biochem. 138:267-284; Ausubel et al. eds, 1995, Current Protocols in Molecular Biology, Chapter 2, Greene Publishing and Wiley-Interscience, New York; and Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization with Nucleic Acid Probes, Part I, Chapter 2, Elsevier, N.Y. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent or highly stringent conditions to a sequence of SEQ ID NO:2, SEQ ID NO:5 SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29 corresponds to a naturally occurring nucleic acid molecule. As used herein, a "naturally occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural polypeptide). In one embodiment, the nucleic acid encodes a naturally occurring Physcomitrella patens, Brassica napus, Glycine max, or Oryza saliva PKSRP.
[0059]Using the above-described methods, and others known to those of skill in the art, one of ordinary skill in the art can isolate homologs of the PKSRPs comprising amino acid sequences shown in SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ if NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30. One subset of these homologs are allelic variants, As used herein, the term "allelic variant" refers to a nucleotide sequence containing polymorphisms that lead to changes in the amino acid sequences of a PKSRP and that exist within a natural population (e.g., a plant species or variety). Such natural allelic variations can typically result in 1-5% variance in a PKSRP nucleic acid. Allelic variants can be identified by sequencing the nucleic acid sequence of interest in a number of different plants, which can be readily carried out by using hybridization probes to identify the same PKSRP genetic locus in those plants. Any and all such nucleic acid variations and resulting amino acid polymorphisms or variations in a PKSRP that are the result of natural allelic variation and that do not alter the functional activity of a PKSRP, are intended to be within the scope of the invention.
[0060]Moreover, nucleic acid molecules encoding PKSRPs from the same or other species such as PKSRP analogs, orthologs, and paralogs, are intended to be within the scope of the present invention. As used herein, the term "analogs" refers to two nucleic acids that have the same or similar function, but that have evolved separately in unrelated organisms. As used herein, the term "orthologs" refers to two nucleic acids from different species, but that have evolved from a common ancestral gene by speciation. Normally, orthologs encode polypeptides having the same or similar functions. As also used herein, the term "paralogs" refers to two nucleic acids that are related by duplication within a genome. Paralogs usually have different functions, but these functions may be related (Tatusov, R. L. et al., 1997, Science 278(5338):631-637). Analogs, orthologs and paralogs of a naturally occurring PKSRP can differ from the naturally occurring PKSRP by post-translational modifications, by amino acid sequence differences, or by both. Post-translational modifications include in vivo and in vitro chemical derivatization of polypeptides, e.g., acetylation, carboxylation, phosphorylation, or glycosylation, and such modifications may occur during polypeptide synthesis or processing or following treatment with isolated modifying enzymes. In particular, orthologs of the invention will generally exhibit at least 80-85%, more preferably, 85-90% or 90-95%, and most preferably 95%, 96%, 97%, 98% or even 99% identity or sequence identity with all or part of a naturally occurring PKSRP amino acid sequence and will exhibit a function similar to a PKSRP. Preferably, a PKSRP ortholog of the present invention functions as a modulator of an environmental stress response in a plant and/or functions as a protein kinase. More preferably, a PKSRP ortholog increases the stress tolerance of a plant. In one embodiment, the PKSRP orthologs maintain the ability to participate in the metabolism of compounds necessary for the construction of cellular membranes in a plant, or in the transport of molecules across these membranes.
[0061]In addition to naturally-occurring variants of a PKSRP sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into a nucleotide sequence of SEQ ID NO:2, SEQ ID NO:5 SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29, thereby leading to changes in the amino acid sequence of the encoded PKSRP, without altering the functional activity of the PKSRP. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in a sequence of SEQ ID NO:2, SEQ ID NO:5 SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of one of the PKSRPs without altering the activity of said PKSRP, whereas an "essential" amino acid residue is required for PKSRP activity. Other amino acid residues, however, (e.g., those that are not conserved or only semi-conserved in the domain having PKSRP activity) may not be essential for activity and thus are likely to be amenable to alteration without altering PKSRP activity.
[0062]Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding PKSRPs that contain changes in amino acid residues that are not essential for PKSRP activity. Such PKSRPs differ in amino acid sequence from a sequence contained in SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30, yet retain at least one of the PKSRP activities described herein. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence at least about 50% identical to an amino acid sequence of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ if) NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30. Preferably, the polypeptide encoded by the nucleic acid molecule is at least about 50-60% identical to one of the sequences of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30, more preferably at least about 60-70% identical to one of the sequences of SEQ ID NO:3, SEQ D) NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ If NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30, even more preferably at least about 70-75%, 75-80%, 80-85%, 85-90%, 90-95% identical to one of the sequences of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO:16, SEQ if NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30, and most preferably at least about 96%, 97%, 98%, or 99% identical to one of the sequences of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30. The preferred PKSRP homologs of the present invention participate in the a stress tolerance response in a plant, or more particularly, participate in the transcription of a polypeptide involved in a stress tolerance response in a plant, and/or function as a protein kinase.
[0063]An isolated nucleic acid molecule encoding a PKSRP having sequence identity with a polypeptide sequence of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:116, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30 can be created by introducing one or more nucleotide substitutions, additions or deletions into a nucleotide sequence of SEQ ID NO:2, SEQ ID NO:5 SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29, respectively, such that one or more amino acid substitutions, additions, or deletions are introduced into the encoded polypeptide. Mutations can be introduced into one of the sequences of SEQ ID NO:2, SEQ ID NO:5 SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
[0064]Families of amino acid residues having similar side chains have been defined in the art These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a PKSRP is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a PKSRP coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for a PKSRP activity described herein to identify mutants that retain PKSRP activity. Following mutagenesis of one of the sequences of SEQ ID NO:2, SEQ ID NO:5 SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29, the encoded polypeptide can be expressed recombinantly and the activity of the polypeptide can be determined by analyzing the stress tolerance of a plant expressing the polypeptide as described in Example 7.
[0065]Additionally, optimized PKSRP nucleic acids can be created. Preferably, an optimized PKSRP nucleic acid encodes a PKSRP that functions as a protein kinase and/or modulates a plant's tolerance to an environmental stress, and more preferably increases a plant's tolerance to an environmental stress upon its overexpression in the plant. As used herein, "optimized" refers to a nucleic acid that is genetically engineered to increase its expression in a given plant or animal. To provide plant optimized PKSRP nucleic acids, the DNA sequence of the gene can be modified to 1) comprise codons preferred by highly expressed plant genes; 2) comprise an A+T content in nucleotide base composition to that substantially found in plants; 3) form a plant initiation sequence; or 4) eliminate sequences that cause destabilization, inappropriate polyadenylation, degradation, and termination of RNA, or that form secondary structure hairpins or RNA splice sites Increased expression of PKSRP nucleic acids in plants can be achieved by utilizing the distribution frequency of codon usage in plants in general or a particular plant. Methods for optimizing nucleic acid expression in plants can be found in EPA 0359472; EPA 0385962; PCT Application No. WO 91/16432; U.S. Pat. No. 5,380,831; U.S. Pat. No. 5,436,391; Perlack et at., 1991, Proc. Natl. Acad. Sci. USA 88:3324-3328; and Murray et al., 1989, Nucleic Acids Res. 17:477-498.
[0066]As used herein, "frequency of preferred codon usage" refers to the preference exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. To determine the frequency of usage of a particular codon in a gene, the number of occurrences of that codon in the gene is divided by the total number of occurrences of all codons specifying the same amino acid in the gene. Similarly, the frequency of preferred codon usage exhibited by a host cell can be calculated by averaging frequency of preferred codon usage in a large number of genes expressed by the host cell. It is preferable that this analysis be limited to genes that are highly expressed by the host cell. The percent deviation of the frequency of preferred codon usage for a synthetic gene from that employed by a host cell is calculated first by determining the percent deviation of the frequency of usage of a single codon from that of the host cell followed by obtaining the average deviation over all codons. As defined herein, this calculation includes unique codons (i.e., ATG and TGG). In general terms, the overall average deviation of the codon usage of an optimized gene from that of a host cell is calculated using the equation 1A=n=1 Z Xn-YnXn times 100 Z where Xn=frequency of usage for codon n in the host cell; Yn=frequency of usage for codon n in the synthetic gene; n represents an individual codon that specifies an amino acid; and the total number of codons is Z. The overall deviation of the frequency of codon usage, A, for all amino acids should preferably be less than about 25%, and more preferably less than about 10%.
[0067]Hence, a PKSRP nucleic acid can be optimized such that its distribution frequency of codon usage deviates, preferably, no more than 25% from that of highly expressed plant genes and, more preferably, no more than about 10%. In addition, consideration is given to the percentage G+C content of the degenerate third base (monocotyledons appear to favor G+C in this position, whereas dicotyledons do not). It is also recognized that the XCG (where X is A, T, C, or G) nucleotide is the least preferred codon in dicots whereas the XTA codon is avoided in both monocots and dicots. Optimized PKSRP nucleic acids of this invention also preferably have CG and TA doublet avoidance indices closely approximating those of the chosen host plant (i.e., Physcomitrella patens, Brassica napus, Glycine max, or Oryza sativa). More preferably these indices deviate from that of the host by no more than about 10-15%.
[0068]In addition to the nucleic acid molecules encoding the PKSRPs described above, another aspect of the invention pertains to isolated nucleic acid molecules that are antisense thereto. Antisense polynucleotides are thought to inhibit gene expression of a target polynucleotide by specifically binding the target polynucleotide and interfering with transcription, splicing, transport, translation, and/or stability of the target polynucleotide. Methods are described in the prior art for targeting the antisense polynucleotide to the chromosomal DNA, to a primary RNA transcript, or to a processed mRNA. Preferably, the target regions include splice sites, translation initiation codons, translation termination codons, and other sequences within the open reading frame.
[0069]The term "antisense," for the purposes of the invention, refers to a nucleic acid comprising a polynucleotide that is sufficiently complementary to all or a portion of a gene, primary transcript, or processed mRNA, so as to interfere with expression of the endogenous gene. "Complementary" polynucleotides are those that are capable of base pairing according to the standard Watson-Crick complementarity rules. Specifically, purines will base pair with pyrimidines to form a combination of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNAi. It is understood that two polynucleotides may hybridize to each other even if they are not completely complementary to each other, provided that each has at least one region that is substantially complementary to the other. The term "antisense nucleic acid" includes single stranded RNA as well as double-stranded DNA expression cassettes that can be transcribed to produce an antisense RNA. "Active" antisense nucleic acids are antisense RNA molecules that are capable of selectively hybridizing with a primary transcript or mRNA encoding a polypeptide having at least 80% sequence identity with the polypeptide of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30.
[0070]The antisense nucleic acid can be complementary to an entire PKSRP coding strand, or to only a portion thereof. In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding a PKSRP. The term "coding region" refers to the region of the nucleotide sequence comprising codons that are translated into amino acid residues (e.g., the entire coding region of PK-3 comprises nucleotides 138-1409 of SEQ ID NO:2, and the entire coding region of PK-4 comprises nucleotides 142-1395 of SEQ ID NO:5). In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding a PKSRP. The term "noncoding region" refers to 5' and 3' sequences that flank the coding region that are not translated into amino acids (ire., also referred to as 5' and 3' untranslated regions). The antisense nucleic acid molecule can be complementary to the entire coding region of PKSRP mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of PKSRP mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of PKSRP mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. Typically, the antisense molecules of the present invention comprise an RNA having 60-100% sequence identity with at least 14 consecutive nucleotides of SEQ ID NO:2, SEQ ID NO:5 SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29 or a polynucleotide encoding SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30. Preferably, the sequence identity will be at least 70%, more preferably at least 75%, 80%, 85%, 90%, 95%, 98% and most preferably 99%.
[0071]An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyluracil, 2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
[0072]In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al., 1987, Nucleic Acids. Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al., 1987, Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-330).
[0073]The antisense nucleic acid molecules of the invention are typically administered to a cell or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a PKSRP to thereby inhibit expression of the polypeptide, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. The antisense molecule can be modified such that it specifically binds to a receptor or an antigen expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecule to a peptide or an antibody which binds to a cell surface receptor or antigen. The antisense nucleic acid molecule can also be delivered to cells using the vectors described herein, To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong prokaryotic, viral, or eukaryotic (including plant) promoter are preferred.
[0074]As an alternative to antisense polynucleotides, ribozymes, sense polynucleotides, or double stranded RNA (dsRNA) can be used to reduce expression of a PKSRP polypeptide. By "ribozyme" is meant a catalytic RNA-based enzyme with ribonuclease activity which is capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which it has a complementary region. Ribozymes (e.g., hammerhead ribozymes described in Haselhoff and Gerlach, 1988, Nature 334:585-591) can be used to catalytically cleave PKSRP mRNA transcripts to thereby inhibit translation of PKSRP mRNA. A ribozyme having specificity for a PKSRP-encoding nucleic acid can be designed based upon the nucleotide sequence of a PKSRP cDNA, as disclosed herein (i.e., SEQ ID NO:2, SEQ ID NO:5 SEQ ID NO:8, SEQ ID NO: 1, SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29) or on the basis of a heterologous sequence to be isolated according to methods taught in this invention. For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a PKSRP-encoding mRNA. See, e.g., U.S. Pat. Nos. 4,987,071 and 5,116,742 to Cech et al. Alternatively, PKSRP mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W., 1993, Science 261:1411-1418. In preferred embodiments, the ribozyme will contain a portion having at least 7, 8, 9, 10, 12, 14, 16, 18 or 20 nucleotides, and more preferably 7 or 8 nucleotides, that have 100% complementarity to a portion of the target RNA. Methods for making ribozymes are known to those skilled in the art. See, e.g., U.S. Pat. Nos. 6,025,167; 5,773,260; and 5,496,698.
[0075]The term "dsRNA," as used herein, refers to RNA hybrids comprising two strands of RNA. The dsRNAs can be linear or circular in structure. In a preferred embodiment, dsRNA is specific for a polynucleotide encoding either the polypeptide of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30 or a polypeptide having at least 70% sequence identity with SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30. The hybridizing RNAs may be substantially or completely complementary. By "substantially complementary," is meant that when the two hybridizing RNAs are optimally aligned using the BLAST program as described above, the hybridizing portions are at least 95% complementary. Preferably, the dsRNA will be at least 100 base pairs in length, Typically, the hybridizing RNAs will be of identical length with no over hanging 5' or 3' ends and no gaps. However, dsRNAs having 5' or 3' overhangs of up to 100 nucleotides may be used in the methods of the invention.
[0076]The dsRNA may comprise ribonucleotides or ribonucleotide analogs, such as 2'-O-methyl ribosyl residues, or combinations thereof. See, e.g., U.S. Pat. Nos. 4,130,641 and 4,024,222. A dsRNA polyriboinosinic acid:polyribocytidylic acid is described in U.S. Pat. No. 4,283,393. Methods for making and using dsRNA are known in the art. One method comprises the simultaneous transcription of two complementary DNA strands, either in vivo, or in a single in vitro reaction mixture. See, e.g., U.S. Pat. No. 5,795,715. In one embodiment, dsRNA can be introduced into a plant or plant cell directly by standard transformation procedures. Alternatively, dsRNA can be expressed in a plant cell by transcribing two complementary RNAs.
[0077]Other methods for the inhibition of endogenous gene expression, such as triple helix formation (Moser et al., 1987, Science 238:645-650 and Cooney et al., 1988, Science 241:456-459) and cosuppression (Napoli et al., 1990, The Plant Cell 2:279-289) are known in the art. Partial and full-length cDNAs have been used for the cosuppression of endogenous plant genes. See, e.g., U.S. Pat. Nos. 4,801,340, 5,034,323, 5,231,020, and 5,283,184; Van der Kroll et al., 1990, The Plant Cell 2:291-299; Smith et al., 1990, Mol. Gen. Genetics 224:477-481 and Napoli et al., 1990, The Plant Cell 2:279-289.
[0078]For sense suppression, it is believed that introduction of a sense polynucleotide blocks transcription of the corresponding target gene. The sense polynucleotide will have at least 65% sequence identity with the target plant gene or RNA. Preferably, the percent identity is at least 80%, 90% Yo, 95% or more. The introduced sense polynucleotide need not be full length relative to the target gene or transcript. Preferably, the sense polynucleotide will have at least 65% sequence identity with at least 100 consecutive nucleotides of SEQ ID NO:2, SEQ ID NO:5 SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29. The regions of identity can comprise introns and and/or exons and untranslated regions. The introduced sense polynucleotide may be present in the plant cell transiently, or may be stably integrated into a plant chromosome or extrachromosomal replicon.
[0079]Alternatively, PKSRP gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of a PKSRP nucleotide sequence (e.g., a PKSRP promoter and/or enhancer) to form triple helical structures that prevent transcription of a PKSRP gene in target cells. See generally, Helene, C., 1991, Anticancer Drug Des. 6(6):569-84; Helene, C. et al., 1992, Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L. J., 1992, Bioassays 14(12):807-15.
[0080]In addition to the PKSRP nucleic acids and polypeptides described above, the present invention encompasses these nucleic acids and polypeptides attached to a moiety. These moieties include, but are not limited to, detection moieties, hybridization moieties, purification moieties, delivery moieties, reaction moieties, binding moieties, and the like. A typical group of nucleic acids having moieties attached are probes and primers. Probes and primers typically comprise a substantially isolated oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, preferably about 25, more preferably about 40, 50 or 75 consecutive nucleotides of a sense strand of one of the sequences set forth in SEQ ID NO:2, SEQ ID NO:5 SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29; an anti-sense sequence of one of the sequences set forth in SEQ ID NO:2, SEQ ID NO5 SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29; or naturally occurring mutants thereof. Primers based on a nucleotide sequence of SEQ ID NO:2, SEQ ID NO:5 SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ If NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ if NO:25, SEQ ID NO:27, or SEQ ID NO:29 can be used in PCR reactions to clone PKSRP homologs. Probes based on the PKSRP nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or substantially identical polypeptides. In preferred embodiments, the probe further comprises a label group attached thereto, e.g. the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a genomic marker test kit for identifying cells which express a PKSRP, such as by measuring a level of a PKSRP-encoding nucleic acid, in a sample of cells, e.g., detecting PKSRP mRNA levels or determining whether a genomic PKSRP gene has been mutated or deleted.
[0081]In particular, a useful method to ascertain the level of transcription of the gene (an indicator of the amount of mRNA available for translation to the gene product) is to perform a Northern blot. For reference, see, for example, Ausubel et al., 1988, Current Protocols in Molecular Biology, Wiley: New York. The information from a Northern blot at least partially demonstrates the degree of transcription of the transformed gene. Total cellular RNA can be prepared from cells, tissues or organs by several methods, all well-known in the art, such as that described in Bormann, E. R. et al., 1992, Mol. Microbiol. 6:317-326. To assess the presence or relative quantity of polypeptide translated from this mRNA, standard techniques, such as a Western blot, may be employed. These techniques are well known to one of ordinary skill in the art. See, for example, Ausubel et al., 1988, Current Protocols in Molecular Biology, Wiley: New York.
[0082]The invention further provides an isolated recombinant expression vector comprising a PKSRP nucleic acid as described above, wherein expression of the vector in a host cell results in increased tolerance to environmental stress as compared to a wild type variety of the host cell. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.
[0083]The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. As used herein with respect to a recombinant expression vector, "operatively linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequencers) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) and Gruber and Crosby, in: Methods in Plant Molecular Biology and Biotechnology, eds, Glick and Thompson, Chapter 7, 89-108, CRC Press: Boca Raton, Fla., including the references therein. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells or under certain conditions. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce polypeptides or peptides, including fusion polypeptides or peptides, encoded by nucleic acids as described herein (e.g., PKSRPs, mutant forms of PKSRPs, fusion polypeptides, etc.).
[0084]The recombinant expression vectors of the invention can be designed for expression of PKSRPs in prokaryotic or eukaryotic cells. For example, PKSRP genes can be expressed in bacterial cells such as C. glutamicum, insect cells (using baculovirus expression vectors), yeast and other fungal cells (See Romanos, M. A. et al., 1992, Foreign gene expression in yeast: a review, Yeast 8:423-488; van den Hondel, C. A. M. J. J. et al., 1991, Heterologous gene expression in filamentous fungi, in: More Gene Manipulations in Fungi, J. W. Bennet & L. L. Lasure, eds., p. 396-428: Academic Press: San Diego; and van den Hondel, C. A. M. J. J. & Punt, P. J., 1991, Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, Peberdy, J. F. et al., eds., p. 1-28, Cambridge University Press: Cambridge), algae Falciatore et al., 1999, Marine Biotechnology 1(3):239-251), ciliates of the types: Holotrichia, Peritrichia, Spirotrichia, Suctoria, Tetrahymena, Paramecium, Colpidium, Glaucoma, Platyophrya, Potomacus, Pseudocohnilembus, Euplotes, Engelmaniella, and Stylonychia, especially of the genus Stylonychia lemnae with vectors following a transformation method as described in PCT Application No. WO 98/01572, and multicellular plant cells (See Schmidt, R. and Willmitzer, L., 1988, High efficiency Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana leaf and cotyledon explants, Plant Cell Rep. 583-586; Plant Molecular Biology and Biotechnology, C Press, Boca Raton, Fla., chapter 6/7, S.71-119 (1993); F. F. White, B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds. Kung und R. Wu, 128-43, Academic Press: 1993; Potrykus, 1991, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42:205-225 and references cited therein) or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press: San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
[0085]Expression of polypeptides in prokaryotes is most often carried out with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polypeptides. Fusion vectors add a number of amino acids to a polypeptide encoded therein, usually to the amino terminus of the recombinant polypeptide but also to the C-terminus or fused within suitable regions in the polypeptides. Such fusion vectors typically serve three purposes: 1) to increase expression of a recombinant polypeptide; 2) to increase the solubility of a recombinant polypeptide; and 3) to aid in the purification of a recombinant polypeptide by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide to enable separation of the recombinant polypeptide from the fusion moiety subsequent to purification of the fusion polypeptide. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin, and enterokinase.
[0086]Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S., 1988, Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding polypeptide, or polypeptide A, respectively, to the target recombinant polypeptide. In one embodiment, the coding sequence of the PKSRP is cloned into a pGEX expression vector to create a vector encoding a fusion polypeptide comprising, from the N-terminus to the C-terminus, GST-thrombin cleavage site-X polypeptide. The fusion polypeptide can be purified by affinity chromatography using glutathione-agarose resin. Recombinant PKSRP unfused to GST can be recovered by cleavage of the fusion polypeptide with thrombin.
[0087]Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., 1988, Gene 69:301-315) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET 11d vector relies on transcription from a T7 gn10-lac fusion promoter mediated by a co-expressed viral RNA polymerase (T7 gn1). This viral polymerase is supplied by host strains BL21 (DE3) or UMS174(DE3) from a resident λ prophage harboring a T7 gn1 gene under the transcriptional control of the lacUV 5 promoter.
[0088]One strategy to maximize recombinant polypeptide expression is to express the polypeptide in a host bacteria with an impaired capacity to proteolytically cleave the recombinant polypeptide (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128). Another strategy is to alter the sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in the bacterium chosen for expression, such as C. glutamicum (Wada et al., 1992, Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
[0089]In another embodiment, the PKSRP expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSec1 (Baldari, et al., 1987, EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, 1982, Cell 30:933-943), pJRY88 (Schultz et al., 1987, Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.). Vectors and methods for the construction of vectors appropriate for use in other fungi, such as the filamentous fungi, include those detailed in: van den Hondel, C. A. M. J. J. & Punt, P. J., 1991, "Gene transfer systems and vector development for filamentous fungi," in: Applied Molecular Genetics of Fungi, J. F. Peberdy, et al., eds., p. 1-28, Cambridge University Press: Cambridge.
[0090]Alternatively, the PKSRPs of the invention can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of polypeptides in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al., 1983, Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow and Summers, 1989, Virology 170:31-39).
[0091]In yet another embodiment a PKSRP nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, B., 1987, Nature 329:840) and pMT2PC (Kaufman et al., 1987, EMBO J. 6:187-195), When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus, and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells, see chapters 16 and 17 of Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
[0092]In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al., 1987, Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton, 1988, Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989, EMBO J. 8:729-733) and immunoglobulins (Banerji et al., 1983, Cell 33:729-740; Queen and Baltimore, 1983, Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989, PNAS 86:5473-5477), pancreas-specific promoters (Edlund et al., 1985, Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example, the murine hox promoters (Kessel and Gruss, 1990, Science 249:374-379) and the fetopolypeptide promoter (Campes and Tilghman, 1989, Genes Dev. 3:537-546).
[0093]For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identity and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin, and methotrexate, or in plants that confer resistance towards a herbicide such as glyphosate or glufosinate. Nucleic acid molecules encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a PKSRP or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid molecule can be identified by, for example, drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
[0094]In a preferred embodiment of the present invention, the PKSRPs are expressed in plants and plants cells such as unicellular plant cells (e.g. algae) (See Falciatore et al., 1999, Marine Biotechnology 1(3):239-251 and references therein) and plant cells from higher plants (e.g., the spermatophytes, such as crop plants). A PKSRP may be "introduced" into a plant cell by any means, including transfection, transformation or transduction, electroporation, particle bombardment, agroinfection, and the like. One transformation method known to those of skill in the art is the dipping of a flowering plant into an Agrobacteria solution, wherein the Agrobacteria contains the PKSRP nucleic acid, followed by breeding of the transformed gametes.
[0095]Other suitable methods for transforming or transfecting host cells including plant cells can be found in Sambrook, et al., Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, and other laboratory manuals such as Methods in Molecular Biology, 1995, Vol. 44, Agrobacterium protocols, ed: Gartland and Davey, Humana Press, Totowa, N.J. As biotic and abiotic stress tolerance is a general trait wished to be inherited into a wide variety of plants like maize, wheat, rye, oat, triticale, rice, barley, soybean, peanut, cotton, rapeseed and canola, manihot, pepper, sunflower and tagetes, solanaceous plants like potato, tobacco, eggplant, and tomato, Vicia species, pea, alfalfa, bushy plants (coffee, cacao, tea), Salix species, trees (oil palm, coconut), perennial grasses, and forage crops, these crop plants are also preferred target plants for a genetic engineering as one further embodiment of the present invention. Forage crops include, but are not limited to, Wheatgrass, Canarygrass, Bromegrass, Wildrye Grass, Bluegrass, Orchardgrass, Alfalfa, Salfoin, Birdsfoot Trefoil, Alsike Clover, Red Clover, and Sweet Clover.
[0096]In one embodiment of the present invention, transfection of a PKSRP into a plant is achieved by Agrobacterium mediated gene transfer. Agrobacterium mediated plant transformation can be performed using for example the GV3101 (pMP90) (Koncz and Schell, 1986, Mol. Gen. Genet. 204:383-396) or LBA4404 (Clontech) Agrobacterium tumefaciens strain. Transformation can be performed by standard transformation and regeneration techniques (Deblaere et al., 1994, Nucl. Acids Res. 13:4777-4788; Gelvin, Stanton B. and Schilperoort, Robert A, Plant Molecular Biology Manual, 2nd Ed.--Dordrecht: Kluwer Academic Publ., 1995.--in Sect., Ringbuc Zentrale Signatur: BT11-P ISBN 0-7923-2731-4; Glick, Bernard R.; Thompson, John E., Methods in Plant Molecular Biology and Biotechnology, Boca Raton: CRC Press, 1993 360 S., ISBN 0-8493-5164-2). For example, rapeseed can be transformed via cotyledon or hypocotyl transformation (Moloney et al., 1989, Plant cell Report 8:238-242; De Block et al., 1989, Plant Physiol. 91:694-701). Use of antibiotics for Agrobacterium and plant selection depends on the binary vector and the Agrobacterium strain used for transformation. Rapeseed selection is normally performed using kanamycin as selectable plant marker. Agrobacterium mediated gene transfer to flax can be performed using, for example, a technique described by Mlynarova et al., 1994, Plant Cell Report 13:282-285. Additionally, transformation of soybean can be performed using for example a technique described in European Patent No. 0424 047, U.S. Pat. No. 5,322,783, European Patent No. 0397 687, U.S. Pat. No. 5,376,543, or U.S. Pat. No. 5,169,770. Transformation of maize can be achieved by particle bombardment, polyethylene glycol mediated DNA uptake or via the silicon carbide fiber technique. (See, for example, Freeling and Walbot "The maize handbook" Springer Verlag: New York (1993) ISBN 3-540-97826-7). A specific example of maize transformation is found in U.S. Pat. No. 5,990,387, and a specific example of wheat transformation can be found in PCT Application No. WO 93/07256.
[0097]According to the present invention, the introduced PKSRP may be maintained in the plant cell stably if it is incorporated into a non-chromosomal autonomous replicon or integrated into the plant chromosomes. Alternatively, the introduced PKSRP may be present on an extra-chromosomal non-replicating vector and be transiently expressed or transiently active.
[0098]In one embodiment, a homologous recombinant microorganism can be created wherein the PKSRP is integrated into a chromosome, a vector is prepared which contains at least a portion of a PKSRP gene into which a deletion, addition, or substitution has been introduced to thereby alter, e.g., functionally disrupt, the PKSRP gene. Preferably, the PKSRP gene is a Physomitrella patens, Brassica napus, Glycine max, or Oryza saliva PKSRP gene, but it can be a homolog from a related plant or even from a mammalian, yeast, or insect source. In one embodiment the vector is designed such that, upon homologous recombination, the endogenous PKSRP gene is functionally disrupted (i.e., no longer encodes a functional polypeptide; also referred to as a knock-out vector). Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous PKSRP gene is mutated or otherwise altered but still encodes a functional polypeptide (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous PKSRP). To create a point mutation via homologous recombination, DNA-RNA hybrids can be used in a technique known as chimeraplasty (Cole-Strauss et al., 1999, Nucleic Acids Research 27(5):1323-1330 and Kmiec, 1999 Gene therapy American Scientist. 87(3):240-247). Homologous recombination procedures in Physcomitrella patens are also well known in the art and are contemplated for use herein.
[0099]Whereas in the homologous recombination vector, the altered portion of the PKSRP gene is flanked at its 5, and 3' ends by an additional nucleic acid molecule of the PKSRP gene to allow for homologous recombination to occur between the exogenous PKSRP gene carried by the vector and an endogenous PKSRP gene, in a microorganism or plant. The additional flanking PKSRP nucleic acid molecule is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several hundreds of base pairs up to kilobases of flanking DNA (both at the 5' and 3' ends) are included in the vector. See, e.g., Thomas, K. R., and Capecchi, M. R., 1987, Cell 51:503 for a description of homologous recombination vectors or Strepp et al., 1998, PNAS, 95 (8):4368-4373 for cDNA based recombination in Physcomitrella patens). The vector is introduced into a microorganism or plant cell (e.g., via polyethylene glycol mediated DNA), and cells in which the introduced PKSRP gene has homologously recombined with the endogenous PKSRP gene are selected using art-known techniques.
[0100]In another embodiment, recombinant microorganisms can be produced that contain selected systems which allow for regulated expression of the introduced gene. For example, inclusion of a PKSRP gene on a vector placing it under control of the lac operon permits expression of the PKSRP gene only in the presence of IPTG. Such regulatory systems are well known in the art.
[0101]Whether present in an extra-chromosomal non-replicating vector or a vector that is integrated into a chromosome, the PKSRP polynucleotide preferably resides in a plant expression cassette. A plant expression cassette preferably contains regulatory sequences capable of driving gene expression in plant cells that are operatively linked so that each sequence can fulfill its function, for example, termination of transcription by polyadenylation signals. Preferred polyadenylation signals are those originating from Agrobacterium tumefaciens t-DNA such as the gene 3 known as octopine synthase of the Ti-plasmid pTiACH5 (Gielen et al., 1984, EMBO J. 3:835) or functional equivalents thereof but also all other terminators functionally active in plants are suitable. As plant gene expression is very often not limited on transcriptional levels, a plant expression cassette preferably contains other operatively linked sequences like translational enhancers such as the overdrive-sequence containing the 5'-untranslated leader sequence from tobacco mosaic virus enhancing the polypeptide per RNA ratio (Gallie et al., 1987, Nucl. Acids Research 15:8693-8711). Examples of plant expression vectors include those detailed in: Becker, D. et al., 1992, New plant binary vectors with selectable markers located proximal to the left border, Plant Mol. Biol. 20: 1195-1197; and Bevan, M. W., 1984, Binary Agrobacterium vectors for plant transformation, Nucl. Acid. Res. 12:8711-8721; and Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds.: Kung and R. Wu, Academic Press, 1993, S. 15-38.
[0102]Plant gene expression should be operatively linked to an appropriate promoter conferring gene expression in a timely, cell or tissue specific manner. Promoters useful in the expression cassettes of the invention include any promoter that is capable of initiating transcription in a plant cell. Such promoters include, but are not limited to, those that can be obtained from plants, plant viruses, and bacteria that contain genes that are expressed in plants, such as Agrobacterium and Rhizobium.
[0103]The promoter may be constitutive, inducible, developmental stage-preferred, cell type-preferred, tissue-preferred, or organ-preferred. Constitutive promoters are active under most conditions. Examples of constitutive promoters include the CaMV 19S and 35 S promoters (Odell et al., 1985, Nature 313:810-812), the sX CaMV 35 S promoter (Kay et al., 1987, Science 236:1299-1302) the Sep1 promoter, the rice actin promoter (McElroy et al., 1990, Plant Cell 2:163-171), the Arabidopsis actin promoter, the ubiquitan promoter (Christensen et al., 1989, Plant Molec Biol 18:675-689); pemu (Last et al., 1991, Theor Appl Genet. 81:581-588), the figwort mosaic virus 35S promoter, the Smas promoter (Velten et al., 1984, EMBO J. 3:2723-2730), the GRP1-8 promoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439), promoters from the T-DNA of Agrobacterium, such as mannopine synthase, nopaline synthase, and octopine synthase, the small subunit of ribulose biphosphate carboxylase (ssuRUBISCO) promoter, and the like.
[0104]Inducible promoters are active under certain environmental conditions, such as the presence or absence of a nutrient or metabolite, heat or cold, light, pathogen attack, anaerobic conditions, and the like, For example, the hsp80 promoter from Brassica is induced by heat shock; the PPDK promoter is induced by light; the PR-1 promoter from tobacco, Arabidopsis, and maize are inducible by infection with a pathogen; and the Adh1 promoter is induced by hypoxia and cold stress. Plant gene expression can also be facilitated via an inducible promoter (For a review, see Gatz, 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:89-108). Chemically inducible promoters are especially suitable if gene expression is wanted to occur in a time specific manner. Examples of such promoters are a salicylic acid inducible promoter (PCT Application No. WO 95/19443), a tetracycline inducible promoter (Gatz et al., 1992, Plant J. 2:397-404), and an ethanol inducible promoter (PCT Application No. WO 93/21334).
[0105]In one preferred embodiment of the present invention, the inducible promoter is a stress-inducible promoter. Stress inducible promoters include, but are not limited to, Cor78 (Chak et al., 2000, Planta 210:875-883; Hovath et al., 1993, Plant Physiol. 103:1047-1053), Cor15a (Artus et al., 1996, PNAS 93(23):13404-09), Rci2A (Medina et al., 2001, Plant Physiol. 125:1655-66; Nylander et al., 2001, Plant Mol. Biol. 45:341-52; Navarre and Goffeau, 2000, EMBO J. 19:2515-24; Capel et al., 1997, Plant Physiol. 115:569-76), Rd22 (Xiong et al., 2001, Plant Cell 13:2063-83; Abe et al., 1997, Plant Cell 9:1859-68; Ivasaki et al., 1995, Mol. Gen. Genet. 247:391-8), cDet6 (Lang and Palve, 1992, Plant Mol. Biol. 20:951-62), ADH1 (Hoeren et al., 1998, Genetics 149:479-90), KAT1 (Nakamura et al., 1995, Plant Physiol. 109:371-4), KST1 (Muller-Rober et al., 1995, EMBO 14:2409-16), Rha1 (Terryn et al., 1993, Plant Cell 5:1761-9; Terryn et al., 1992, FEBS Lett. 299(3):287-90), ARSK1 (Atkinson et al., 1997, GenBank Accession # L22302, and PCT Application No. WO 97/20057), PtxA (Plesch et al., GenBank Accession # X67427), SbHRGP3 (Ahn et al., 1996, Plant Cell 8:1477-90), GH3 (Liu et al., 1994, Plant Cell 6:645-57), the pathogen inducible PRP1-gene promoter (Ward et al, 1993, Plant. Mol. Biol. 22:361-366), the heat inducible hsp80-promoter from tomato (U.S. Pat. No. 5,187,267), cold inducible alpha-amylase promoter from potato (PCT Application No. WO 96/12814), or the wound-inducible pinII-promoter (European Patent No. 375091). For other examples of drought, cold, and salt-inducible promoters, such as the RD29A promoter, see Yamaguchi-Shinozalei et al., 1993, Mol. Gen. Genet. 236:331-340.
[0106]Developmental stage-preferred promoters are preferentially expressed at certain stages of development. Tissue and organ preferred promoters include those that are preferentially expressed in certain tissues or organs, such as leaves, roots, seeds, or xylem. Examples of tissue preferred and organ preferred promoters include, but are not limited to fruit-preferred, ovule-preferred, male tissue-preferred, seed-preferred, integument-preferred, tuber-preferred, stalk-preferred, pericarp-preferred, and leaf-preferred, stigma-preferred, pollen-preferred, anther-preferred, a petal-preferred, sepal-preferred, pedicel-preferred, silique-preferred, stem-preferred, root-preferred promoters, and the like. Seed preferred promoters are preferentially expressed during seed development and/or germination. For example, seed preferred promoters can be embryo-preferred, endosperm preferred, and seed coat-preferred. See Thompson et al., 1989, BioEssays 10:108. Examples of seed preferred promoters include, but are not limited to, cellulose synthase (ce1A), Cim1, gamma-zein, globulin-1, maize 19 kD zein (cZ19B1), and the like.
[0107]Other suitable tissue-preferred or organ-preferred promoters include the napin-gene promoter from rapeseed (U.S. Pat. No. 5,608,152), the USP-promoter from Vicia faba (Baeumlein et al., 1991, Mol Gen Genet. 225(3):459-67), the oleosin-promoter from Arabidopsis PCT Application No. WO 98/45461), the phaseolin-promoter from Phaseohis vulgaris (U.S. Pat. No. 5,504,200), the Bce-4-promoter from Brassica (PCT Application No. WO 91/13980), or the legumin B4 promoter (LeB4; Baeumlein et al. 1992, Plant Journal, 2(2):233-9) as well as promoters conferring seed specific expression in monocot plants like maize, barley, wheat, rye, rice, etc. Suitable promoters to note are the Ipt2 or Ipt1-gene promoter from barley (PCT Application No. WO 95/15389 and PCT Application No. WO 95/23230) or those described in PCT Application No. WO 99/16890 (promoters from the barley hordein-gene, rice glutelin gene, rice oryzin gene, rice prolamin gene, wheat gliadin gene, wheat glutelin gene, oat glutelin gene, Sorghum kasirin-gene, and rye secalin gene).
[0108]Other promoters useful in the expression cassettes of the invention include, but are not limited to, the major chlorophyll a/b binding protein promoter, histone promoters, the Ap3 promoter, the β-conglycin promoter, the napin promoter, the soybean lectin promoter, the maize 15 kD zein promoter, the 22 kD zein promoter, the 27 kD zein promoter, the g-zein promoter, the waxy, shrunken 1, shrunken 2 and bronze promoters, the Zm13 promoter (U.S. Pat. No. 5,086,169), the maize polygalacturonase promoters (PG) (U.S. Pat. Nos. 5,412,085 and 5,545,546), and the SGB6 promoter (U.S. Pat. No. 5,470,359), as well as synthetic or other natural promoters.
[0109]Additional flexibility in controlling heterologous gene expression in plants may be obtained by using DNA binding domains and response elements from heterologous sources (i.e., DNA binding domains from non-plant sources). An example of such a heterologous DNA binding domain is the LexA DNA binding domain (Brent and Ptashne, 1985, Cell 43:729-736).
[0110]The invention further provides a recombinant expression vector comprising a PKSRP DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to a PKSRP mRNA. Regulatory sequences operatively linked to a nucleic acid molecule cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types. For instance, viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific, or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid, or attenuated virus wherein antisense nucleic acids are produced under the control of a high efficiency regulatory region. The activity of the regulatory region can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes, see Weintraub, H. et at., 1986, Antisense RNA as a molecular toot for genetic analysis, Reviews--Trends in Genetics, Vol. 1(1), and Mol et al., 1990, FEBS Letters 268:427-430.
[0111]Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but they also apply to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not) in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. A host cell can be any prokaryotic or eukaryotic cell. For example, a PKSRP can be expressed in bacterial cells such as C. glutamicum, insect cells, fungal cells, or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells), algae, ciliates, plant cells, fungi, or other microorganisms like C. glutamicum. Other suitable host cells are known to those skilled in the art.
[0112]A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) a PKSRP. Accordingly, the invention further provides methods for producing PKSRPs using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding a PKSRP has been introduced, or into which genome has been introduced a gene encoding a wild-type or altered PKSRP) in a suitable medium until PKSRP is produced. In another embodiment, the method further comprises isolating PKSRPs from the medium or the host cell.
[0113]Another aspect of the invention pertains to isolated PKSRPs, and biologically active portions thereof. An "isolated" or "purified" polypeptide or biologically active portion thereof is free of some of the cellular material when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of PKSRP in which the polypeptide is separated from some of the cellular components of the cells in which it is naturally or recombinantly produced. In one embodiment, the language "substantially free of cellular material" includes preparations of a PKSRP having less than about 30% (by dry weight) of non-PKSRP material (also referred to herein as a "contaminating polypeptide"), more preferably less than about 20% of non-PKSRP material, still more preferably less than about 10% of non-PKSRP material, and most preferably less than about 5% non-PKSRP material.
[0114]When the PKSRP or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the polypeptide preparation. The language "substantially free of chemical precursors or other chemicals" includes preparations of PKSRP in which the polypeptide is separated from chemical precursors or other chemicals that are involved in the synthesis of the polypeptide. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of a PKSRP having less than about 30% (by dry weight) of chemical precursors or non-PKSRP chemicals, more preferably less than about 20% chemical precursors or non-PKSRP chemicals, still more preferably less than about 10% chemical precursors or non-PKSRP chemicals, and most preferably less than about 5% chemical precursors or non-PKSRP chemicals. In preferred embodiments, isolated polypeptides, or biologically active portions thereof, lack contaminating polypeptides from the same organism from which the PKSRP is derived. Typically, such polypeptides are produced by recombinant expression of, for example, a Physcomitrella patens, Brassica napus, Glycine max, or Oryza saliva PKSRP in plants other than Physcomitrella patens, Brassica napus, Glycine max, or Oryza sativa, or microorganisms such as C. glutamicum, ciliates, algae or fungi.
[0115]The nucleic acid molecules, polypeptides, polypeptide homologs, fusion polypeptides, primers, vectors, and host cells described herein can be used in one or more of the following methods: identification of Physcomitrella patens, Brassica napus, Glycine max, or Oryza sativa and related organisms; mapping of genomes of organisms related to Physcomitrella patens, Brassica napus, Glycine max, or Oryza sativa; identification and localization of Physcomitrella patens, Brassica napus, Glycine max, or Oryza sativa sequences of interest; evolutionary studies; determination of PKSRP regions required for function; modulation of a PKSRP activity; modulation of the metabolism of one or more cell functions; modulation of the transmembrane transport of one or more compounds; modulation of stress resistance; and modulation of expression of PKSRP nucleic acids.
[0116]The moss Physcomitrella patens represents one member of the mosses. It is related to other mosses such as Ceratodon purpureus which is capable of growth in the absence of light. Mosses like Ceratodon and Physcomitrella share a high degree of sequence identity on the DNA sequence and polypeptide level allowing the use of heterologous screening of DNA molecules with probes evolving from other mosses or organisms, thus enabling the derivation of a consensus sequence suitable for heterologous screening or functional annotation and prediction of gene functions in third species. The ability to identify such functions can therefore have significant relevance, e.g., prediction of substrate specificity of enzymes. Further, these nucleic acid molecules may serve as reference points for the mapping of moss genomes, or of genomes of related organisms.
[0117]The PKSRP nucleic acid molecules of the invention have a variety of uses. Most importantly, the nucleic acid and amino acid sequences of the present invention can be used to transform plants, thereby inducing tolerance to stresses such as drought, high salinity and cold or lodging. The present invention therefore provides a transgenic plant transformed by a PKSRP nucleic acid, wherein expression of the nucleic acid sequence in the plant results in increased tolerance to environmental stress or increased resistance to lodging as compared to a wild type variety of the plant. The transgenic plant can be a monocot or a dicot. The invention further provides that the transgenic plant can be selected from maize, wheat, rye, oat, triticale, rice, barley, soybean, peanut, cotton, rapeseed, canola, manihot, pepper, sunflower, tagetes, solanaceous plants, potato, tobacco, eggplant, tomato, Vicia species, pea, alfalfa, coffee, cacao, tea, Salix species, oil palm, coconut, perennial grass, and forage crops, for example.
[0118]In particular, the present invention describes using the expression of PK-3, PK-4, PK-10, and PK-11 of Physcomitrella patens; the BnPK-1, BnPK-2, BnPK-3, and BnPK-4 of Brassica napus; the mPK-1, GmPK-2, GmPK-3, and GmPK-4 of Glycine max; and the OsPK-1 of Oryza sativa to engineer drought-tolerant salt-tolerant, cold-tolerant, and/or lodging-resistant plants. This strategy has herein been demonstrated for Arabidopsis thaliana, Rapeseed/Canola soybeans, corn, and wheat, but its application is not restricted to these plants. Accordingly, the invention provides a transgenic plant containing a PKSRP such as PK-3 as defined in SEQ ID NO:3, PK-4 as defined in SEQ ID NO:6, PK-10 as defined in SEQ ID NO:9, PK-11 as defined in SEQ ID NO:12, BnPK-1 as defined in SEQ ID NO:14, BnPK-2 as defined in SEQ ID NO:16, BnPK-3 as defined in SEQ ID NO:18, BnPK-4 as defined in SEQ If) NO:20, GmPK-1 as defined in SEQ ID NO:22, GmPK-2 as defined in SEQ ID NO:24, GmPK-3 as defined in SEQ if NO:26, GmPK-4 as defined in SEQ ID NO:28, and OsPK-1 as defined in SEQ ID NO:30, wherein the plant has an increased tolerance to an environmental stress selected from drought, increased salt, decreased or increased temperature, or lodging. In preferred embodiments, the environmental stress is drought or decreased temperature.
[0119]Accordingly, the invention provides a method of producing a transgenic plant with a PKSRP coding nucleic acid, wherein expression of the nucleic acid in the plant results in increased tolerance to environmental stress as compared to a wild type variety of the plant comprising: (a) introducing into a plant cell an expression vector comprising a PKSRP nucleic acid, and (b) generating from the plant cell a transgenic plant with an increased tolerance to environmental stress as compared to a wild type variety of the plant. Also included within the present invention are methods of increasing a plant's resistance to lodging, comprising transforming a plant cell with an expression cassette comprising a nucleic acid encoding a PKSRP and generating a transgenic plant from the transformed plant cell. The plant cell includes, but is not limited to, a protoplast, gamete producing cell, and a cell that regenerates into a whole plant. As used herein, the term "transgenic" refers to any plant, plant cell, callus, plant tissue, or plant part, that contains all or part of at least one recombinant polynucleotide. In many cases, all or part of the recombinant polynucleotide is stably integrated into a chromosome or stable extra-chromosomal element, so that it is passed on to successive generations. In preferred embodiments, the PKSRP nucleic acid encodes a protein comprising SEQ ID NO:3, SEQ It) NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:118, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ D) NO:28, or SEQ ID NO:30.
[0120]The present invention also provides a method of modulating a plant's tolerance to an environmental stress comprising, modifying the expression of a PKSRP coding nucleic acid in the plant. The plant's tolerance to the environmental stress can be increased or decreased as achieved by increasing or decreasing the expression of a PKSRP, respectively. Preferably, the plant's tolerance to the environmental stress is increased by increasing expression of a PKSRP. Expression of a PKSRP can be modified by any method known to those of skill in the art. The methods of increasing expression of PKSRPs can be used wherein the plant is either transgenic or not transgenic. In cases when the plant is transgenic, the plant can be transformed with a vector containing any of the above described PKSRP coding nucleic acids, or the plant can be transformed with a promoter that directs expression of native PKSRP in the plant, for example. The invention provides that such a promoter can be tissue specific, developmentally regulated, or stress-inducible. Alternatively, non-transgenic plants can have native PKSRP expression modified by inducing a native promoter. The expression of PK-3 as defined in SEQ ID NO:2, PK-4 as defined in SEQ ID NO:5, PK-10 as defined in SEQ ID NO:8, PK-11 as defined in SEQ ID NO:11, BnPK-1 as defined in SEQ ID NO:13, BnPK-2 as defined in SEQ ID NO:15, BnPK-3 as defined in SEQ ID NO:17, BnPK-4 as defined in SEQ ID NO:19, GmPK-1 as defined in SEQ ID NO:21, GmPK-2 as defined in SEQ ID NO:23, GmPK-3 as defined in SEQ ID NO:25, GmPK-4 as defined in SEQ ID NO:27, and OsPK-1 as defined in SEQ ID NO:29 in target plants can be accomplished by, but is not limited to, one of the following examples: (a) constitutive promoter, (b) stress-inducible promoter, (c) chemical-induced promoter, and (d) engineered promoter overexpression with, for example, zinc-finger derived transcription factors (Greisman and Pabo, 1997, Science 275:657).
[0121]In a preferred embodiment, transcription of the PKSRP is modulated using zinc-finger derived transcription factors (ZFPs) as described in Greisman and Pabo, 1997, Science 275:657 and manufactured by Sangamo Biosciences, Inc. These ZFPs comprise both a DNA recognition domain and a functional domain that causes activation or repression of a target nucleic acid such as a PKSRP nucleic acid. Therefore, activating and repressing ZFPs can be created that specifically recognize the PKSRP promoters described above and used to increase or decrease PKSRP expression in a plant, thereby modulating the stress tolerance of the plant. The present invention also includes identification of the homologs of SEQ ID NO:2, PK-4 as defined in SEQ ID NO:5, PK-10 as defined in SEQ ID NO:8, PK-11 as defined in SEQ ID NO: 11, BNPK-1 as defined in SEQ ID NO: 13, BnPK-2 as defined in SEQ ID NO:15, BnPK-3 as defined in SEQ ID NO:17, BnPK-4 as defined in SEQ ID NO:19, GmPK-1 as defined in SEQ ID NO:21, GmPK-2 as defined in SEQ ID NO:23, GmPK-3 as defined in SEQ ID NO:25, GmPK-4 as defined in SEQ ID NO:27, and OsPK-1 as defined in SEQ ID NO:29 in a target plant as well as the homolog's promoter. The invention also provides a method of increasing expression of a gene of interest within a host cell as compared to a wild type variety of the host cell, wherein the gene of interest is transcribed in response to a PKSRP, comprising: (a) transforming the host cell with an expression vector comprising a PKSRP coding nucleic acid, and (b) expressing the PKSRP within the host cell, thereby increasing the expression of the gene transcribed in response to the PKSRP, as compared to a wild type variety of the host cell.
[0122]In addition to introducing the PKSRP nucleic acid sequences into transgenic plants, these sequences can also be used to identify an organism as being Physcomitrella patens, Brassica napus, Glycine max, Oryza saliva, or a close relative thereof. Also, they may be used to identify the presence of Physcomitrella patens, Brassica napus, Glycine max, Oryza saliva, or a relative thereof in a mixed population of microorganisms. The invention provides the nucleic acid sequences of a number of Physcomitrella patens, Brassica napus, Glycine max, and Oryza saliva genes; by probing the extracted genomic DNA of a culture of a unique or mixed population of microorganisms under stringent conditions with a probe spanning a region of a Physcomitrella patens, Brassica napus, Glycine max, or Oryza saliva gene which is unique to this organism, one can ascertain whether this organism is present.
[0123]Further, the nucleic acid and polypeptide molecules of the invention may serve as markers for specific regions of the genome. This has utility not only in the mapping of the genome, but also in functional studies of Physcomitrella patens, Brassica napus, Glycine max, and Oryza saliva polypeptides. For example, to identify the region of the genome to which a particular Physcomitrella patens DNA-binding polypeptide binds, the Physcomitrella patens genome could be digested, and the fragments incubated with the DNA-binding polypeptide. Those fragments that bind the polypeptide may be additionally probed with the nucleic acid molecules of the invention, preferably with readily detectable labels. Binding of such a nucleic acid molecule to the genome fragment enables the localization of the fragment to the genome map of Physcomitrella patens, and, when performed multiple times with different enzymes, facilitates a rapid determination of the nucleic acid sequence to which the polypeptide binds. Further, the nucleic acid molecules of the invention may be sufficiently identical to the sequences of related species such that these nucleic acid molecules may serve as markers for the construction of a genomic map in related mosses.
[0124]The PKSRP nucleic acid molecules of the invention are also useful for evolutionary and polypeptide structural studies. The metabolic and transport processes in which the molecules of the invention participate are utilized by a wide variety of prokaryotic and eukaryotic cells; by comparing the sequences of the nucleic acid molecules of the present invention to those encoding similar enzymes from other organisms, the evolutionary relatedness of the organisms can be assessed. Similarly, such a comparison permits an assessment of which regions of the sequence are conserved and which are not, which may aid in determining those regions of the polypeptide that are essential for the functioning of the enzyme. This type of determination is of value for polypeptide engineering studies and may give an indication of what the polypeptide can tolerate in terms of mutagenesis without losing function.
[0125]Manipulation of the PKSRP nucleic acid molecules of the invention may result in the production of PKSRPs having functional differences from the wild-type PKSRPs. These polypeptides may be improved in efficiency or activity, may be present in greater numbers in the cell than is usual, or may be decreased in efficiency or activity.
[0126]There are a number of mechanisms by which the alteration of a PKSRP of the invention may directly affect stress response and/or stress tolerance. In the case of plants expressing PKSRPs, increased transport can lead to improved salt and/or solute partitioning within the plant tissue and organs. By either increasing the number or the activity of transporter molecules which exportionic molecules from the cell, it may be possible to affect the salt tolerance of the cell.
[0127]The effect of the genetic modification in plants, C. glutamicum, fungi, algae, or ciliates on stress tolerance can be assessed by growing the modified microorganism or plant under less than suitable conditions and then analyzing the growth characteristics and/or metabolism of the plant. Such analysis techniques are well known to one skilled in the art, and include dry weight, wet weight, polypeptide synthesis, carbohydrate synthesis, lipid synthesis, evapotranspiration rates, general plant and/or crop yield, flowering, reproduction, seed setting, root growth, respiration rates, photosynthesis rates, etc. (Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, vol. 17; Rehm et al., 1993 Biotechnology, vol 3, Chapter III: Product recovery and purification, page 469-714, VCH: Weinheim; Belter, P. A. et al., 1988, Bioseparations: downstream processing for biotechnology, John Wiley and Sons; Kennedy, J. F. and Cabral, J. M. S., 1992, Recovery processes for biological materials, John Wiley and Sons; Shaeiwitz, J. A. and Henry, J. D., 1988, Biochemical separations, in: Ulmann's Encyclopedia of Industrial Chemistry, vol. B3, Chapter 1, page 1-27, VCH: Weinheim; and Dechow, F. J., 1989, Separation and purification techniques in biotechnology, Noyes Publications).
[0128]For example, yeast expression vectors comprising the nucleic acids disclosed herein, or fragments thereof, can be constructed and transformed into Saccharomyces cerevisiae using standard protocols. The resulting transgenic cells can then be assayed for fail or alteration of their tolerance to drought, salt, and temperature stress. Similarly, plant expression vectors comprising the nucleic acids disclosed herein, or fragments thereof, can be constructed and transformed into an appropriate plant cell such as Arabidopsis, soy, rape, maize, wheat, Medicago truncatula, etc., using standard protocols. The resulting transgenic cells and/or plants derived therefrom can then be assayed for fail or alteration of their tolerance to drought, salt, temperature stress, and lodging.
[0129]The engineering of one or more PKSRP genes of the invention may also result in PKSRPs having altered activities which indirectly impact the stress response and/or stress tolerance of algae, plants, ciliates, or fingi, or other microorganisms like C. glutamicum. For example, the normal biochemical processes of metabolism result in the production of a variety of products (e.g., hydrogen peroxide and other reactive oxygen species) which may actively interfere with these same metabolic processes. For example, peroxynitrite is known to nitrate tyrosine side chains, thereby inactivating some enzymes having tyrosine in the active site (Groves, J. T., 1999, Curr. Opin. Chem. Biol. 3(2):226-235). While these products are typically excreted, cells can be genetically altered to transport more products than is typical for a wild-type cell. By optimizing the activity of one or more PKSRPs of the invention which are involved in the export of specific molecules, such as salt molecules, it may be possible to improve the stress tolerance of the cell.
[0130]Additionally, the sequences disclosed herein, or fragments thereof, can be used to generate knockout mutations in the genomes of various organisms, such as bacteria, mammalian cells, yeast cells, and plant cells (Girke, T., 1998, The Plant Journal 15:39-48). The resultant knockout cells can then be evaluated for their ability or capacity to tolerate various stress conditions, their response- to various stress conditions, and the effect on the phenotype and/or genotype of the mutation. For other methods of gene inactivation, see U.S. Pat. No. 6,004,804 "Non-Chimeric Mutational Vectors" and Puttaraju et al., 1999, Spliceosome-mediated RNA trans-splicing as a tool for gene therapy, Nature Biotechnology 17:246-252.
[0131]The aforementioned mutagenesis strategies for PKSRPs resulting in increased stress resistance are not meant to be limiting; variations on these strategies will be readily apparent to one skilled in the art. Using such strategies, and incorporating the mechanisms disclosed herein, the nucleic acid and polypeptide molecules of the invention may be utilized to generate algae, ciliates, plants, fungi, or other microorganisms like C. glutamicum expressing mutated PKSRP nucleic acid and polypeptide molecules such that the stress tolerance is improved.
[0132]The present invention also provides antibodies that specifically bind to a PKSRP, or a portion thereof, as encoded by a nucleic acid described herein. Antibodies can be made by many well-known methods (See, e.g. Harlow and Lane, "Antibodies; A Laboratory Manual," Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1988)). Briefly, purified antigen can be injected into an animal in an amount and in intervals sufficient to elicit an immune response. Antibodies can either be purified directly, or spleen cells can be obtained from the animal. The cells can then fused with an immortal cell line and screened for antibody secretion. The antibodies can be used to screen nucleic acid clone libraries for cells secreting the antigen. Those positive clones can then be sequenced. See, for example, Kelly et al., 1992, Bio/Technology 10:163-167; Bebbington et al., 1992, Bio/Technology 10:169-175.
[0133]The phrases "selectively binds" and "specifically binds" with the polypeptide refer to a binding reaction that is determinative of the presence of the polypeptide in a heterogeneous population of polypeptides and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bound to a particular polypeptide do not bind in a significant amount to other polypeptides present in the sample. Selective binding of an antibody under such conditions may require an antibody that is selected for its specificity for a particular polypeptide. A variety of immunoassay formats may be used to select antibodies that selectively bind with a particular polypeptide. For example, solid-phase ELISA immunoassays are routinely used to select antibodies selectively immunoreactive with a polypeptide. See Harlow and Lane, "Antibodies, A Laboratory Manual," Cold Spring Harbor Publications, New York, (1988), for a description of immunoassay formats and conditions that could be used to determine selective binding.
[0134]In some instances, it is desirable to prepare monoclonal antibodies from various hosts. A description of techniques for preparing such monoclonal antibodies may be found in Stites et al., eds., "Basic and Clinical Immunology," (Lange Medical Publications, Los Altos, Calif., Fourth Edition) and references cited therein, and in Harlow and Lane, "Antibodies, A Laboratory Manual," Cold Spring Harbor Publications, New York, (1988).
[0135]Throughout this application, various publications are referenced. The disclosures of all of these publications and those references cited within those publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
[0136]It should also be understood that the foregoing relates to preferred embodiments of the present invention and that numerous changes may be made therein without departing from the scope of the invention. The invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof, which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention and/or the scope of the appended claims.
EXAMPLES
Example 1
Growth of Physcomitrella patens Cultures
[0137]For this study, plants of the species Physcomitrella patens (Hedw.) B.S.G. from the collection of the genetic studies section of the University of Hamburg were used. They originate from the strain 16/14 collected by H. L. K. Whitehouse in Gransden Wood, Huntingdonshire (England), which was subcultured from a spore by Engel (1968, Am. J. Bot. 55, 438-446). Proliferation of the plants was carried out by means of spores and by means of regeneration of the gametophytes. The protonema developed from the haploid spore as a chloroplast-rich chloronema and chloroplast-low caulonema, on which buds formed after approximately 12 days. These grew to give gametophores bearing antheridia and archegonia. After fertilization, the diploid sporophyte with a short seta and the spore capsule resulted, in which the meiospores matured.
[0138]Culturing was carried out in a climatic chamber at an air temperature of 25° C. and light intensity of 55 micromol s-1 m-2 (white light; Philips TL 65W/25 fluorescent tube) and a light/dark change of 16/8 hours. The moss was either modified in liquid culture using Knop medium according to Reski and Abel (1985, Planta 165:354-358) or cultured on Knop solid medium using 1% oxoid agar (Unipath, Basingstoke, England). The protonemas used for RNA and DNA isolation were cultured in aerated liquid cultures. The protonemas were comminuted every 9 days and transferred to fresh culture medium.
Example 2
Total DNA Isolation from Plants
[0139]The details for the isolation of total DNA relate to the working up of one gram fresh weight of plant material. The materials used include the following buffers: CTAB buffer: 2% (w/v) N-cethyl-N,N,N-trimethylammonium bromide (CTAB); 100 mM Tris HCl pH 8,0; 1.4 M NaCl; 20 mM EDTA; N-Laurylsarcosine buffer: 10% (w/v) N-laurylsarcosine; 100 mM Tris HCl pH 8.0; 20 mM EDTA.
[0140]The plant material was triturated under liquid nitrogen in a mortar to give a fine powder and transferred to 2 ml Eppendorf vessels. The frozen plant material was then covered with a layer of 1 ml of decomposition buffer (1 ml CTAB buffer, 100 μl of N-laurylsarcosine buffer, 20 μl of β-mercaptoethanol and 10 μl of proteinase K solution, 10 mg/ml) and incubated at 60° C. for one hour with continuous shaking. The homogenate obtained was distributed into two Eppendorf vessels (2 ml) and extracted twice by shaking with the same volume of chloroform/isoamyl alcohol (24:1). For phase separation, centrifugation was carried out at 8000×g and room temperature for 15 minutes in each case. The DNA was then precipitated at -70° C. for 30 minutes using ice-cold isopropanol. The precipitated DNA was sedimented at 4° C. and 10,000×g for 30 minutes and resuspended in 180 μl of TE buffer (Sambrook et al., 1989, Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6). For further purification, the DNA was treated with NaCl (1.2 M final concentration) and precipitated again at -70° C. for 30 minutes using twice the volume of absolute ethanol. After a washing step with 70% ethanol, the DNA was dried and subsequently taken up in 50 μl of H2O+RNAse (50 mg/ml final concentration). The DNA was dissolved overnight at 4° C. and the RNAse digestion was subsequently carried out at 37° C. for 1 hour. Storage of the DNA took place at 4° C.
Example 3
Isolation of Total RNA and poly-(A)+ RNA and cDNA Library Construction from Physcomitrella patens
[0141]For the investigation of transcripts, both total RNA and poly-(A).sup.+ RNA were isolated. The total RNA was obtained from wild-type 9 day old protonemata following the GTC-method (Reski et al., 1994, Mol. Gen. Genet., 244:352-359). The Poly(A)+ RNA was isolated using Dyna BeadsR (Dynal, Oslo, Norway) following the instructions of the manufacturer's protocol. After determination of the concentration of the RNA or of the poly(A)+ RNA, the RNA was precipitated by addition of 1/10 volumes of 3 M sodium acetate pH 4.6 and 2 volumes of ethanol and stored at -70° C.
[0142]For cDNA library construction, first strand synthesis was achieved using Murine Leukemia Virus reverse transcriptase (Roche, Mannheim, Germany) and oligo-d(T)-primers, second strand synthesis by incubation with DNA polymerase I, Klenow enzyme and RNAseH digestion at 12° C. (2 hours), 16° C. (1 hour), and 22° C. (1 hour). The reaction was stopped by incubation at 65° C. (10 minutes) and subsequently transferred to ice. Double stranded DNA molecules were blunted by T4-DNA-polymerase (Roche, Mannheim) at 37° C. (30 minutes). Nucleotides were removed by phenol/chloroform extraction and Sephadex G50 spin columns. EcoRI adapters (Pharmacia, Freiburg, Germany) were ligated to the cDNA ends by T4-DNA-ligase (Roche, 12° C., overnight) and phosphorylated by incubation with polynucleotide kinase TRoche, 37° C., 30 minutes). This mixture was subjected to separation on a low melting agarose gel. DNA molecules larger than 300 base pairs were eluted from the gel, phenol extracted, concentrated on Elutip-D-columns (Schleicher and Schuell, Dassel, Germany), and were ligated to vector arms and packed into lambda ZAPII phages or lambda ZAP-Express phages using the Gigapack Gold Kit (Stratagene, Amsterdam, Netherlands) using material and following the instructions of the manufacturer.
Example 4
Sequencing and Function Annotation of Physconitirella patens ESTs
[0143]cDNA libraries as described in Example 3 were used for DNA sequencing according to standard methods, and in particular, by the chain termination method using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Weiterstadt, Germany). Random Sequencing was carried out subsequent to preparative plasmid recovery from cDNA libraries via in vivo mass excision, retransformation, and subsequent plating of DH10B on agar plates (material and protocol details from Stratagene, Amsterdam, Netherlands). Plasmid DNA was prepared from overnight grown E. coli cultures grown in Luria-Broth medium containing ampicillin (See Sambrook et al., 1989, Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6) on a Qiagene DNA preparation robot (Qiagen, Hilden) according to the manufacturer's protocols. Sequencing primers with the following nucleotide sequences were used:
TABLE-US-00001 5'-CAGGAAACAGCTATGACC-3' SEQ ID NO: 31 5'-CTAAAGGGAACAAAAGCTG-3' SEQ ID NO: 32 5'-TGTAAAACGACGGCCAGT-3' SEQ ID NO: 33
[0144]Sequences were processed and annotated using the software package EST-MAX commercially provided by Bio-Max (Munich, Germany). The program incorporates practically all bioinformatics methods important for functional and structural characterization of polypeptide sequences. The most important algorithms incorporated in EST-MAX are: FASTA (Very sensitive sequence database searches with estimates of statistical significance; Pearson W. R., 1990, Rapid and sensitive sequence comparison with FASTP and FASTA, Methods Enzymol. 183:63-98); BLAST (Very sensitive sequence database searches with estimates of statistical significance; Altschul S. F. et al., Basic local alignment search tool, Journal of Molecular Biology 215:403-10); PREDATOR (High-accuracy secondary structure prediction from single and multiple sequences, Frishman, D. and Argos, P., 1997, 75% accuracy in polypeptide secondary structure prediction, Polypeptides, 27:329-335); CLUSTALW (Multiple sequence alignment; Thompson, J. D. et al., 1994, CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22:4673-4680); TMAP (Transmembrane region prediction from multiply aligned sequences; Persson, B. and Argos, P., 1994, Prediction of transmembrane segments in polypeptides utilizing multiple sequence alignments, J. Mol. Biol. 237:182-192); ALOM2 (Transmembrane region prediction from single sequences; Klein, P. et al., Prediction of polypeptide function from sequence properties: A discriminate analysis of a database. Biochim. Biophys. Acta 787:221-226 (1984). Version 2 by Dr. K. Nakai); PROSEARCH (Detection of PROSITE polypeptide sequence patterns; Kolakowski L. F. Jr. et al., 1992, ProSearch: fast searching of polypeptide sequences with regular expression patterns related to polypeptide structure and function, Biotechniques 13:919-921); BLIMPS (Similarity searches against a database of ungapped blocks; J. C. Wallace and Henikoff S., 1992); and PATMAT (A searching and extraction program for sequence, pattern and block queries and databases, CABIOS 8:249-254. Written by Bill Alford.).
Example 5
Identification of Physcomitrella Patens ORFs corresponding to PK-3, PK-4, PK-10, and PK-11
[0145]The Physcomitrella patens partial cDNAs (ESTs) for partial PK-3 (SEQ ID NO:1), partial PK-4 (SEQ ID NO:4), partial PK-10 (SEQ ID NO:7), and partial PK-11 (SEQ ID NO: 10) were identified in the Physcomitrella patens EST sequencing program using the program EST-MAX through BLAST analysis. These particular clones, which were found to encode Protein Kinases, were chosen for further analyses.
TABLE-US-00002 TABLE 1 Degree of Amino Acid Identity and Similarity of PK-3 and Other Kinases (Pairwise Comparison was used: gap penalty: 10; gap extension penalty: 0.1; score matrix: blosum62) Swiss-Prot # P51139 Q40518 P43288 P43289 Q9LYJ6 Protein Glycogen Shaggy- Shaggy- Shaggy- Protein name Synthase Related Related Related Kinase MSK- Kinase-3 Protein Protein Protein 3-Like Homolog Kinase Kinase Kinase MSK-3 NTK-1 Alpha Gamma Species Medicago sativa Nicotiana Arabidopsis Arabidopsis Arabidopsis (Alfalfa) tabacum thaliana thaliana thaliana (Common (Mouse-ear (Mouse-ear (Mouse-ear tobacco) cress) cress) cress) Identity % 78% 79% 79% 80% 79% Similarity % 86% 86% 86% 87% 87%
TABLE-US-00003 TABLE 2 Degree of Amino Acid Identity and Similarity of PK-4 and Other Kinases (Pairwise Comparison was used: gap penalty: 10; gap extension penalty: 0.1; score matrix: blosum62) Swiss-Prot # Q9SZI1 Q9ZUP4 P42158 Q39050 Q9LW62 Polypeptide COL-0 Putative Casein Casein Casein name Casein Casein Kinase I, Kinase I Kinase Kinase I- Kinase I Delta Like Protein Isoform Like Species Arabidopsis Arabidopsis Arabidopsis Arabidopsis Arabidopsis thaliana thaliana thaliana thaliana thaliana (Mouse-ear cress) (Mouse-ear (Mouse-ear (Mouse-ear (Mouse-ear cress) cress) cress) cress) Identity % 35% 35% 37% 35% 35% Similarity % 46% 44% 47% 45% 44%
TABLE-US-00004 TABLE 3 Degree of Amino Acid Identity and Similarity of PK-10 and a Similar Protein (Pairwise Comparison was used: gap penalty: 10; gap extension penalty: 0.1; score matrix: blosum62) Public Sequence Database Sequence Similarity Gene Name Sequence Protein Name Species Identity (%) (%) PK-10 AAG51974 Putative Leucine- Arabidopsis 45% 57% Rich Repeat thaliana Transmembrane Protein Kinase I
TABLE-US-00005 TABLE 4 Degree of Amino Acid Identity and Similarity of PK-11 and a Similar Protein (Pairwise Comparison was used: gap penalty: 10; gap extension penalty: 0.1; score matrix: blosum62) Public Sequence Database Sequence Similarity Gene Name Sequence Protein Name Species Identity (%) (%) PK-11 AAK72257.1 CBL-Interacting Arabidopsis 64% 76% Protein Kinase thaliana 24
Example 6
Cloning of the Full-Length Physcomitrella patens cDNA Encoding for PK-3, PK-4, PK-10, and PK-11
[0146]To isolate the clone encoding full-length PK-3 (SEQ ID NO:2), PCR was performed (as described below in Full-Length Amplification) using the original ESTs described in Example 5 as template. The primers used for amplification are listed below in Table 5.
[0147]To isolate the clones encoding PK-4 (SEQ ID NO:5), PK-10 (SEQ ID NO:8), and PK-11 (SEQ ID NO: 1) from Physcomitrella patens, cDNA libraries were created with SMART RACE cDNA Amplification kit (Clontech Laboratories) following the manufacturer's instructions. Total RNA isolated as described in Example 3 was used as the template. The cultures were treated prior to RNA isolation as follows: Salt Stress: 2, 6, 12, 24, 48 hours with 1-M NaCl-supplemented medium; Cold Stress: 4° C. for the same time points as for salt; Drought Stress: cultures were incubated on dry filter paper for the same time points as for salt.
5' RACE Protocol
[0148]The EST sequences of PK-4 (SEQ ID NO:4), PK-10 (SEQ ID NO:7), and PK-11 (SEQ ID NO:10) identified from the database search as described in Example 5 were used to design oligos for RACE (See Table 5). The extended sequence for these genes were obtained by performing Rapid Amplification of cDNA Ends polymerase chain reaction (RACE PCR) using the Advantage 2 PCR kit (Clontech Laboratories) and the SMART RACE cDNA amplification kit (Clontech Laboratories) using a Biometra T3 Thermocycler following the manufacturer's instructions. The sequences obtained from the RACE reactions corresponded to full-length coding region of PK-4, PK-10, and PK-11 and were used to design oligos for full-length cloning of the respective gene (See below Full-Length Amplification).
Full-Length Amplification
[0149]A full-length clone corresponding to PK-3 (SEQ ID NO:2) was obtained by performing polymerase chain reaction (PCR) with gene-specific primers (See Table 5) and the original EST as the template. The conditions for the reaction were standard conditions with PWO DNA polymerase (Roche). PCR was performed according to standard conditions and to manufacturer's protocols (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., Biometra T3 Thermocycler). The parameters for the reaction were: five minutes at 94° C. followed by five cycles of one minute at 94° C., one minute at 50° C., and 1.5 minutes at 72° C. This was followed by twenty five cycles of one minute at 94° C., one minute at 65° C., and 1.5 minutes at 72° C.
[0150]Full-length clones for PK-4 (SEQ ID NO:5), PK-10 (SEQ ID NO:8), and PK-11 (SEQ ID NO:11) were isolated by repeating the RACE method but using the gene-specific primers as given in Table 5.
[0151]The amplified fragments were extracted from agarose gel with a QIAquick Gel Extraction Kit (Qiagen) and ligated into the TOPO pCR 2.1 vector (Invitrogen) following manufacturer's instructions. Recombinant vectors were transformed into Top10 cells (Invitrogen) using standard conditions (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual. 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Transformed cells were selected for on LB agar containing 100 μg/ml carbenicillin, 0.8 mg X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside), and 0.8 mg IPTG (isopropylthio-β-D-galactoside) grown overnight at 37° C. White colonies were selected and used to inoculate 3 ml of liquid LB containing 100 μg/ml ampicillin and grown overnight at 37° C. Plasmid DNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen) following manufacturer's instructions. Analyses of subsequent clones and restriction mapping was performed according to standard molecular biology techniques (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
TABLE-US-00006 TABLE 5 Scheme and Primers Used for Cloning of Full-Length Clones Sites in the Isolation Primers Primers Gene final product Method Race RT-PCR PK-3 XmaI/SacI PCR of original RC021: EST clone 5'ATCCCGGGCGAG TCTTCTATGGCATC TGCGACT3' (SEQ ID NO: 34) RC022: 5'ATGAGCTCAATA TCAGGAGTTGCAC CCTTCAAC3' (SEQ ID NO: 35) PK-4 XmaI/EcoRV 5' RACE and RC072: RC133N: RT-PCR for FL 5'TGTGTCTACGT 5'ATCCCGGGAGGC clone GTCGCGGGGTC ATTGAACTACCTG GAT3' GAGTGAG3' (SEQ ID NO: 36) (SEQ ID NO: 37) RC134N: 5'GCGATATCGTTG AACTAGTAATCTG TGTTAACTT3' (SEQ ID NO: 38) PK-10 XmaI/SacI 5' RACE and NVT: RC580: RT-PCR for FL 5'CTGCGACGGA 5'ATCCCGGGTGTC clone AAACTCTCTTGC GGAATTCGGTCAC TGT3' AATGAGCT3' (SEQ ID NO: 39) RC834: 5'GCGAGCTCGTG CGAATCATGTACT CCCATCACAC3' (SEQ ID NO: 40) PK-11 XmaI/SacI 5' RACE and RC253: RC1158: RT-PCR for FL 5'GCAGCGGTATA 5'ATCCCGGGTTTC clone TCCTTGCTCCTCA TGGAATAGCTCAG TC3' AAGCGT3' RC520: RC1159: 5'CGAT 5'CGGAGCTCGATGC GTGAGACGCCCT AGCGGTATATCCT TGCTGTGGCA3' TGCTCCT3' RC721: (SEQ ID NO: 42) 5'GCAACGA CTTGCCAGAACCT CGTGC3' (SEQ ID NO: 41)
Tissue Harvest, RNA Isolation, and cDNA Library Construction
[0152]Canola, soybean, and rice plants were grown under a variety of conditions and treatments, and different tissues were harvested at various developmental stages. Plant growth and harvesting were done in a strategic manner such that the probability of harvesting all expressable genes in at least one or more of the resulting libraries is maximized. The mRNA was isolated as described in Example 3 from each of the collected samples, and cDNA libraries were constructed. No amplification steps were used in the library production process in order to minimize redundancy of genes within the sample and to retain expression information. All libraries were 3' generated from mRNA purified on oligo dT columns. Colonies from the transformation of the cDNA library into E. coli were randomly picked and placed into microtiter plates.
Probe Hybridization
[0153]Plasmid DNA was isolated from the E. coli colonies and then spotted on membranes. A battery of 288 33P radiolabeled 7-mer oligonucleotides were sequentially hybridized to these membranes. To increase throughput, duplicate membranes were processed, After each hybridization, a blot image was captured during a phosphorimage scan to generate a hybridization profile for each oligonucleotide. This raw data image was automatically transferred via LIMS to a computer. Absolute identity was maintained by barcoding for the image cassette, filter, and orientation within the cassette. The filters were then treated using relatively mild conditions to strip the bound probes and returned to the hybridization chambers for another round of hybridization. The hybridization and imaging cycle was repeated until the set of 288 oligomers was completed.
[0154]After completion of the hybridizations, a profile was generated for each spot (representing a cDNA insert), as to which of the 288 33P radiolabeled 7-mer oligonucleotides bound to that particular spot (cDNA insert), and to what degree. This profile is defined as the signature generated from that clone. Each clone's signature was compared with all other signatures generated from the same organism to identify clusters of related signatures. This process "sorts" all of the clones from an organism into clusters before sequencing.
Gene Isolation
[0155]The clones were sorted into various clusters based on their having identical or similar hybridization signatures. A cluster should be indicative of the expression of an individual gene or gene family. A by-product of this analysis is an expression profile for the abundance of each gene in a particular library. One-path sequencing from the 5' end was used to predict the function of the particular clones by similarity and motif searches in sequence databases.
[0156]The full-length DNA sequence of the Physconritrella patens PK-3 (SEQ If) NO:8) or PK-10 (SEQ ID NO: 11) was blasted against proprietary contig databases of canola, rice, and soybean at E value of E-10, (Altschul, Stephen et al. Gapped BLAST and PSI BLAST: a new generation of protein database search program. Nucleic Acids Res. 25: 3389-3402). All the contig hits were analyzed for the putative full length sequences, and the longest clones representing the putative full length contigs were fully sequenced. Nine such contigs isolated from the proprietary contig databases are BnPK-1, BnPK-2, BnPK-3, BnPK-4, GmPK-1, GmPK-2, GmPK-3, GmPK-4, and OsPK-1. The homology of the BnPK-1, BnPK-2, BnPK-3, BnPK-4, GmPK-1, GmPK-2, GmPK-3, GmPK-4, and OsPK-1 amino acid sequences to the closest prior art is indicated in Tables 6-14.
TABLE-US-00007 TABLE 6 Degree of Amino Acid Identity and Similarity of BnPK-1 and a Similar Protein (Pairwise Comparison was used: gap penalty: 10; gap extension penalty: 0.1; score matrix: blosum62) Public Sequence Database Sequence Similarity Gene Name Sequence Protein Name Species Identity (%) (%) BnPK-1 CAA55866 Shaggy/Glycogen Arabidopsis 93% 95% Synthase Kinase-3 thaliana Homologue
TABLE-US-00008 TABLE 7 Degree of Amino Acid Identity and Similarity of BnPK-2 and a Similar Protein (Pairwise Comparison was used: gap penalty: 10; gap extension penalty: 0.1; score matrix: blosum62) Public Sequence Gene Database Protein Sequence Similarity Name Sequence Name Species Identity (%) (%) BnPK-2 CAB78873 Shaggy- Arabidopsis 98% 99% Like thaliana Protein Kinase Etha
TABLE-US-00009 TABLE 8 Degree of Amino Acid Identity and Similarity of BnPK-3 and a Similar Protein (Pairwise Comparison was used: gap penalty: 10; gap extension penalty: 0.1; score matrix: blosum62) Public Sequence Gene Database Protein Sequence Similarity Name Sequence Name Species Identity (%) (%) BnPK-3 CAA11903 Shaggy- Arabidopsis 92% 94% Like thaliana Kinase Beta
TABLE-US-00010 TABLE 9 Degree of Amino Acid Identity and Similarity of BnPK-4 and a Similar Protein (Pairwise Comparison was used: gap penalty: 10; gap extension penalty: 0.1; score matrix: blosum62) Public Sequence Database Sequence Similarity Gene Name Sequence Protein Name Species Identity (%) (%) BnPK-4 AAG51974 Putative Leucine- Arabidopsis 87% 92% Rich Repeat thaliana Transmembrane Protein Kinase 1
TABLE-US-00011 TABLE 10 Degree of Amino Acid Identity and Similarity of GmPK-1 and a Similar Protein (Pairwise Comparison was used: gap penalty: 10; gap extension penalty: 0.1; score matrix: blosum62) Public Sequence Database Sequence Similarity Gene Name Sequence Protein Name Species Identity (%) (%) GmPK-1 AAL36376 Putative Shaggy Arabidopsis 80% 87% Protein Kinase thaliana dzeta
TABLE-US-00012 TABLE 11 Degree of Amino Acid Identity and Similarity of GmPK-2 and a Similar Protein (Pairwise Comparison was used: gap penalty: 10; gap extension penalty: 0.1; score matrix: blosum62) Public Sequence Gene Database Sequence Similarity Name Sequence Protein Name Species Identity (%) (%) GmPK-2 AAG50665 Putative Glycogen Arabidopsis 85% 92% Synthase Kinase thaliana
TABLE-US-00013 TABLE 12 Degree of Amino Acid Identity and Similarity of GmPK-3 and a Similar Protein (Pairwise Comparison was used: gap penalty: 10; gap extension penalty: 0.1; score matrix: blosum62) Public Sequence Database Sequence Similarity Gene Name Sequence Protein Name Species Identity (%) (%) GmPK-3 AAK93730 Putative Shaggy Arabidopsis 85% 89% Kinase thaliana
TABLE-US-00014 TABLE 13 Degree of Amino Acid Identity and Similarity of GmPK-4 and a Similar Protein (Pairwise Comparison was used: gap penalty: 10; gap extension penalty: 0.1; score matrix: blosum62) Public Sequence Database Sequence Similarity Gene Name Sequence Protein Name Species Identity (%) (%) GmPK-4 AAL59961 Putative Leucine- Arabidopsis 58% 68% Rich Repeat thaliana Transmembrane Protein Kinase
TABLE-US-00015 TABLE 14 Degree of Amino Acid Identity and Similarity of OsPK-1 and a Similar Protein (Pairwise Comparison was used: gap penalty: 10; gap extension penalty: 0.1; score matrix: blosum62) Public Sequence Gene Database Protein Sequence Similarity Name Sequence Name Species Identity (%) (%) OsPK-1 CAA48474 Protein Medicago 89% 90% Kinase sativa
Example 7
Engineering Stress-Tolerant Arabidopsis Plants by Over-Expressing the Genes PK-3 and PK-4
[0157]Binary Vector Construction: pBPS-JH001
[0158]The plasmid construct pLMNC53 (Mankin, 2000, Ph.D. thesis, University of North Carolina) was digested with HindIII (Roche) and blunt-end filled with Klenow enzyme and 0.1 mM dNTPs according to manufacturer's instructions. This fragment was purified by agarose gel and extracted via the QIAquick Gel Extraction kit (Qiagen) according to manufacturer's instructions. The purified fragment was then digested with EcoRI (Roche), purified by agarose gel, and extracted via the QIAquick Gel Extraction kit (Qiagen) according to manufacturer's instructions. The resulting 1.4 kilobase fragment, the gentamycin cassette, included the nos promoter, aacCI gene, and the g7 terminator.
[0159]The vector pBlueScript was digested with EcoRI and SmaI (Roche) according to manufacturer's instructions, and the resulting fragment was extracted from agarose gel with a QIAquick Gel Extraction Kit (Qiagen) according to manufacturer's instructions. The digested pBlueScript vector and the gentamycin cassette fragments were ligated with T4 DNA Ligase (Roche) according to manufacturer's instructions, joining the two respective EcopI sites and joining the blunt-ended HindIII site with the SmaI site.
[0160]The recombinant vector (pGGMBS) was transformed into Top10 cells (Invitrogen) using standard conditions. Transformed cells were selected for on LB agar containing 100 μg/ml carbenicillin, 0.8 mg X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) and 0.8 mg IPTG (isopropylthio-β-D-galactoside), grown overnight at 37° C. White colonies were selected and used to inoculate 3 ml of liquid LB containing 100 μg/ml ampicillin and grown overnight at 37° C. Plasmid DNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen) following manufacturer's instructions. Analyses of subsequent clones and restriction mapping were performed according to standard molecular biology techniques (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual. 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
[0161]Both the pGMBS vector and p1bxSuperGUS vector were digested with XbaT and KpnI (Roche) according to manufacturer's instructions, excising the gentamycin cassette from pGMBS and producing the backbone from the p1bxSuperGUS vector. The resulting fragments were extracted from agarose gel with a QIAquick Gel Extraction Kit (Qiagen) according to manufacturer's instructions. These two fragments were ligated with T4 DNA ligase (Roche) according to manufacturer's instructions.
[0162]The resulting recombinant vector (pBPS-JH001) was transformed into Top10 cells (Invitrogen) using standard conditions. Transformed cells were selected for on LB agar containing 100 μg/ml carbenicillin, 0.8 mg X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) and 0.8 mg IPTG (isopropylthio-J)-D-galactoside), grown overnight at 37° C. White colonies were selected and used to inoculate 3 ml of liquid LB containing 100 μg/ml ampicillin and grown overnight at 37° C. Plasmid DNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen) following manufacturer's instructions. Analyses of subsequent clones and restriction mapping were performed according to standard molecular biology techniques (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
Binary Vector Construction: pBPS-SC022
[0163]The plasmid construct pACGH101 was digested with PstI (Roche) and FseI (NEB) according to manufacturers' instructions. The fragment was purified by agarose gel and extracted via the Qiaex II DNA Extraction kit (Qiagen). This resulted in a vector fragment with the Arabidopsis Actin2 promoter with internal intron and the OCS3 terminator.
[0164]Primers for PCR amplification of the NPTH gene were designed [5'NPT-Pst: GCG-CTG-CAG-ATT-TCA-TTT-CGA-GAG-GAC-ACG (SEQ ID NO:39); 3'NPT-Fse: CGC-GGC-CGG-CCT-CAG-AAG-AAC-TCG-TCA-AGA-AGG-CG (SEQ ID NO:40)]. The 0.9 kilobase NPTIH gene was amplified via PCR from pCambia 2301 plasmid DNA using the following parameters: 94° C. 60 sec, {94° C. 60 sec, 61° C. (-01° C. per cycle) 60 sec, 72° C. 2 min}×25 cycles, 72° C. 10 min on Biometra T-Gradient machine. The amplified product was purified via the Qiaquick PCR Extraction kit (Qiagen) following manufacturer's instructions. The PCR DNA was then subcloned into the pCR-BluntII TOPO vector (Invitrogen) following manufacturer's instructions (NPT-Topo construct). These ligations were transformed into Top10 cells (Invitrogen) and grown on LB plates with 50 μg/ml kanamycin sulfate overnight at 37° C. Colonies were then used to inoculate 2 ml LB media with 50 μg/ml kanamycin sulfate and grown overnight at 37° C. Plasmid DNA was recovered using the Qiaprep Spin Miniprep kit (Qiagen) and sequenced in both the 5' and 3' directions using standard conditions. Subsequent analysis of the sequence data using Vector NTI software revealed that there were not any PCR errors introduced in the NPTII gene sequence.
[0165]The NPT-Topo construct was then digested with PstI (Roche) and FseI (NEB) according to manufacturers' instructions. The 0.9 kilobase fragment was purified on agarose gel and extracted by Qiaex II DNA Extraction kit (Qiagen). The Pst/Fse insert fragment from NPT-Topo and the Pst/Fse vector fragment from pACHQ101 were then ligated together using T4 DNA Ligase (Roche) following manufacturer's instructions. The ligation reaction was then transformed into Top10 cells (Invitrogen) under standard conditions, creating pBPS-sc019 construct. Colonies were selected on LB plates with 50 μg/ml kanamycin sulfate and grown overnight at 37° C. These colonies were then used to inoculate 2 ml LB media with 50 μg/ml kanamycin sulfate and grown overnight at 37° C. Plasmid DNA was recovered using the Qiaprep Spin Miniprep kit (Qiagen) following the manufacturer's instructions.
[0166]The pBPS-SC019 construct was digested with KpnI and BsaI (Roche) according to manufacturer's instructions. The fragment was purified via agarose gel and then extracted via the Qiaex II DNA Extraction kit (Qiagen) as per its instructions, resulting in a 3 kilobase Act-NPT cassette, which included the Arabidopsis Actin2 promoter with internal intron, the NPTII gene, and the OCS3 terminator.
[0167]The pBPS-JH001 vector was digested with SpeI and ApaI (Roche) and blunt-end filled with Klenow enzyme and 0.1 mM dNTPs (Roche) according to manufacturer's instructions. This produced a 10.1 kilobase vector fragment minus the Gentamycin cassette, which was recircularized by self-ligating with T4 DNA Ligase (Roche), and transformed into Top10 cells (Invitrogen) via standard conditions. Transformed cells were selected for on LB agar containing 50 μg/ml kanmycin sulfate and grown overnight at 37° C. Colonies were then used to inoculate 2 ml of liquid LB containing 50 μg/ml kanamycin sulfate and grown overnight at 37° C. Plasmid DNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen) following manufacturer's instructions. The recircularized plasmid was then digested with KpnI (Roche) and extracted from agarose gel via the Qiaex II DNA Extraction kit (Qiagen) according to manufacturers' instructions.
[0168]The Act-NPT Kpn-cut insert and the Kpn-cut pBPS-JH001 recircularized vector were then ligated together using T4 DNA Ligase (Roche) and transformed into Top10 cells (Invitrogen) according to manufacturers' instructions, The resulting construct, pBPS-SC022, now contained the Super Promoter, the GUS gene, the NOS terminator, and the Act-NPT cassette. Transformed cells were selected for on LB agar containing 50 μg/ml kanmycin sulfate and grown overnight at 37° C. Colonies were then used to inoculate 2 ml of liquid LB containing 50 μg/ml kanamycin sulfate and grown overnight at 37° C. Plasmid DNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen) following manufacturer's instructions. After confirmation of ligation success via restriction digests, pBPS-sc022 plasmid DNA was further propagated and recovered using the Plasmid Midiprep Kit (Qiagen) following the manufacturer's instructions.
[0169]Analyses of clones by restriction mapping was performed according to standard molecular biology techniques (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory),
Subcloning of PK-3, PK-4, PK-10, and PK-11 into the Binary Vectors
[0170]The fragments containing the different Physcomitrella patens polypeptide kinases were subcloned from the recombinant PCR2.1 TOPO vectors by double digestion with restriction enzymes (See Table 15) according to manufacturer's instructions. The subsequent fragment was excised from agarose gel with a QIAquick Gel Extraction Kit (Qiagen) according to manufacturer's instructions and ligated into the binary vector pBPS-JH001 or pBPS-SC022 which was cleaved with appropriate enzymes (See Table 15) and dephosphorylated prior to ligation. The resulting recombinant vectors (See Table 15) contained the corresponding Polypeptide Kinase in the sense orientation under the constitutive super promoter.
TABLE-US-00016 TABLE 15 Listed are the names of the various constructs of the Physcomitrella patens Polypeptide Kinases used for plant transformation Enzymes Enzymes Used Used to Binary to Generate Restrict the Binary Vector Gene Vector Gene Fragment Binary Vector Construct PK-3 pBPS-JH001 XmaI/SacI XmaI/SacI pBPS-LVM071 PK-4 pBPS-JH001 XmaI/EcoRV XmaI/Ecl136 pBPS-LVM015 PK-10 pBPS-SC022 XmaI/SacI XmaI/SacI pBPS-ERG015 PK-11 pBPS-SC022 XmaI/SacI XmaI/SacI pBPS-LVM230
Agrobacterium Transformation
[0171]The recombinant vectors were transformed into Agrobacterium tumefaciens C58C1 and PMP90 according to standard conditions (Hoefgen and Wilimitzer, 1990).
Plant Transformation
[0172]Arabidopsis thaliana ecotype C24 were grown and transformed according to standard conditions (Bechtold, 1993, Acad. Sci. Paris. 316:1194-1199; Bent et al., 1994, Science 265:1856-1860).
Screening of Transformed Plants
[0173]T1 seeds were sterilized according to standard protocols (Xiong et al., 1999, Plant Molecular Biology Reporter 17: 159-170). Seeds were plated on 1/2 Murashige and Skoog media (MS) pH 5.7 with KOH (Sigma-Aldrich), 0.6% agar and supplemented with 1% sucrose, 2 μg/ml benomyl (Sigma-Aldrich), and 150 μg/ml gentamycin (Sigma-Aldrich)(pBPS-JH001 transformants) or 50 μg/ml kanamycin (pBPS-SC022 transformants). Seeds on plates were vernalized for four days at 4° C. The seeds were germinated in a climatic chamber at an air temperature of 22° C. and light intensity of 40 micromol s-1 m2 (white light; Philips TL 65W/25 fluorescent tube) and 16 hours light and 8 hours dark day length cycle. Transformed seedlings were selected after 14 days and transferred to 12 MS media pH 5.7 with KOH 0.6% agar plates supplemented with 1% sucrose, 0.5 g/L US (Sigma-Aldrich), and 2 μg/ml benomyl (Sigma-Aldrich) and allowed to recover for five to seven days.
Drought Tolerance Screening
[0174]T1 seedlings were transferred to dry, sterile filter paper in a petri dish and allowed to desiccate for two hours at 80% RH (relative humidity) in a Sanyo Growth Cabinet MLR-350H, micromole s-1 m-2 (white light; Philips TL 65W/25 fluorescent tube). The RH was then decreased to 60%, and the seedlings were desiccated further for eight hours. Seedlings were then removed and placed on 1 MS 0.6% agar plates supplemented with 2 μg/ml benomyl (Sigma-Aldrich) and 0.5 g/L MES (Sigma-Aldrich) and scored after five days.
[0175]Under drought stress conditions, PK-3 over-expressing Arabidopsis thaliana plants showed a 54% survival rate (7 survivors from 13 stressed plants) to the stress screening, whereas the untransformed control only showed a 6% survival rate (1 survivor from 18 stressed plants). It is noteworthy that the analyses of these transgenic lines were performed with T1 plants, and therefore, the results will be better when a homozygous, strong expresser is found.
[0176]Transgenic plants overexpressing the PKSRP are screened for their improved drought tolerance, demonstrating that transgene expression confers drought tolerance.
TABLE-US-00017 TABLE 16 Summary of the drought stress tests Drought Stress Test Total number of Percentage of Gene Name Number of survivors plants survivors PK-3 7 13 54% Control 1 18 6%
"In-Soil" Drought Tolerance Screening
[0177]Ti seeds were sterilized in 100% bleach, 0.01% TritonX for five minutes two times and rinsed five times with sterile ddH2O. The sterile seeds were plated onto selection plates (1/2 MS, 0.6% phytagar, 0.5 g/L MES, 1% sucrose, 2 μg/ml benamyl, 50 μg/ml kanamycin, 0.6% agar). Plates were incubated at 4° C. for 4 days in the dark.
[0178]Plates were then moved for to 22° C. tinder continuous light for 10 days for germination and concomitant selection for transgenic plants. Seedlings were transplanted at the 4-5-leaf stage into 5.5 cm diameter pots filled with loosely packed soil (Metromix 360, Scotts) wetted with 1 g/L 20-20-20 fertilizer (Peters Professional, Scotts), Pots were placed randomly on trays with 5 control plants (transformed lines with empty vector) in each tray. Trays were placed randomly in the growth chamber.
[0179]Plants were grown (22° C., continuous light) for approximately seven days, watering as needed. Watering was stopped at the time when the majority of the plants was about to bolt, and this point was denoted day "0" of the assay. After this day, trays were turned 180° every other day to minimize local drying patterns. The assay was stopped approximately at day 12-19, depending on the speed of drying of the pots containing the controls. Pots were then watered and survival rates were determined after 5 days.
[0180]PK-10 overexpressing Arabidopsis thaliana plants showed a 60% survival rate (6 survivors from 10 stressed plants) to the stress screening. PK-11 over-expressing Arabidopsis thaliana plants showed a 65% survival rate (11 survivors from 17 stressed plants) to the stress screening. This survival rate is significantly higher, 99% confidence interval, than that of the control. It is noteworthy that these analyses were performed with T1 plants. The results should be better when a homozygous, strong expresser is found.
TABLE-US-00018 TABLE 17 Summary of the drought stress tests Drought Test Summay Gene Number of Total Number of Percentage of Name survivors plants survivors PpPK-10 6 10 60% PpPK-11 11 17 65%
HS=significant difference with 99% confidence interval on a z-test
Freezing Tolerance Screening
[0181]Seedlings are moved to petri dishes containing 1/2 MS 0.6% agar supplemented with 2% sucrose and 2 μg/ml benomyl. After four days, the seedlings are incubated at 4° C. for 1 hour and then covered with shaved ice. The seedlings are then placed in an Environmental Specialist ES2000 Environmental Chamber and incubated for 3.5 hours beginning at -1.0° C., decreasing 1° C./hour. The seedlings are then incubated at -5.0° C. for 24 hours and then allowed to thaw at 5° C. for 12 hours. The water is poured off and the seedlings are scored after 5 days. Transgenic plants over-expressing PK-3 and PK-4 are screened for their improved freezing tolerance demonstrating that transgene expression confers freezing tolerance,
Salt Tolerance Screening
[0182]Seedlings are transferred to filter paper soaked in 1/2 MS and placed on 1/2 MS 0.6% agar supplemented with 2 μg/ml benomyl the night before the salt tolerance screening. For the salt tolerance screening, the filter paper with the seedlings is moved to stacks of sterile filter paper, soaked in 50 mM NaCl, in a petri dish. After two hours, the filter paper with the seedlings is moved to stacks of sterile filter paper, soaked with 200 mM NaCl, in a petri dish. After two hours, the filter paper with the seedlings is moved to stacks of sterile filter paper, soaked in 600 mM NaCl, in a petri dish. After 10 hours, the seedlings are moved to petri dishes containing 1 MS 0.6% agar supplemented with 2 μg/ml benomyl. The seedlings are scored after 5 days. The transgenic plants are screened for their improved salt tolerance demonstrating that transgene expression confers salt tolerance.
Example 8
Detection of the PK-3 and PK-4 Transgenes in the Transgenic Arabidopsis Lines
[0183]To check for the presence of the PK-3 and PK-4 transgenes in transgenic Arabidopsis lines, PCR was performed on genomic DNA which contaminates the RNA samples taken as described in Example 9 below. Two and one half microliters of the RNA sample was used in a 50 μl PCR reaction using Taq DNA polymerase (Roche Molecular Biochemicals) according to the manufacturer's instructions.
[0184]Binary vector plasmid with each gene cloned in was used as positive control, and the wild-type C24 genomic DNA was used as negative control in the PCR reactions. Ten μl of the PCR reaction was analyzed on 0.8% agarose--ethidium bromide gel.
PK-3: The primers used in the reactions were:
TABLE-US-00019 5'CGAGAGCTGCAGATCATGCGACTGTTG3' (SEQ ID NO: 41) 5'GCTCTGCCATCACGCAACCCATCGAC 3' (SEQ ID NO: 42)
[0185]The PCR program was as following: 35 cycles of 1 minute at 94° C., 30 seconds at 62° C., and 1 minute at 72° C., followed by 5 minutes at 72° C. A 0.45 kilobase fragment was produced from the positive control and the transgenic plants.
[0186]PK-4: The primers used in the reactions were:
TABLE-US-00020 (SEQ ID NO: 37) 5'ATCCCGGGAGGCATTGAACTACCTGGAGTGAG3' (SEQ ID NO: 43) 5'GCGATATCGTTGAACTAGTAATCTGTGTTAACTTTATC3'
[0187]The PCR program was as following: 30 cycles of 1 minute at 94° C., 1 minute at 62° C., and 4 minutes at 72° C., followed by 10 minutes at 72° C. A 1.7 kilobase fragment was produced from the positive control and the transgenic plants.
[0188]The transgenes were successfully amplified from the T1 transgenic lines, but not from the wild type C24. This result indicates that the T1 transgenic plants contain at least one copy of the transgenes. There was no indication of existence of either identical or very similar genes in the untransformed Arabidopsis thaliana control which could be amplified by this method from the wild-type plants.
Example 9
Detection of the PK-3 and PK-4 Transgene mRNA in transgenic Arabidopsis Lines
[0189]Transgene expression was detected using RT-PCR. Total RNA was isolated from stress-treated plants using a procedure adapted from Verwoerd et alt, 1989, NAR 17:2362). Leaf samples (50-100 mg) were collected and ground to a fine powder in liquid nitrogen. Ground tissue was resuspended in 500 μl of a 80° C., 1:1 mixture, of phenol to extraction buffer (100 mM LiCl, 100 mM Tris pH8, 10 mM EDTA, 1% SDS), followed by brief vortexing to mix. After the addition of 250 μl of chloroform, each sample was vortexed briefly. Samples were then centrifuged for 5 minutes at 12,000×g. The upper aqueous phase was removed to a fresh eppendorf tube. RNA was precipitated by adding 1/10th volume 3 M sodium acetate and 2 volumes 95% ethanol. Samples were mixed by inversion and placed on ice for 30 minutes. RNA was pelleted by centrifugation at 12,000×g for 10 minutes. The supernatant was removed and pellets briefly air-dried. RNA sample pellets were resuspended in 10 μl DEPC treated water. To remove contaminating DNA from the samples, each was treated with RNase-free DNase (Roche) according to the manufacturer's recommendations. cDNA was synthesized from total RNA using the Superscript First-Strand Synthesis System for RT-PCR (Gibco-BRL) following manufacturer's recommendations.
[0190]PCR amplification of a gene-specific fragment from the synthesized cDNA was performed using Taq DNA polymerase (Roche) and gene-specific primers described in Example 8 in the following reaction: 1×PCR buffer, 1.5 mM MgCl2, 0.2 μM each primer, 0.2 μM dNTPs, 1 unit polymerase, 5 μl cDNA from synthesis reaction. Amplification was performed under the following conditions: denaturation, 95° C., 1 minute; annealing, 62° C., 30 seconds; extension, 72° C., 1 minute, 35 cycles; extension, 72° C., 5 minutes; hold, 4° C., forever. PCR products were run on a 1% agarose gel, stained with ethidium bromide, and visualized tinder UV light using the Quantity-One gel documentation system (Bio-Rad).
[0191]Expression of the transgenes was detected in the T1 transgenic line. This result indicated that the transgenes are expressed in the transgenic lines and suggested that their gene product improved plant stress tolerance in the transgenic line. In agreement with the previous statement, no expression of identical or very similar endogenous genes could be detected by this method. These results are in agreement with the data from Example 8.
Example 10
Engineering Stress-Tolerant Soybean Plants by Over-Expressing the PK-3, PK-4, PK-10, and PK-11 Genes
[0192]The constructs pBPS-LVM071, pBPS-LVM015, pBPS-ERG015, and pBPS-LVM230 are used to transform soybean as described below.
[0193]Seeds of soybean are surface sterilized with 70% ethanol for 4 minutes at room temperature with continuous shaking, followed by 20% (v/v) Clorox supplemented with 0.05% (v/v) Tween for 20 minutes with continuous shaking. Then, the seeds are rinsed 4 times with distilled water and placed on moistened sterile filter paper in a Petri dish at room temperature for 6 to 39 hours. The seed coats are peeled off, and cotyledons are detached from the embryo axis. The embryo axis is examined to make sure that the meristematic region is not damaged. The excised embryo axes are collected in a half-open sterile Petri dish and air-dried to a moisture content less than 20% (fresh weight) in a scaled Petri dish until further use.
[0194]Agrobacterium tumefaciens culture is prepared from a single colony in LB solid medium plus appropriate antibiotics (e.g. 100 mg/l streptomycin, 50 mg/l kanamycin) followed by growth of the single colony in liquid LB medium to an optical density at 600 nm of 0.8. Then, the bacteria culture is pelleted at 7000 rpm for 7 minutes at room temperature, and resuspended in MS (Murashige and Skoog, 1962) medium supplemented with 100 μM acetosyringone. Bacteria cultures are incubated in this pre-induction medium for 2 hours at room temperature before use. The axis of soybean zygotic seed embryos at approximately 15% moisture content are imbibed for 2 hours at room temperature with the pre-induced Agrobacterium suspension culture. The embryos are removed from the imbibition culture and transferred to Petri dishes containing solid MS medium supplemented with 2% sucrose and incubated for 2 days, in the dark at room temperature.
[0195]Alternatively, the embryos are placed on top of moistened (liquid MS medium) sterile filter paper in a Petri dish and incubated under the same conditions described above. After this period, the embryos are transferred to either solid or liquid MS medium supplemented with 500 mg/L carbenicillin or 300 mg/L cefotaxime to kill the agrobacteria. The liquid medium is used to moisten the sterile filter paper. The embryos are incubated during 4 weeks at 25° C., under 150 μmol m-2 sec-1 and 12 hours photoperiod. Once the seedlings have produced roots, they are transferred to sterile metromix soil. The medium of the in vitro plants is washed off before transferring the plants to soil. The plants are kept under a plastic cover for 1 week to favor the acclimatization process. Then the plants are transferred to a growth room where they are incubated at 25° C., under 150 μmol m-2 sec-1 light intensity and 12 hours photoperiod for about 80 days.
[0196]The transgenic plants are then screened for their improved drought, salt, and/or cold tolerance according to the screening method described in Example 7, demonstrating that transgene expression confers stress tolerance.
Example 11
Engineering Stress-Tolerant Rapeseed/Canola Plants by Over-Expressing the PK-3, PK-4, PK-10, and PK-11 Genes
[0197]The constructs pBPS-LVM071, pBPS-LVM015, pBPS-ERG015, and pBPS-LVM230 are used to transform rapeseed/canola as described below.
[0198]The method of plant transformation described herein is applicable to Brassica and other crops. Seeds of canola are surface sterilized with 70% ethanol for 4 minutes at room temperature with continuous shaking, followed by 20% (v/v) Clorox supplemented with 0.05% (v/v) Tween for 20 minutes, at room temperature with continuous shaking. Then, the seeds are rinsed 4 times with distilled water and placed on moistened sterile filter paper in a Petri dish at room temperature for 18 hours. Then the seed coats are removed, and the seeds are air dried overnight in a half-open sterile Petri dish. During this period, the seeds lose approximately 85% of its water content. The seeds are then stored at room temperature in a sealed Petri dish until further use. DNA constricts and embryo imbibition are as described in Example 10. Samples of the primary transgenic plants (T0) are analyzed by PCR to confirm the presence of T-DNA. These results are confirmed by Southern hybridization in which DNA is electrophoresed on a 1% agarose get and transferred to a positively charged nylon membrane (Roche Diagnostics). The PCR DIG Probe Synthesis Kit (Roche Diagnostics) is used to prepare a digoxigenin-labelled probe by PCR, and used as recommended by the manufacturer.
[0199]The transgenic plants are then screened for their improved stress tolerance according to the screening method described in Example 7, demonstrating that transgene expression confers stress tolerance.
Example 12
Engineering Stress-Tolerant Corn Plants by Over-Expressing the PK-3, PK-4, PK-10, and PK-11 Genes
[0200]The constructs pBPS-LVM071, pBPS-LVM015, pBPS-ERG015, and pBPS-LVM230 are used to transform corn as described below.
[0201]Transformation of maize (Zea Mays L.) is performed with the method described by Ishida et al., 1996, Nature Biotech. 14745-50. Immature embryos are co-cultivated with Agrobacterium tumefaciens that carry "super binary" vectors, and transgenic plants are recovered through organogenesis. This procedure provides a transformation efficiency of between 2.5% and 20%. The transgenic plants are then screened for their improved drought, salt, and/or cold tolerance according to the screening method described in Example 7, demonstrating that transgene expression confers stress tolerance.
Example 13
[0202]Engineering Stress-Tolerant Wheat Plants by Over-Expressing the PK-3, PK-4, PK-10, and PK-11 Genes
[0203]The constructs pBPS-LVM071, pBPS-LVM015, pBPS-ERG015, and pBPS-LVM230 are used to transform wheat as described below.
[0204]Transformation of wheat is performed with the method described by Ishida et al., 1996, Nature Biotech. 14745-50, Immature embryos are co-cultivated with Agrobacterium tumefaciens that carry "super binary" vectors, and transgenic plants are recovered through organogenesis. This procedure provides a transformation efficiency between 2.5% and 20%. The transgenic plants are then screened for their improved stress tolerance according to the screening method described in Example 7, demonstrating that transgene expression confers stress tolerance.
Example 14
Identification of Identical and Heterologous Genes
[0205]Gene sequences can be used to identify identical or heterologous genes from cDNA or genomic libraries. Identical genes (e.g. full-length cDNA clones) can be isolated via nucleic acid hybridization using for example cDNA libraries. Depending on the abundance of the gene of interest, 100,000 up to 1,000,000 recombinant bacteriophages are plated and transferred to nylon membranes. After denaturation with alkali, DNA is immobilized on the membrane by e.g. UV cross linking. Hybridization is carried out at high stringency conditions. In aqueous solution, hybridization and washing is performed at an ionic strength of 1 M NaCl and a temperature of 68° C. Hybridization probes are generated by e.g. radioactive (32P) nick transcription labeling (High Prime, Roche, Mannheim, Germany). Signals are detected by autoradiography.
[0206]Partially identical or heterologous genes that are related but not identical can be identified in a manner analogous to the above-described procedure using low stringency hybridization and washing conditions. For aqueous hybridization, the ionic strength is normally kept at 1 M NaCl while the temperature is progressively lowered from 68 to 42° C.
[0207]Isolation of gene sequences with homology (or sequence identity/similarity) only in a distinct domain of (for example 10-20 amino acids) can be carried out by using synthetic radio labeled oligonucleotide probes. Radiolabeled oligonucleotides are prepared by phosphorylation of the 5-prime end of two complementary oligonucleotides with T4 polynucleotide kinase. The complementary oligonucleotides are annealed and ligated to form concatemers. The double stranded concatemers are than radiolabeled by, for example, nick transcription. Hybridization is normally performed at low stringency conditions using high oligonucleotide concentrations.
Oligonucleotide Hybridization Solution:
6×SSC
[0208]0.01 M sodium phosphate
11 nM EDTA (pH 8)
0.5% SDS
[0209]100 μg/ml denatured salmon sperm DNA0.1% nonfat dried milk
[0210]During hybridization, temperature is lowered stepwise to 5-10° C. below the estimated oligonucleotide Tm or down to room temperature followed by washing steps and autoradiography. Washing is performed with low stringency such as 3 washing steps using 4×SSC. Further details are described by Sambrook, J. et al., 1989, "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor Laboratory Press or Ausubel, F. M. et al., 1994, "Current Protocols in Molecular Biology," John Wiley & Sons.
Example 15
Identification of Identical Genes by Screening Expression Libraries with Antibodies
[0211]cDNA clones can be used to produce recombinant polypeptide for example in E. coli (e.g. Qiagen QIAexpress pQE system). Recombinant polypeptides are then normally affinity purified via Ni-NTA affinity chromatography (Qiagen). Recombinant polypeptides are then used to produce specific antibodies for example by using standard techniques for rabbit immunization. Antibodies are affinity purified using a Ni-NTA column saturated with the recombinant antigen as described by Gu et al., 1994, BioTechniques 17:257-262. The antibody can than be used to screen expression cDNA libraries to identify identical or heterologous genes via an immunological screening (Sambrook, J. et al., 1989, "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor Laboratory Press or Ausubel, F. M. et al., 1994, "Current Protocols in Molecular Biology", John Wiley & Sons).
Example 16
[0212]In Vivo Mutagenesis
[0213]In vivo mutagenesis of microorganisms can be performed by passage of plasmid (or other vector) DNA through E. coli or other microorganisms (e.g. Bacillils spp. or yeasts such as Saccharomyces cerevisiae) which are impaired in their capabilities to maintain the integrity of their genetic information. Typical mutator strains have mutations in the genes for the DNA repair system (e.g., mutHLS, mutD, mutT, etc.; for reference, see Rupp, W. D., 1996, DNA repair mechanisms, in: Escherichia coli and Salmonella, p. 2277-2294, ASM: Washington.) Such strains are well known to those skilled in the art. The use of such strains is illustrated, for example, in Greener, A. and Callahan, M., 1994, Strategies 7: 32-34. Transfer of mutated DNA molecules into plants is preferably done after selection and testing in microorganisms. Transgenic plants are generated according to various examples within the exemplification of this document.
Example 17
In vitro Analysis of the Function of Physcomitrella Genes in Transgenic Organisms
[0214]The determination of activities and kinetic parameters of enzymes is well established in the art. Experiments to determine the activity of any given altered enzyme must be tailored to the specific activity of the wild-type enzyme, which is well within the ability of one skilled in the art, Overviews about enzymes in general, as well as specific details concerning structure, kinetics, principles, methods, applications and examples for the determination of many enzyme activities may be found, for example, in the following references: Dixon, M., and Webb, E. C., 1979, Enzymes. Longmans: London; Fersht, 1985, Enzyme Structure and Mechanism. Freeman: New York; Walsh, 1979, Enzymatic Reaction Mechanisms. Freeman: San Francisco; Price, N. C., Stevens, L., 1982, Fundamentals of Enzymology. Oxford Univ. Press: Oxford; Boyer, P. D., ed., 1983, The Enzymes, 3rd ed. Academic Press: New York; Bisswanger, H., 1994, Enzymkinetik, 2nd cd. VCH: Weinheim (ISBN 3527300325); Bergmeyer, H. U., Bergmeyer, J., GraβI, M., eds., 1983-1986, Methods of Enzymatic Analysis, 3rd ed., vol. I-XII, Verlag Chemie: Weinheim; and Ullmann's Encyclopedia of Industrial Chemistry, 1987, vol. A9, Enzymes. VCH: Weinheim, p. 352-363.
[0215]The activity of proteins which bind to DNA can be measured by several well-established methods, such as DNA band-shift assays (also called gel retardation assays). The effect of such proteins on the expression of other molecules can be measured using reporter gene assays (such as that described in Kolmar, H. et al., 1995, EMBO J. 14: 3895-3904 and references cited therein). Reporter gene test systems are well known and established for applications in both prokaryotic and eukaryotic cells, using enzymes such as O-galactosidase, green fluorescent protein, and several others.
[0216]The determination of activity of membrane-transport proteins can be performed according to techniques such as those described in Gennis, R. B., 1989, Pores, Channels and Transporters, in Biomembranes, Molecular Structure and Function, pp. 85-137, 199-234 and 270-322, Springer: Heidelberg.
Example 18
Purification of the Desired Product from Transformed Organisms
[0217]Recovery of the desired product from plant material (i.e., Physcomitrella patens or Arabidopsis thaliana), fungi, algae, ciliates, C. glutamicin cells, or other bacterial cells transformed with the nucleic acid sequences described herein, or the supernatant of the above-described cultures can be performed by various methods well known in the art. If the desired product is not secreted from the cells, the cells can be harvested from the culture by low-speed centrifugation, and the cells can be lysed by standard techniques, such as mechanical force or sonification. Organs of plants can be separated mechanically from other tissue or organs. Following homogenization, cellular debris is removed by centrifugation, and the supernatant fraction containing the soluble proteins is retained for further purification of the desired compound. If the product is secreted from desired cells, then the cells are removed from the culture by low-speed centrifugation, and the supernatant fraction is retained for further purification.
[0218]The supernatant fraction from either purification method is subjected to chromatography with a suitable resin, in which the desired molecule is either retained on a chromatography resin while many of the impurities in the sample are not, or where the impurities are retained by the resin while the sample is not. Such chromatography steps may be repeated as necessary, using the same or different chromatography resins. One skilled in the art would be well-versed in the selection of appropriate chromatography resins and in their most efficacious application for a particular molecule to be purified. The purified product may be concentrated by filtration or ultrafiltration, and stored at a temperature at which the stability of the product is maximized.
[0219]There is a wide array of purification methods known to the art and the preceding method of purification is not meant to be limiting. Such purification techniques are described, for example, in Bailey, J. E. & Ollis, D. F., 1986, Biochemical Engineering Fundamentals, McGraw-Hill: New York. Additionally, the identity and purity of the isolated compounds may be assessed by techniques standard in the art. These include high-performance liquid chromatography (HPLC), spectroscopic methods, staining methods, thin layer chromatography, NIRS, enzymatic assay, or microbiologically. Such analysis methods are reviewed in: Patek et at., 1994, Appl. Environ. Microbiol. 60:133-140; Malakhova et al., 1996, Biotekhnologiya 11:27-32; Schmidt et al., 1998, Bioprocess Engineer 19:67-70; Ulmann's Encyclopedia of Industrial Chemistry, 1996, vol. A27, VCH: Weinheim, p. 89-90, p. 521-540, p. 540-547, p. 559-566, 575-581, and p. 581-587; Michal, G., 1999, Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. et al., 1987, Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, vol. 17.
APPENDIX
TABLE-US-00021 [0220](SEQ ID NO: 1) Nucleoticle sequence of the partial PK-3 from Physcomitrella patens CGGCACCAGCATCTTCGCGAGGCATGTGATGTGTGGTCGGTGGAGTTAGCTTCTA CGGGCAACTGGAAATCCAGGGAATTCTGCCAGAATTATACGTACTAAAGTAGAA ATTTACGTTTCGGGGACTTCGAGTCTTCTATGGCATCTGCGACTGCGGGTATTAT CAACAGCACAAACATGATCGGAGGAGGAATAGCTCCAACTAAAGCTGGCTCAAG CGGAGTAGAATTGTTACCGAAAGAAATGCACGACATGAAGCTCAGCGATGACAA GGTTGACCACAGCGACGACAAGGAAATTGAGGCTTCAATAGTAGATGGAAACGG TACCGAAACTGGCCACATCATAGCTACTACTATTGGAGGGCGAAATGGACAACC TAAGCAGACGATCAGCTATTCGGCAGAACGTGTTGTTGGCACTGGATCATTCGG GATTGTCTTCCAGGCAAAATGCATCGAAACTGGGGAGACGGTGGCTATAAAGAA AGTGTTGCAGGACAAAAGATACAAGAATCGAGAGCTGCAGATCATGCGACTGTT GGACCACCCGAATATTGTAGCTTTGAAGCATTGCTTCTTCTCGACGACGGATAAA GACGAATTGTACTTAAACCTGGTGCTGGAGTATGTACCCGAGACGGTGTATCGTA TTGCAAAGCACTACAATCGCATGAATCAGCGAATGCCCCTTGTTTACGTGAAACT GTACACGTATCAGATATGCCGATCACTGGCATATATCCACAATGGCATCGGTGTC TGCCACCGCGACATCAAGCCCCAGAACCTGCTGGTGGAATCCTCATACGCACCA GCTGAAACTGTGTGATTTTGGGAAGTGCGAAAGTGCTGGTGAAAGGGGAGCCCA ATATCTCGTACATTTGTTCGCGGTACTACCGTGCTCCGGGAGCTTATTTTTGGAGC GACGGAGTACACGACTGCCATAGATATATGGTCGATGGGTTGCGTGATGGCAGA GCTTCTACTAGGACAGCCTTTGTTTCCTGGAGAGAGTGGAGTGGATCAATTGGTG GAAATCATCAAGGTTTTGGGGACACCGACTCGTGAGGAGATCAAGTGCATGAAT CCGAACTACAC (SEQ ID NO: 2) Nucleotide sequence of the full-length PK-3 from Physcomitrella patens GGCACCAGCATCTTCGCGAGGCATGTGATGTGTGGTCGGTGGAGTTAGCTTCTAC GGGCAACTGGAAATCCAGGGAATTCTGCCAGAATTATACGTACTAAAGTAGAAA TTTACGTTTCGGGGACTTCGAGTCTTCTATGGCATCTGCGACTGCGGGTATTATC AACAGCACAAACATGATCGGAGGAGGAATAGCTCCAACTAAAGCTGGCTCAAGC GGAGTAGAATTGTTACCGAAAGAAATGCACGACATGAAGCTCAGGGATGACAAG GTTGACCACAGCGACGACAAGGAAATTGAGGCTTCAATAGTAGATGGAAACGGT ACCGAAACTGGCCACATCATAGCTACTACTATTGGAGGGCGAAATGGACAACCT AAGCAGACGATCAGCTATTCGGCAGAACGTGTTGTTGGCACTGGATCATTCGGG ATTGTCTTCCAGGCAAAATGCATCGAAACTGGGGAGACGGTGGCTATAAAGAAA GTGTTGCAGGACAAAAGATACAAGAATCGAGAGCTGCAGATCATGCGACTGTTG GACCACCCGAATATTGTAGCTTTGAAGCATTGCTTCTTCTCGACGACGGATAAAG ACGAATTGTACTTAAACCTGGTGCTGGAGTATGTACCCGAGACGGTGTATCGTAT TGCAAAGCACTACAATCGCATGAATCAGCGAATGCCCCTTGTTTACGTGAAACTG TACACGTATCAGATATGCCGATCACTGGCATATATCCACAATGGCATCGGTGTCT GCCACCGCGACATCAAGCCCCAGAACCTGCTGGTGAATCCTCATACGCACCAGC TGAAACTGTGTGATTTTGGAAGTGCGAAAGTGCTGGTGAAAGGGGAGCCCAATA TCTCGTACATTTGTTCGCGGTACTACCGTGCTCCGGAGCTTATTTTTGGAGCGAC GGAGTACACGACTGCCATAGATATATGGTCGATGGGTTGCGTGATCGCAGAGCT TCTACTAGGACAGCCTTTGTTTCCTGGAGAGAGTGGAGTGGATCAATTGGTGGAA ATCATCAAGGTTTTGGGGACACCGACTCGTGAGGAGATCAAGTGCATGAATCCG AACTACACAGAGTTCAAGTTTCCACAAATCAAGGCGCACCCGTGGCACAAAGTT TTCCACAAACGCATGCCACCTGAAGCAGTTGACTTGGTGTCAAGGCTCCTTCAGT ACTCTCCAAATCTGCGGTGCAACGCTCTGGAAGCGTGTGTGCACCCGTTCTTTGA TGAGCTAAGGGATCCTAACTGCCGGCTTCCGAATGGGCGGCCACTGCCCTCTCTG TTCAACTTCAAAACCCAAGAGTTGAACGGTGCAACTCCTGATATTCTGCAGCGTT TGATACCCGAGCACGCGAGGAAGCAGAATCCGATGCTGGCGCTGTGAGGGGTGC CTGGAAAGAGATCGGAAGAGTCTACTGCGTGAAAGGTTTTCCTCTGTTTGGAGG AGTGGTCCGCTTTGTGGAGGGCTTCATAGGCACTCTGTATCATTGCTTAAACACG TAAAGTCAACCAATTTGCTATGGATCCCTGCTTTCGCTGTGATTGGAGGAAGACT TAGTAGACGATTAGCATGCCACTTTTAGGAACGGCAATTCTCCTGTAGTGAAGGT TACGATTCTATTGTACTTCAGAACGGTAAAGGTATTTAGGGGTTCTCAGTGCTTC CTGATTTGGGTACGTGATGTACCATTGGAAAGGCTTCAAACGCATGTATATCTAT GAGACTTTGACGTTACTTTTTATCGTCAGTACTCAGGAAGCTCCTCTCTGGATGG GATTATCCATTCGTGCCGTTCGAATCGCAATAAAAAAAAAAAAAAAAAA (SEQ ID NO: 3) Deduced amino acid sequence of PK-3 from Physcomitrella patens MASATAGIINSTNMIGGGIAPTKAGSSGVELLPKEMHDMKLRDDKVDHSDDKEIEASI VDGNGTETGHIIATTIGGRNGQPKQTISYSAERVVGTGSFGIVFQAKCIETGETVAIKK VLQDKRYKNRELQIMRLLDHPNIVALKHCFFSTTDKDELYLNLVLEYVPETVYRIAK HYNRMNQRMPLVYVKLYTYQICRSLAYIHNGIGVCHRDIKPQNLLVNPHTHQLKLC DFGSAKVLVKGEPNISYICSRYYRAPELIFGATEYTTAIDIWSMGCVMAELLLGQPLFP GESGVDQLVEIIKVLGTPTREEIKCMNPNYTEFKFPQIKAHPWHKVFHKRMPPEAVD LVSRLLQYSPNLRCNALEACVHPFFDELRDPNCRLPNGRPLPSLFNFKTQELKGATPD ILQRLIPEHARKQNPMLAL* (SEQ ID NO: 4) Nucleotide sequence of the partial PK-4 from Physcomitrella patens GCACGAGGATCGACCGGGTGGAGTACGTGCACTCCCGAGGTCTAATTCATCGTG ACTTGAAACCAGATAATTTCCTCATGGGCTGCGGCCGGCAAGGGAACCAAGTGT TCATTATTGACTTTGGCTTGGCAAAAGAGTACATCGACCCCGCGACACGTAGACA CATTCCTTACCGAGATAGAAAGAGCTTTACAGGAACAGCGCGGTATGCTAGTAG GAATCNCCACNAAGGAATCGAACACAGCAGGAGAGATGACATANAATCNCTTG GTTACATTCTTATGTACTTTCTTAGGGGGAATTTACCATGGCAAGGTCAAGGGGG GCAACGTTTCACCGATCAGAAGCAACATGAGTACATGCNCAACAAAATTAAGAT GGAGACTANCATCNAGGATCTCTGCGATGGGTACCCAGACA (SEQ ID NO: 5) Nucleotide sequence of the full-length PK-4 from Physcomitrella patens GCCCTTATCCCGGGAGGCATTGAACTACCTGGAGTGAGATTTTTTTGGGAATTTG AAAGAGAATTACATATATACAAGGTTGAGGCTCACCGAGAACAAGTCTGCTGAT AGCTTCTTCACTCTTGAAATAGATAGTTCATCATGGATTCAGGAGGTGACCGCGT GCGAGCTCCTCAGAAGCAGTCTCGCGAGGAGGATCAGTACCGTTCATTGAACAT TGCTACAGAGCATCGTCAGCATATACAGAAGCACCAACAACACCAACAGCAGCC GGGGACTGGATTGGTTGTTGAAACGCTTCAAAAAACACTATGTAACGTGACTGT GACCTCACCTACAAGCAGTCCGGAGGGGGGTAGATTACGTACTGTTGCGAACAA GTATGCAGTGGAAGGAATGGTCGGCAGTGGCGCATTTTGCAAGGTGTACCAGGG TTCTGACTTAACCAACCATGAGGTTGTGGGCATCAAGCTCGAGGATACAAGAAC AGAGCACGCACAATTGATGCACGAGTCGCGATTATACAACATTTTGCGGGGTGG AAAGGGAGTGCCCAACATGAGATGGTTTGGGAAAGAGCAAGACTACAATGTGAT GGTGCTAGATTTGCTGGGGCCTAACCTACTGCACCTTTTCAAGGTGTGTGGGCAA AGATTTTCGTTGAAGACGGTGATCATGTTGGGGTACCAAATGATCGACCGGGTG GAGTACGTGCACTCGCGAGGTCTAGTTCATCGTGACTTGAAACCAGATAATTTCC TCATGGGCTGCGGCCGGCAAGGGAACCAAGTGTTCATTATTGACTTTGGCTTGGC AAAAGAGTACATCGACCCCGCGACACGTAGACACATTCCTTACCGAGATAGAAA GAGCTTTACAGGAACAGCGCGGTATGCTAGTAGGAATCAGCACAAAGGAATCGA ACACAGCAGGAGAGATGAGATAGAATCACTTGGTTACATTCTTATGTACTTTCTT AGGGGGAATTTACCATGGCAAGGTCAAGGGGGGCAACGTTTCACCGATCAGAAG CAACATGAGTACATGCACAACAAAATTAAGATGGAGACTACCATCGAGGATCTC TGCGATGGGTACCCCAGACAATTTGCCGACTTTTTACACCACCCGCGCGAGTTGG GATTCTATGAGCAGCCTGACTACTCGTACCTTCGCAGCCTGTTCCGTGATCTTTTC ATTCAGAAGAAATTCCAGCTTGACCATGTCTACGACTGGACAGTGTACACTCAAC CTCCTCAGAATGGCTCTGCACAAACAGTTCGAAGCCCGGCTGCCGGTCCACAGA CTCACTTACAAAGTCGCCCTTCCAATGTATCATATTGTCCACCTCTGACTAAACC AGAGTTCCGCCGTGAGGTAGTTGCGGCGAATTAGGGTTTACACAGGAAGAGATG TGGTAAAGCATCTCATCTTCTTCGTTCTGGTGCCAAAATGGTACAAGGTCGTCTG CTGTCTCTTTCTCGCAAGCCCTCACATATAGATGAAGGTTTGTGAAGTTAGAGAT GCAACTACCAAGCAAAGGCTAGGAAAAGAGCTGTAGACTTTCTAGTGTGTAGTG TGTAAATCAAGGCTTCTGGCATGGTATCGGCAGTGAGGTGCATGGAGCAGAATA GAAATTACTTCGTGCATGACAAGATTTTTTTTCTTGCAGAGCTCTCGACGGTTCTG CGATCTCACTTCTCTACACAACCAGCGCTCCTTTAATTGAAAAGAGGATCTGGTA CGAGTATGATAAAGTTAACACAGATTACTAGTTCAACGATATCGCAAGGGC (SEQ ID NO: 6) Deduced amino acid sequence of PK-4 from Physcomitrella patens MDSGGDRVRAPQKQSREEDQYRSLNIATEHRQHIQKHQQHQQQPGTGLVVETLQKT LCNVTVTSPTSSPEGGRLRTVANKYAVEGMVGSGAFCKVYQGSDLTNHEVVGIKLE DTRTEHAQLMHESRLYNILRGGKGVPNMRWFGKEQDYNVMVLDLLGPNLLHLFKV CGQRFSLKTVIMLGYQMIDRVEYVHSRGLVHRDLKPDNFLMGCGRQGNQVFIIDFGL AKEYIDPATRRHIPYRDRKSFTGTARYASRNQHKGIEHSRRDDIESLGYILMYFLRGN LPWQGQGGQRFTDQKQHEYMHNKIKMETTIEDLCDGYPRQFADFLHHARELGFYEQ PDYSYLRSLFRDLFIQKKFQLDHVYDWTVYTQPPQNGSAQTVRSPAAGPQTHLQSRP SNVSYCPPLTKPEFRREVVAAN* (SEQ ID NO: 7) Nucleotide sequence of the partial PK-10 from Physcomitrella patens GCACGAGCGCACTTGGTTTCTGCCACTTATTCCAGCTGGTAAAGAAAAACCACCT AAAATGAAAGTGTTTGAAGCAGATACATTTGAGAAGGAAGTGGAAGAACCGAA GATCAAGGCCTTACCTCCATTGAAGTCACTTAAAGTACCTCCAGCTTTGAAGGTT
GAGGAAGCTACCTACAAGGTTGAAAGTGAAGGGAAGGTGAACAAGAGCAACAT TACAGCAAGAGAGTTTTCCGTCGCAGAACTTCAGGCGGCTACGGACAGTTTCTCA GAGGATAATTTACTTGGCGAAGGTTCGCTTGGTTGTGTTTACCGCGCGGAGTTCC CCGACGGTGAGGTTCTAGCTGTCAGAAACTTGATACAACAGCCTCCATGGTTCGG AATGAAGATGATTTCTTGAGCGTTGTCGATGGCTTGGCCCGGCTACAATACCAAT TCTAATGAACTCGTAGGCTACTGTGCCGAGCATGGGCAACGACTTCTGGTCTACA AGTTCATCAGTCGAGGGACACTCCATGAACTGCTTCATGGCTCAGCCG (SEQ ID NO: 8) Nucleotide sequence of the full-length PK-10 from Physcomitrella patens TTTCTGGAATAGCTCAGAAGCGTTGCAAAATTTATCAGGACGTTTGCAGACATGG TGATGAGGAAAGTCGGCAAGTATGAAGTGGGGCGAACTATTGGTGAGGGAACCT TCGCCAAGGTGAAATTTGCCCAGAACACCGAGACAGGGGAGAGCGTGGCCATGA AGGTGCTAGATCGTCAGACGGTGCTCAAGCACAAGATGGTAGAGCAGATCAGGC GAGAAATATCCATAATGAAGCTGGTTAGGCATCCTAATGTTGTCCGATTGCACGA GGTTCTGGCAAGTCGTTGCAAGATTTACATCATTTTGGAGTTTGTAACGGGCGGG GAGCTTTTTGACAAAATTGTGCATCAAGGAAGGCTTAATGAGAACGACTCTCGC AAATATTTTCAGCAGCTCATGGATGGAGTTGATTATTGCCACAGCAAGGGCGTCT CACATCGAGATTTGAAGCCTGAAAATCTCCTTCTTGATTCACTGGACAATCTCAA AATATCAGATTTTGGTCTGAGTGCTCTTCCTCAGCAAGTGAGGGAAGATGGACTT TTGCACACCACTTGTGGTACTCCCAATTATGTTGCACCTGAGGTTCTTAATGATA AGGGCTACGATGGTGCAGTGGCTGATATCTGGTCTTGCGGTGTCATCTTGTTTGT ATTAATGGCTGGATTTCTCCCATTTGATGAGGCTGACTTGAATACTCTTTACAGC AAGATACGAGAGGCAGATTTTACTTGTCCACCTTGGTTTTCCTCCGGCGCCAAAA CACTGATTACTAATATTCTGGATCCCAATCCCCTAACACGTATCAGGATGAGAGG AATTCGGGATGACGAATGGTTCAAAAAGAACTATGTTCCTGTTCGTATGTATGAC GATGAAGATATTAATCTTGATGATGTGGAGACTGCTTTTGATGATTCTAAGGAAC AATTTGTGAAAGAGCAGAGGGAGGTGAAAGACGTGGGTCCGTCGTTGATGAATG CCTTTGAACTCATAAGCCTATCTCAAGGACTAAACCTCTCTGCGTTGTTTGATAG ACGTCAGGACCATGTAAAGCGCCAAACTCGTTTCACTTCAAAGAAACCAGCTCG AGATATAATTAATAGAATGGAAACCGCTGCGAAGTCGATGGGCTTTGGTGTTGG AACGCGTAACTACAAGATGAGACTCGAGGCAGCTAGTGAGTGCAGAATATCACA GCACTTGGCTGTGGCTATCGAAGTGTACGAGGTGGCTCCTTCTTTATTCATGATT GAAGTGCGGAAGGCTGCGGGTGATACTTTGGAATATCACAAGTTCTATAAAAGC TTTTGTACCCGGTTGAAAGATATCATATGGACAACGGCAGTTGATAAGGACGAA GTTAAGACATTGACGCCATCTGTAGTTAAGAATAAATAATTCTGCTCCAGCATTA ACTTGGATGAGGAGCAAGGATATACCGCTGCATCGAGCTCCGAAGGGC (SEQ ID NO: 9) Deduced amino acid sequence of PK-10 from Physcomitrella patens MVMRKVGKYEVGRTIGEGTFAKVKFAQNTETGESVAMKVLDRQTVLKHKMVEQIR REISIMKLVRHPNVVRLHEVLASRCKIYIILEFVTGGELFDKIVHQGRLNENDSRKYFQ QLMDGVDYCHSKGVSHRDLKPENLLLDSLDNLKISDFGLSALPQQVREDGLLHTTCG TPNYVAPEVLNDKGYDGAVADIWSCGVILFVLMAGFLPFDEADLNTLYSKIREADFT CPPWFSSGAKTLITNILDPNPLTRIRMRGIRDDEWFKKNYVPVRMYDDEDINLDDVET AFDDSKEQFVKEQREVKDVGPSLMNAFELISLSQGLNLSALFDRRQDHVKRQTRFTS KKPARDIINRMETAAKSMGFGVGTRNYKMRLEAASECRISQHLAVAIEVYEVAPSLF MIEVRKAAGDTLEYHKFYKSFCTRLKDIIWTTAVDKDEVKTLTPSVVKNK* (SEQ ID NO: 10) Nucleotide sequence of the partial PK-11 from Physcomitrella patens GGCACGAGATTTGGTTGCAAAATAGGTAACTACAACTTAAGAAGAAAAACAATC TCTCTCTTTCCCCACACAAGATACAACTTCGCTTTTTCCATCACTTACACCAGAAA GCCCAAAGTAGGGTAGATTGTCACACATCGCTATGATCCCAATTAAGCATCTACT ACTTTTCATCAGATCAGCAAACTACCAATCATAGAAACTAGGTGATGAATATTAC GATACTTTCAGGTTCAATGCGAAATCCAAGGTTAACAGTAATGAATGTATTCAAG CTCTGTACATGCATTAATTTTATGCTACCAGTAGAAAACTTCATTTGACGATGCA GCGGTATATCCTTGCTCCTCATCCAAGTTAATGCTGGAGCAGAATTATTTATTCTT AACTACAGATGGCGTCAATGTCTTAACTTCGTCCTTATCAACTGCCGTTGTCCAT ATGATATCTTTCAACCGGGTACAAAAGCTTTTATAGAACTTGTGATATTCCAAAG TATCACCCGCAGCCTTCCGCACTTCAATCATGAATAAAGAAGGAGCCACCTCGTA CACTTCGATAGCCCAGCCAAGTGCTGTGATATTCTGCCTCACTACTGCCTCGAGC (SEQ ID NO: 11) Nucleotide sequence of the full-length PK-11 from Physcomitrella patens ATCCCGGGTGTCGGAATTCGGTCACAATGAGCTAGTGTGTTGTTTGATTGTGGCC TCAGCTGGAGAGGCTTTGGTATCGTTAGCAGCGAGTGACGCTGTTGAAGGATTGT ATCCATCCACAAGCGAGAAGCCTTGCCTAATTTTTGGGAGGGAAAGGTGGTTCTC ACATGAGAGGAGGAGTTGTCGATGCCCCAATGAAGGGTGACAGGAGAGCATGCA TTTTGGGAGGAATGGGAAGACCTAATGGTGGAACCATCTTGTACGTGTTGGTGAT TTCATTCATTGCTTTGGTGAATGGAGCCACCGATCCGAACGATGTGTCTGCTTTG AATACTATGTTCACTGGCTTCAACAGCGATCCTAAGCTCACGAACTGGGTGCAAA ACGCGGGTGATCCCTGCGGAACCAACTGGCTGGGCGTTACTTGTGATGGGACCTT CGTCACCTCAATCAAGCTATCCAACATGGGACTGAATGGGAAGGTGGACGGATG GGTGTTGCAGAAGTTTCAACACCTCTCTGTGCTTGACCTTAGCCATAATAATCTT GCTAGCGGAATTCCTGAGATGTTTCCTCCCAAGTTGACTGAACTAGATTTGTCTT ACAACCAGCTCACGGGTAGTTTTCCTTATTTGATAATCAACATCCCTACTTTGAC AAGCATAAAACTGAATAACAACAAGCTGAGTGGAACGCTCGATGGGCAGGTTTT CAGTAAACTCACAAACTTAATCACCCTCGATATTTCCAACAACGCAATTACAGGG CCGATTCCCGAGGGCATGGGTGACATGGTCAGCCTAAGATTTTTGAACATGCAA AATAATAAGCTGACTGGACCAATCCCAGACACATTGGCTAATATTCCATCTCTAG AAACATTGGACGTATCTAACAACGCGCTTACTGGCTTTCTCCCACCAAACCTGAA CCCAAAGAATTTCAGATATGGAGGCAATCCACTCAACACCCAAGCCCCTCCTCC ACCACCGTTTACACCACCGCCACCTTCAAAGAATCCAAAGCCTATTCCTCCTCCA CCCCACCCTGGTAGCCGAACACCAGATACTGCTCCTAAGGCTGAAGGCGGCATC GTATCAGGCGCAGCAATTGCTGGGATTGTCGTGGGAGCAATTTTGGTGCTTGCAG CAATTTTCATAGCTGTATGGTTCTTTGTCGTCCGTAAAAGATCTGAGCTTACCAA ACCTTTGGATTTAGAGGCTAATCACAGCAGCCGACGCACTTGGTTTCTGCCACTT ATTCCAGCTGGTAAAGAAAAACCACCTAAAATGAAAGTGTTTGAAGCAGATACA TTTGAGAAGGAAGTGGAAGAGCCGAAGATCAAGGCCTTACCTCCATTGAAGTCA CTTAAAGTACCTCCAGCATTGAAGGTTGAGGAAGCTACCTACAAGGTTGAAAGT GAAGGGAAGGTGAACAAGAGCAACATTACAGCAAGAGAGTTTTCCGTCGCAGA ACTTCAGGCGGCTACGGACAGTTTCTCAGAGGATAATTTACTTGGCGAAGGTTCG CTTGGTTGTGTTTACCGCGCGGAGTTCCCCGACGGIGAGGTTCTAGCTGTCAAGA AACTTGATACAACAGCCTCCATGGTTCGGAATGAAGATGATTTCTTGAGCGTTGT CGATGGCTTGGCCCGGCTACAACATACCAATTCTAATGAACTCGTAGGCTACTGT GCCGAGCATGGGCAACGACTTCTGGTCTACAAGTTCATCAGTCGAGGGACACTC CATGAACTGCTTCATGGCTCAGCCGATAGCCCCAAGGAGTTGTCATGGAATGTCC GTGTGAAGATTGCACTTGGTTGTGCGCGGGCTCTTGAGTATTTGCATGAAATCGT TTCGCAGCCGGTTGTGCACCGCAACTTTAGATCCTCAAACATTCTTTTGGATGAT GAGCTGAACCCACATGTGTCGGATTGTGGTTTGGCTGCTTTTACCCCATCCAGTG CTGAACGGCAGGTCTCTGCCCAAGTGTTGGGATCTTTTGGACACAGTCCCCCTGA ATTCAGCACATCTGGAATGTATGATGTGAAAAGCGACGTTTATAGCTTTGGTGTT GTGATGCTTGAGCTTATGACAGGACGCAAGCCTTTAGACAGCTCAAGACCAAGA TCCGAGCAAAACCTGGTGCGATGGGCAACACCACAACTGCATGATATTGATGCA CTCGCAAGAATGGTGGATCCAGCGTTAGAGGGTGCTTACCCTGCCAAGTCCCTCT CCCGGTTCGCCGACATCGTTGCCTTGTGTGTCCAGCCCGAACCCGAATTCCGACC TCCTATATCTGAAGTAGTGCAGTCCCTGGTAAGGCTTATGCAGCGTGCAGCTTTA AGTAAACGCCGGCATGAGTACAACGCAGGCGTTCCTCAGACTGATATGGAGGAC CCTAGTGATTACTTGTGACAGAAGTAAGTATCCTGGTCGATACTTCCCAATTTCA AGCATAGAGAACCTCCCGCGCGTCTACTCCCACTTGATTTTCAAAGCTGGCGAAA AGTGGCCAAATTTGTGGATTTGTGACACCTTGCAACTAAATCGGGGAGATATTCA GCTTCTTTGCAATTCCAGACCATGATGGCACAGACTTTGGCTTGCATCCTCCTCAT TATTACTGAAGCTTTTGCTTCTAATGGCGGATTACTGATTATGGATGACTATCCC GTTTCCAGGCAGACGTGAAGAGAAGTGTTGGCTTCCGAAGTTGTTAAATTGTATC GACGGCTGAAAGCTTTTTTAAGAGCTTACTTCTGGGTCCTAGTTAGTGATATTAA GGTCCCTGTGCCTTAAGAGTAATGTGCAATTCCTGTTGTGTTGCAAACTCGGGTA ACGCTTTGTCTTGTAGTTTTGGCACATTACAAGGTTAGTTCGACAGTGAACTCAC AATTTGAACAGATTAGTTAGGGAGTGTAACTCTAGCAAAAGTTGATTCCTTGTGG TTACCCAATTTTTTGAATGTGAACTCCCACTCATTGGTGTGATGGAGTACATGAT TCGCACGAGCTCGC (SEQ ID NO: 12) Deduced amino acid sequence of PK-11 from Physcomitrella patens MRGAVVDAPMKGDRRACILGGMGRPNGGTILYVLVISFIALVNGATDPNDVSALNT MFTGFNSDPKLTNWVQNAGDPCGTNWLGVTCDGTFVTSIKLSNMGLNGKVEGWVL QKFQHLSVLDLSHNNLASGIPEMFPPKLTELDLSYNQLTGSFPYLIINIPTLTSIKLNNN KLSGTLDGQVFSKLTNLITLDISNNAITGPIPEGMGDMVSLRFLNMQNNKLTGPIPDTL ANIPSLETLDVSNNALTGFLPPNLNPKNFRYGGNPLNTQAPPPPPFTPPPPSKNPKPIPP PPHPGSRTPDTAPKAEGGIVSGAAIAGIVVGAILVLAAIFIAVWFFVVRKRSELTKPLD LEANHSSRRTWFLPLIPAGKEKPPKMKVFEADTFEKEVEEPKIKALPPLKSLKVPPAL KVEEATYKVESEGKVNKSNITAREFSVAELQAATDSFSEDNLLGEGSLGCVYRAEFP DGEVLAVKKLDTTASMVRNEDDFLSVVDGLARLQHTNSNELVGYCAEHGQRLLVY
KFISRGTLHELLHGSADSPKELSWNVRYKIALGCARALEYFHEIVSQPVVHRNFRSSNI LLDDELNPHVSDCGLAAFTPSSAERQVSAQVLGSFGHSPPEFSTSGMYDVKSDVYSF GVVMLELMTGRKPLDSSRPRSEQNLVRWATPQLHDIDALARMVDPALEGAYPAKSL SRFADIVALCVQPEPEFRPPISEVVQSLNRLMQRAALSKRRHEYNAGVPQTDMEDPS DYL* (SEQ ID NO: 13) Nucleotide sequence of BnPK-1 from Brossica napus AACAAAAAAAAATCTAAGGTTTATCTTTTTCTTCTTCTATCTGATCATCAATCATC GAGAGAGAAAAAAGTATACTTTTTTAGATGTGAAGAAGCTCATCAATCGAAGAA GACAATCATCAAATGCTTCACTTTGGTTCCCTTTCTTCATCAGAAAACTCGAGGT AGATCAGTTCTTTGATGGGATGGGACACCAAATCGCTAAGTGTTATGATACCAGC AACTACTAGTTACGTGCTATCTCCAGACCAAATACCATGCCTTGAAACGGAGTA GGCAGTTCGAGATCTTCCAAAGGTGTGAAGGCCTCTTCTAGCTCAGTCGATTGGT TGACCAGAGATTTGGTTGAGATGAGGATAAGGGACAAGGTCGAGACTGATGATG AGAGGGATAGTGAACCAGATATTATTGATGGCGCTGGCACTGAACCTGGCCATG TGATTAGAACCACAGTCCGTGGACGCAATGGTCAATCAAGACAGACAGTCAGTT ACATATCAGAGCATGTAGTTGGTACTGGTTCCTTTGGCATGGTTTTTCAAGCCAA GTGTAGGGAAACTGGGGAGATTGTTGCAATCAAGAAGGTTCTACAAGACAAGCG TTACAAGAACAGGGAGCTACAAATTATGCAGATGCTAGACCACCCCAATGTCGT TGCTCTAAAGCATAGCTTCTACACGAGAGCTGATAACGAAGAGGTTTATTTGAAT CTTGTCCTTGAGTTTGTGCCTGAGACCGTCAATAGGGCTGCAAGAAGTTACACTA GGACGAACCAGCTAATGCCTTTAATATACGTTAAACTCTACACCTATCAGATTTG CAGGGCGCTTGCTTACATCCATAATTGCTTTGGTCTTTGTCACCGTGATATTAAGC CTCAAAACTTGCTAGTGAACCCACATACGCATCAGCTGAAAATCTGTGACTTCGG GAGTGCAAAAGTGTTGGTGAAAGGAGAACCCAATGTTTCTTACATCTGTTCTAGA TACTATCGTGCTCCAGAACTCATTTTTGGCGCCAGCGAATACACACCTGCAATTG ATATATGGTCAACTGGTTGTGTGATGGCTGAATTGCTTCTTGGACAGCCTCTGTT CCCTGGTGAAAGCGGAGTCGATCAGCTTGTTGAAATCATTAAGGTTTTAGGTACA CCAACGAGGGAGGAAATCAAGTGCATGAATCCAAACTATACAGAATTTAAATTC CCCCAGATAAAACCTCACCCATGGCACAAGGTCTTCCAAAAACGTTTACCGCCA GAAGCGGTTGATCTTCTATGTAGGTTCTTCCAATATTCCCCTAATCTGAGATGCA CAGCTTTGGAAGCGTGTATTCATCCGTTATTTGATGAGCTAAGGGACCCGAACAC TCGTCTTCCCAATGGCCGGCCACTTCCTCCGCTTTTCAACTTCAAACCTCAAGAG CTATCTGGCATCCCTTCTGAAATCGTGAACAGGCTTGTACCAGAACATGCCCGTA AGCAGAACTTCTTCATGGCGTTGGATGCCTAAGCGCTTATCCTGTTTCTTTTCTTT TTCTTGCTTATGTATAAACTCTCTAGATATCGGGTATTTGGAGCAGCCAGAAGGC ATTACACGCCCTCTTTGGCTTTTTTTTATCAGTGAGTTGTTTGGTTATCGGGACAC GATGATGCATGAATACAAACAGTACTTGAGGTCGCTGCTGGCTTATAAGACCAC TTGTTTGTTTCACAACCAGTTCTTATATATATTATTATACAAAAAAAAAAAAAAA AAA (SEQ ID NO: 14) Deduced amino acid sequence of BnPK-1 from Brassica napus MASNGVGSSRSSKGVKASSSSVDWLTRDLVEMRIRDKVETDDERDSEPDIIDGAGTE PGHVIRTTVRGRNGQSRQTVSYISEHVVGTGSFGMVFQAKCRETGEIVAIKKVLQDK RYKNRELQIMQMLDHPNVVALKHSFYTRADNEEVYLNLVLEFVPETVNRAARSYTR TNQLMPLIYVKLYTYQICRALAYIHNCFGLCHRDIKPQNLLVNPHTHQLKICDFGSAK VLVKGEPNVSYICSRYYRAPELIFGASEYTPAIDIWSTGCVMAELLLCQPLFPGESGV DQLVEIIKVLGTPTREEIKCMNPNYTEFKFPQIKPHPWHKVFQKRLPPEAVDLLCRFFQ YSPNLRCTALEACIHPLFDELRDPNTRLPNGRPLPPLFNFKPQELSGIPSEIVNRLVPEH ARKQNFFMALDA* (SEQ ID NO: 15) Nucleotide sequence of BnPK-2 from Brassica napus TTTTCTCTCTCTCTCTCTCTCTCCACATTTGATGATCATTACCAACCAAACTAATT GAAATCCATTTGTTCTCTCTCTCTCTCTCTCTCTCTCACACTCTCTTCTCTGCTCTT CTCTGCGCCTCTAACGTCATGGCTGACGATAGGGAGATGCCGCCGGCTGCTGTAG TTGATGGACATGACCAAGTCACTGGCCACATAATCTCCACCACCATCGGTGGTAA AAACGGAGAACCAAAACAGACAATAAGTTACATGCCGGAGCGAGTTGTCGGTAC AGGCTCCTTCGGGATAGTGTTCCAGGCGAAGTGTCTGGAGACTGGAGAAACCGT GGCGATAAAGAAGGTTTTGCAAGACAGGAGGTACAAGAACCGAGAGCTTCAGCT GATGCGTGTGATGGACCATCCGAATGTTGTTTGTTTGAAGCATTGCTTCTTCTCG ACCACGAGCAAAGACGAGCTGTTTCTGAACTTGGTTATGGAGTATGTCCCTGAG AGCTTGTACCGAGTTCTGAAACATTACAGCACTGCTAACCAGAGGATGCCGCTTG TTTATGTTAAACTCTATATGTACCAGATCTTCAGAGGACTTGCTTACATTCACAAT GTTGCTGGAGTTTGTCACAGAGATCTAAAGCCTCAAAATCTTCTGGTTGATCCTC TGACTCATCAAGTGAAGATCTGTGATTTTGGCAGTGCGAAACAGCTTGTTAAAGG TGAAGCCAACATCTCTTACATATGTTCAAGATTCTACCGTGCACCTGAACTTATA TTCGGTGCCACTGAGTACACAACTTCCATTGATATTTGGTCTGCTGGTTGTGTTCT CGCTGAGCTTCTTCTTGGTCAGCCACTATTCCCTGGAGAAAATGCTGTGGGTCAG CTCGTTGAAATCATCAAAGTTCTTGGTACACCAACTCGAGAAGAGATCCGTTGTA TGAATCCACACTACACAGACTTTAGGTTCCCGCAGATAAAGGCACATCCTTGGCA CAAGATTTTCCACAAAAGGATGCCTCCAGAAGCCATTGATTTTGCATCAAGGCTG CTTCAGTACTCTCCAAGTCTTAGATGCACAGCGCTTGAAGCTTGTGCACATCCGT TCTTTGATGAGCTTAGAGAACCAAATGCTCGTTTACCAAACGGACGGCCTTTCCC GCCGCTCTTCAACTTCAAACAAGAGGTAGCTGGAGCTTCACCTGAGCTGGTCAAC AAGTTGATTCCAGACCATATCAAGACGCAGTTGGGTCTAAGCTTCTTGAATCAGT CTGGAACTTAAACAAACGATCAAAAAGACAAGAACTTTTTTATATATAATTGTAC CATTACTCAGAACCAGAAGAAGGTTAGTTGAAGGCACGTGGAGGACACAGTTAG AGGTTTTGCCTCCTCAAAACTCGTTCCAGGAATGAAGGTCAAAAAAGACAAGCT TCTCTACAACCTGACTTCCCCCAAGCCTGCAAGAAAAGCTACTCAGTTGTATCTT CTTCTTCTTCTTTTGTCCTTTTTTAAAAATGTTTGGTTAAAGCAAAGAACAAAATC TTCTCTTTTTGCTTTATTCTTACTGCATCTGTAAATGAGTTTAGTCAGAGATTTTTA TATAGTAAAAAAAAAAAAAAAAAA (SEQ ID NO: 16) Deduced amino acid sequence of BnPK-2 from Brassica napus MADDREMPPAAVVDGHDQVTGHIISTTIGGKNGEPKQTISYMAERVVGTGSFGIVFQ AKCLETGETVAIKKVLQDRRYKNRELQLMRVMDHPNVVCLKHCFFSTTSKDELFLN LVMEYVPESLYRVLKHYSTANQRMPLVYVKLYMYQIFRGLAYIHNVAGVCHRDLK PQNLLVDPLTHQVKICDFGSAKQLVKGEANISYICSRFYRAPELIFGATEYTTSIDIWS AGCVLAELLLGQPLFPGENAVGQLVEIIKVLGTPTREEIRCMNPHYTDFRFPQIKAHP WHKIFHKRMPPEAIDFASRLLQYSPSLRCTALEACAHPFFDELREPNARLPNGRPFPPL FNFKQEVAGASPELVNKLIPDHIKTQLGLSFLNQSGT* (SEQ ID NO: 17) Nucleotide sequence of BnPK-3 from Brassica napus CGTCGTCGTCTCTCTCTCTTTCTTTCTCTTCTCCGTGAATCATCATCATCATCATCA TCTTCGTGTTTTCTCGTTAAGCCCATTTTGTTTTTTTTTTTTCTCTGGGGAAAAACT CGGCTCAAAACGATGAATGTGATGCGTAGATTGACGAGTATCGCTTCTGGACGC GGTTTCGTCTCTTCTGATAACGTAGGAGAGACCGAGACGCCGAGATCGAAGCCT AACCAAATTTGTGAAGAGATAGAAGAGACTACACGAGAAGACTCTGTTTCTAAA ACAGAGGATTCTGATTCATTACCAAAAGAGATGGGAATCGGTGATGACGACAAG GATAAGGACGGTGGGATTATCAAGGGTAATGGGACAGAGTCTGGTCGGATCATT ACCACCACAAAGAAGGGTCTGAACGATCAAAGAGACAAGACAATCTCGTACAG AGCTGAACATGTGATTGGCACTGGCTCATTCGGTGTTGTCTTTCAGGCTAAGTGC TTAGAGACAGAAGAAAAAGTAGCTATCAAGAAAGTGTTGCAAGACAAGAGATA CAAGAACAGAGAGCTTCAGATCATGCGGATGCTTGATCATCCTAATGTTGTTGAC CTCAAGCATTCTTTCTTCTCCACCACTGAGAAAGATGAGCTTTATCTTAACCTTGT TCTTGAGTATGTACCTGAGACTATATACCGTTCTTCAAGATCTTACACCAAGATG AATCAACACATGCCCTTGATCTATATTGAGCTCTATACATATCAGATTTGCCGCG CAATGAACTATCTACATAGAGTTGTTGGAGTGTGTCACCGTGACATTAAACCTCA GAATCTATTGGTCAATAATGTTACACATGAGGTGAAGGTATGCGATTTTGGGAGC GCCAAGATGCTGATTCCGGGAGAACCCAATATATCTTACATATGCTCAAGGTATT ACAGAGCTCCTGAACTCATATTTGGGGTAACTGAGTACACAACCGCCATCGATAT GTGGTCTGTTGGCTGTGTCATGGCTGAACTTTTTCTTGGACATCCTCTGTTCCCTG GAGAGACTAGTGTTGATCAATTGGTTGAGATCATTAAGATTTTGGGAACACCAGC AAGAGAAGAGATCAGAAACATGAATCCTCGTTACAATGATTTTAAGTTCCCTCA GATCAAAGCTCAGCCATGGCACAAGATTTTCCGGAGACAGGTATCTCCAGAAGC AATGGATCTTGCCTCTAGACTCCTCCAGTACTCACCAAACCTGAGATGTTCAGCG CTTGAAGCATGTGCACACCCCTTCTTCGATGATCTGAGAGACCCGAGAGCATCCT TGCCTAATGGAAGAGCACTTCCTCCACTGTTTGATTTCACACCTCAAGAACTGGC TGGTGCATCTGTTGAATTGCGTCATCGCTTAATCCCTGAACATGCAAGGAAATAA CTTACTTTGTCTAACGAGACCGCTTCTTCTCTACACAGATGTTGATATCTAAATTC CTTTTTTTTTGGCATTGTTCTGGTTATGAACACCCTCATTGACCTCTGCAACCACC TTGCACTAGCAGTTCCAAAAGTGTATGATTTGTTAAGTTTGTAACTTTGTAGACTC CATTGTTGCAGACAGAAAATGCAGAATTTTCCGAGTTTGTCTCAAAAAAAAAAA AAAAAAA (SEQ ID NO: 18) Deduced amino acid sequence of BnPK-3 from Brassica napus MNVMRRLTSIASGRGFVSSDNVGETETPRSKPNQICEEIEETTREDSVSKTEDSDSLPK EMGIGDDDKDKDGGIIKGNGTESGRIITTTKKGLNDQRDKTISYRAEHVIGTGSFGVV FQAKCLETEEKVAIKKVLQDKRYKNRELQIMRMLDHPNVVDLKHSFFSTTEKDELYL NLVLEYVPETIYRSSRSYTKMNQHMPLIYIQLYTYQICRAMNYLHRVVGVCHRDIKP QNLLVNNVTHEVKVCDFGSAKMLIPGEPNISYICSRYYRAPELIFGVTEYTTAIDMWS
VGCVMAELFLGHPLFPGETSVDQLVEIIKILGTPAREEIRNMNPRYNDFKFPQIKAQP WHKIFRRQVSPEAMDLASRLLQYSPNLRCSALEACAHPFFDDLRDPRASLPNGRALP PLFDFTAQELAGASVELRHRLIPEHARK* (SEQ ID NO: 19) Nucleotide sequence of BnPK-4 from Brassica napus GTTTTGGCATCTGGAGAGGGAGAGAGAGAGAGAGAAAGGGGAATAAGATGATG GAGAATCGAGTGGTGGTGGTGGCTGCTCTGTTGCGGTCTGCATTGTAGGATTTG AGTTTAGCTTCATCCATGGAGCCACTGATGCATCAGACACTTCAGCATTGAACAT GTTGTTCACCAGTATGCATTCACCAGGACAGTTAACACAATGGACTGCATCAGGT GGGGATCCTTGTGTTCAGAACTGGAGAGGCGTTACTTGCTCCAAATCACGAATTA CTCAATTAAAGTTATCAGGTCTTGAGCTCTCTGGAACACTTGGGTACATGCTTGA TAAATTGACTTCTCTTACAGAGCTTGATCTAAGCAGCAATAATCTTGGAGGTGAT TTACCATATCAGCTTCCTCCAAATCTGCAACGGTTGAATCTTGCAAACAACCAAT TCACTGGAGCTGCTCAATACTCCATTTCTAATATGGCATCACTTAAGTATCTTAAT CTTGGTCACAACCAGTTTAAGGGGCAAGTAGCTGTGGACTTCTCCAAGCTCACCT CTCTTACAACCTTGGACTTCTCTTTCAACTCTTTCACATCGTCTCTACCGGGAACT TTTACTTCTCTTACAAGTTTAAAGTCCCTATACCTTCAGAACAATCAGTTCTCAGG AACACTCAATGTATTAGCCGGTCTTCCTCTTGAGACCCTGAACATTGCAAACAAT GACTTCACCGGCTGGATCCCCAGTACCTTAAAGGGTACTAATTTAATAAAAGATG GTAACTCGTTCAATAATGGACCTGCACCACCACCACCACCTGGTACACCTCCAAT CCACCGCTCACCGAGCCATAAATCCGGAGGAGGTTCAAACCGTGATTCTACCAG CAATGGAGATTCCAAGAAATCAGGAATTGGAGCTGGTGCTATAGCAGGTATAAT CATTTCATTACTAGTAGTTACAGCTCTTGTGGCTTTCTTCTTAGTCAAAAGAAGA AGAAGATCAAAGAGATCATCATCTATGGACATTGAGAAAACTGACAACCAGCCT TTCACTCTTCCTCCAAGCGACTTTCACGAAAACAATTCTATTCAGAGTTCTTCATC AATTGAGACAAAGAAACTTGATACTTCCTTGTCTATTAATCTCCGTCCTCCACCA GCTGATCGATCATTTGATGATGATGAGGATTCTACGAGAAAGCCTATAGTTGTCA AGAAATCCACCGTGGCTGTTCCCTCGAATGTGAGAGTTTACTCAGTTGCTGATCT TCAGATTGCCACTGCCAGTTTCAGTGTTGATAATCTTCTTGGAGAAGGCACTTTT GGAAGAGTATACAGAGCTGAGTTTAACAATGGAAAGGTTCTTGCTGTGAAGAAG ATTGATTCATCTGCTCTTCCACATAGCATGACTGATGATTTCACCGAAATAGTAT CGAAAATAGCCGTTTTGGATCATCCAAATGTGACAAAGCTTGTTGGCTACTGTGC TGAACACGGACAACATCTCCTGGTCTATGAGTTCCACAGCAAAGGATCGTTACAT GACTTCCTACACTTATCAGAAGAAGAAAGCAAAGCATTGGTGTGGAACTCGCGA GTCAAGGTCGCACTTGGGACTGCACGGGCAATAGAGTACTTGCATGAAGTTTGTT CACCGTCTATAGTTGACAAGAACATCAAATCAGCCAATATTTTGCTTGATTCGGA GATGAATCCTCACTTATCAGACACAGGTCTCGCAAGCTTCCTCCCCACAGCAAAT GAGTTACTAAACGAAACCGATGAAGGTTATAGCGCACCGGAAGTATCAATGTCA GGTCAATACTCTTTGAAGAGTGATGTTTACAGTTTTGGAGTAGTGATGCTTGAAC TTTTAACCGGGAGGAAACCATTCGACAGCACAAGGTCAAGATCTGAGCAGTCAT TGGTTAGATGGGCGACACCACAGCTTCATGACATTGATGCTTTAGGCAAAATGGT TGATCCAGCTCTTGAAGGACTTTATCCGGTTAAATCTCTTTCTCGGTTTGCAGATG TTATTGCTCTCTGCGTCCAGCCAGAGCCAGAGTTTAGACCACCAATGTCTGAAGT TGTGCAGTCACTGGTTGTGTTAGTGCAGAGAGCTAACATGAGCAAGAGAACTGT TGGAGTTGATCCATCACAGCGTTCTGGTAGTGCTGAGCCAAGCAACGATTACATG TAAACCCATTACCACAGAGAGAGAAAAAAAGAACACTTTGCTCCCTATGGGATG AAGTCATTGTTTTTATTGTAATATGTTTGATAAACCTTCACACAGTATATTATCCC CATTGTATTTTGTTGTAATGTGTTTCCAAATTTGTAGCTTTTAGATCATTGAAATG AACAAATATTCTTTCTTGTGTAAAAAAAAAAAAAAAAAA (SEQ ID NO: 20) Deduced amino acid sequence of BnPK-4 from Brassica napus MMENRVVVVAALFAVCIVGFEFSFIHGATDASDTSALNMLFTSMHSPGQLTQWTAS GGDPCVQNWRGVTCSKSRITQLKLSGLELSGTLGYMLDKLTSLTELDLSSNNLGGDL PYQLPPNLQRLNLANNQFTGAAQYSISNMASLKYLNLGHNQFKGQVAVDFSKLTSLT TLDFSFNSFTSSLPGTFTSLTSLKSLYLQNNQFSGTLNVLAGLPLETLNIANNDFTGWIP STLKGTNLIKDGNSFNNGPAPPPPPGTPPIHRSPSHKSGGGSNRDSTSNGDSKKSGIGA GAIAGIIISLLVVTALVAFFLVKRRRRSKRSSSMDIEKTDNQPFTLPPSDFHENNSIQSSS SIETKKLDTSLSINLRPPPADRSFDDDEDSTRKPIVVKKSTVAVPSNVRVYSVADLQIA TASFSVDNLLGEGTFGRVYRAEFNNGKVLAVKKIDSSALPHSMTDDFTEIVSKIAVLD HPNVTKLVGYCAEHGQHLLVYEFHSKGSLHDFLHLSEEESKALVWNSRVKVALGTA RAIEYLHEVCSPSIVDKNIKSANILLDSEMNPHLSDTGLASFLPTANELLNQTDEGYSA PEVSMSGQYSLKSDVYSFGVVMLELLTGRKPFDSTRSRSEQSLVRWATPQLHDIDAL GKMVDPALEGLYPVKSLSRFADVIALCVQPEPEFRPPMSEVVQSLVVLVQRANMSK RTVGVDPSQRSGSAEPSNDYM* (SEQ ID NO: 21) Nucleotide sequence of GmPK-1 from Glycine max TTTAGAGAGAGAAAGAGTGTGAGTGTTGTGTTGAGTGCAGTTTCTTTCTCACATG GCCTCTATGCCGTTGGGGCCGCAGCAACAGCTTCCACCGCCGCCGCCGCAACAA CCGCCGCCAGCGGAGAATGACGCGATGAAAGTGGACTCTCGCGGCGGCTCCGAC GCCGGCACCGAAAAGGAAATGTCAGCTCCTGTCGCAGATGGTAATGATGCACTC ACTGGTCACATAATCTCAACCACAATTGCAGGCAAAAATGGCGAACCTAAACAA ACCATCAGTTACATGGCCGAACGTGTTGTTGGCACTGGATCATTTGGCATTGTTT TCCAGGCGAAGTGCTTGGAGACTGGCGAGGCAGTGGCTATAAAGAAGGTCTTGC AGGACAGGCGATACAAAAATCGTGAACTGCAGTTAATGCGCGTGATGGATCACC CAAATATAATTTCCTTGAGTAACTATTTCTTCTCTACAACAAGTAGAGATGAACT TTTTCTGAACTTGGTGATGGAATATGTCCCTGAGACGATCTTCCGTGTTATAAAG CACTACAGTAGCATGAAACAGAGAATGCCCCTAATCTATGTGAAATTATATACA TATCAAATCTTTAGGGGACTGGCGTATATCCATACTGTACCAGGAATCTGCCATA GGGATTTGAAGCCTCAAAATCTTTTGGTTGATCGACTCACACACCAAGTCAAGCT CTGTGATTTTGGGAGTGCAAAAGTTCTGGTGGAGGGTGAATCAAACATTTCATAC ATATGTTCACGGTACTATCGTGCCCCAGAGCTAATATTTGGTGCGGCAGAATACA CAACTTCTGTTGATATTTGGTCCGCTGGTTGTGTCCTTGCGGAACTTCTTCTAGGC CAGCCTTTGTTCCCAGGAGAAAATCAGGTTGACCAACTCGTGGAAATTATCAAG ATTCTTGGCACTCCTACTCGAGAAGAAATTCGATGCATGAATCCTAATTATACAG ATTTCAGATTCCCCCATATCAAAGCTCATCCTTGGCATAAGGTTTTTCACAAGCG AATGCCTCCTGAAGCAATTGACCTTGCATCAAGGCTTCTCCAATATTCCCCAAAA CTTCGTTACAGTGCAGTGGAAGCAATGGCACATCCTTTCTTTGACGAGCTTCGCG AGCCCAATGCCCGGCTACCTAATGGTCGTCCACTGCCTCCACTTTTCAACTTTAA ACAGGAATTAGATGGAGCGCCCCCTGAACTGCTTCCTAAGCTCATCCCAGAGCA TGTCAGCCGGCAAACCCAAATGTAAAGAGATAGTAAAACATAGAGTGAACTGTT CTAGTGGATTAGTGTGAAATACATGAGAGCTTGCTTGTGGTCAATAGAACAGGG GTTAGGCCCAAATATGCAGTTTTTCTCCCCCTTGTGAAGATGTATACATGTGCTG GAAAACTCAGTGTAACCCGGAAATGTAGATTATGTCTAATGTCTAATATTTCATT CTAGTTAAAAAAAAAAAAAAAAAA (SEQ ID NO: 22) Deduced amino acid sequence of GmPK-1 from Glycine max MASMPLGPQQQLPPPPPQQPPPAENDAMKVDSRGGSDAGTEKEMSAPVADGNDAL TGHIISTTIAGKNGEPKQTISYMAERVVGTGSFGIVFQAKCLETGEAVAIKKVLQDRR YKNRELQLMRVMDHPNIISLSNYFFSTTSRDELFLNLVMEYVPETIFRVIKHYSSMKQ RMPLIYVKLYTYQIFRGLAYIHTVPGICHRDLKPQNLLVDRLTHQVKLCDFGSAKVL VEGESNISYICSRYYRAPELIFGAAEYTTSVDIWSAGCVLAELLLGQPLFPGENQVDQL VEIIKILGTPTREEIRCMNPNYTDFRFPHIKAHPWHKVFHKRMPPEAIDLASRLLQYSP KLRYSAVEAMAHPFFDELRIPNARLPNGRPLPPLFNFKQELDGAPPELLPKLIPEHVR RQTQM* (SEQ ID NO: 23) Nucleotide sequence of GmPK-2 from Glycine max AGACACCACAAAGTGTAACTTGAGTGATTATATCTGATGAGTGCAGAAAGAAGG GAGGATTGTTGGTGATCGATCATCGATCATCGATCATCGATCATCGATGGCGTCT GCTAGCCTTGGAAGTGGTGGGGTGGGCAGTTCCAGGTCTGTTAATGGTGGCTTCA GGGGTTCTTCCAGTTCCGTCGATTGGCTTGGCAGAGAGATGCTTGAGATGTCTTT GAGAGACCACGAGGACGATAGAGATAGTGAGCCTGACATCATTGATGGTTTGGG TGCTGAGACTGGTCACGTGATAAGAACCAGCGTTGGTGGCCGAAATGGTCAATC TAAGCAGAATGTTAGTTATATTTCTGAGCATGTTGTGGGAACAGGCTCTTTTGGT GTTGTTTTTCAAGCCAAATGTAGAGAAACGGGAGAAATTGTGGCCATCAAGAAA GTTCTCCAGGACAAGCGCTACAAGAATAGAGAGTTACAAATTATGCAAATGCTG GATCATCCAAATATTGTTGCCCTTAGGCATTGTTTCTATTCAACGACTGACAAAG AAGAAGTTTACTTGAATCTTGTACTTGAATATGTTCCTGAAACTGTGAATCGCAT CGCCAGGAGCTATAGCAGGATTAACCAGCGAATGCCTTTAATATATGTAAAGCT TTATACCTACCAGATTTGCAGGGCCCTTGCTTATATACATAACTGCATTGGTATA TGTCATCGTGACATCAAACCTCAGAACCTACTTGTGAACCCGCACACTCATCAGC TGAAACTATGTGATTTTGGGAGTGCAAAAGTGTTGGTGAAAGGAGAACCTAATG TTTCTTACATCTGTTCAAGATACTACCGTGCTCCGGAACTTATATTTGGGGCCACT GAATATACAACTGCCATAGATATATGGTCAACTGGTTGTGTAATGGCTGAATTAC TTCTTGGACAGCCCTTGTTTCCTGGAGAGAGTGGAGTTGATCAGCTAGTTGAAAT CATCAAGGTTTTGGGAACTCCAACCAGGGAGGAGATAAAGTGCATGAACCCAAA TTATACTGAATTTAAGTTTCCACAGATAAAACCTCATCCATGGCACAAGGTTTT CAGAAACGTTTACCCCCAGAAGCAGTGGACCTTGTCTGTAGGTTCTTTCAGTACT CTCCCAATTTGAGATGCACTGCATTGGAAGCTTGCATTCATCCATTTTTTGATGA ATTGAGGGACCCAAACACCCGCCTTCCTAATGGTCGACCACTTCCTCCACTGTTT AATTTTAAACCTCAGGAACTTTCTGGTGTACCCCCTGATGTCATCAATCGGCTTA TTCCAGAGCATGCGCGTAAACAGAACTTATTTATGGCTTTGCACACCTAGCAATT
CCCGTACCCTCCTAAGTTGTCGTCACTTACTAGCAGGTTGTAAATTATCCGGTTTA TCCGAGAAAAACTCCACAGAAAGAGTTACTAGGATTATATTATTATTATATAATA TGAAAAGTTTCTTTTTTCTTTTTTGGAAAAAAAAAAAAAAAAAA (SEQ ID NO: 24) Deduced amino acid sequence of GmPK-2 from Glycine max MASASLGSCGVGSSRSVNGGFRGSSSSVDWLGREMLEMSLRDHEDDRDSEPDIIDGL GAETGHVIRTSVGGRNGQSKQNVSYISEHVVGTGSFGVVFQAKCRETGEIVAIKKVL QDKRYKNRELQIMQMLDHPNIVALRHCFYSTTDKEEVYLNLVLEYVPETVNRIARSY SRINQRMPLIYVKLYTYQICRALAYIHNCIGICHRDIKPQNLLVNPHTHQLKLCDFGSA KVLVKGEPNVSYICSRYYRAPELIFGATEYTTAIDIWSTGCVMAELLLGQPLFPGESG VDQLVEIIKVLGTPTREEIKCMNPNYTEFKFPQIKPHPWHKVFQKRLPPEAVDLVCRF FQYSPNLRCTALEACIHPFFDELRDPNTRLPNGRPLPPLFNFKPQELSGVPPDVINRLIP EHARKQNLFMALHT* (SEQ ID NO: 25) Nucleotide sequence of GmPK-3 from Glycine max AGAGAGAGAAACGAAGAAGAAGAGTGTTTCTCACATCACATGGCCTCCTTGCCC TTGGGGCACCACCACCACCACCACAAACCGGCGGCGGCGGCTATACATCCGTCG CAACCGCCGCAGTCTCAGCCGCAACCCGAAGTTCCTCGCCGGAGCTCCGATGTG GAGACCGATAAGGATATGTCAGCTACTGTCATTGAGGGGAATGATGCTGTCACT GGCCACATAATCTCCACCACAATTGGAGGCAAAAATGGGGAACCTAAAGAGACC ATCAGTTACATGGCAGAACGTGTTGTTGGCACTGGATCATTTGGAGTTGTTTTTC AGGCAAAGTGCTTGGAGACTGGAGAAGCAGTGGCTATTAAAAAGGTCTTGCAAG ACAGGCGGTACAAAAATCGTGAATTGCAGTTAATGCGCTTAATGGATCACCCTA ATGTAATTTCCCTGAAGCACTGTTTCTTCTCCACAACAAGCAGAGATGAACTTTT TCTAAACTTGGTAATGGAATATGTTCCCGAATCAATGTACCGAGTTATAAAGCAC TACACTACTATGAACCAGAGAATGCCTCTCATCTATGTGAAACTGTATACATATC AAATCTTTAGGGGATTAGCATATATCCATACCGCACTGGGAGTTTGCCATAGGGA TGTGAAGCCTCAAAATCTTTTGGTTCATCCTCTTACTCACCAAGTTAAGCTATGTG ATTTTGGGAGTGCCAAAGTTCTGGTCAAGGGTGAATCAAACATTTCATACATATG TTCACGTTACTATCGGGCTCCAGAACTAATATTTGGTGCAACAGAATACACAGCT TCTATTGATATCTGGTCAGCTGGTTGTGTTCTTGCTGAACTTCTTCTAGGACAGCC ATTATTTCCTGGAGAAAACCAAGTGGACCAACTTGTGGAAATTATCAAGGTTCTT GGTACTCCAACACGCGAGGAAATCCGTTGTATGAACCCAAATTATACAGAGTTT AGATTCCCTCAGATTAAAGCTCATCCTTGGCACAAGGTTTTCCACAAGCGAATGC CTCCTGAAGCAATTGACCTTGCATCAAGGCTTCTCCAATATTCACCTAGTCTCCG CTGCACTGCGCTGGAAGCATGTGCACATCCTTTCTTTGATGAGCTTCGCGAACCA AATGCCCGGCTACCTAATGGCCGTCCACTGCCCCCACTTTTCAACTTCAAACAGG AGTTAGCTGGAGCATCACCTGAACTGATCAATAGGCTCATCCCAGAGCATATTA GGCGGCAGATGGGTCTCAGCTTCCCGCATTCTGCCGGTACATAGATGTAAAGGG ATAATGAAACGATGAGTCAACCTACATAGTGATCGATGTGAATCAACAGAAGGG CTGTTTGAGGCCTATGTATAACTGGGAGTCCCAACATAATATGCAGTTTTTCCTC CCCCTTGTGAAGATGTATACATGTGTTGGTTCCTCGGTAAAGCTTGAAAGTTGGT GATTCTGTGTAGTATTTCATTCAAGTTAAAGCATACTTATCCCTGCATCTGTATAT TGTTTTGGTCAGATTTCAGAAAGCTAGGAGTATAAAATGATAGCAATCATGTCTT CATAGGTAGAGGGGCCCAGCTGAATTGAGGGGCCCCTATAGTAGTTTGGCTTGC TTTTTATGAGATTAAATTCAGGATGTCGTTTATATTATGTTTATAACAATCTCTTG ATTCAAAACAAGAAATTTTCTCGTTGTTGAAAAAAAAAAAAAAAAAA (SEQ ID NO: 26) Deduced amino acid sequence of GmPK-3 from Glycine max MASLPLGHHHHHHKPAAAAIHPSQPPQSQPQPEVPRRSSDVETDKDMSATVIEGNDA VTGHIISTTIGGKNGEPKETISYMAERVVGTGSFGVVFQAKCLETGEAVAIKKVLQDR RYKNRELQLMRLMDHPNVISLKHCFFSTTSRDELFLNLVMEYVPESMYRVIKHYTT MNQRMPLIYVKLYTYQIFRGLAYIHTALGVCHRDVKPQNLLVHPLTHQVKLCDFGS AKVLVKGESNISYICSRYYRAPELIFGATEYTASIDIWSAGCVLAELLLGQPLFPGENQ VDQLVEIIKVLGTPTREEIRCMNPNYTEFRFPQIKAHPWHKVFHKRMPPEAIDLASRL LQYSPSLRCTALEACAHPFFDELREPNARLPNGRPLPPLFNFKQELAGASPELINRLIPE HIRRQMGLSFPHSAGT* (SEQ ID NO: 27) Nucleotide sequence of GmPK-4 from Glycine max GAGTTTCAAAGGTTGTTGGTGTGCATCACCACCTGCATTCTATGTTGGATGCCCA ATGGTGCCACTGCCGCCACAGATCCAAATGATGCTGCTGCTGTGAGATTTTTGTT TCAAAATATGAACTCACCACCCCAGCTAGGTTGGCCTCCTAATGGTGATGATCCA TGTGGACAATCTTGGAAAGGCATTACTTGCTCTGGCAATCGTGTTACAGAGATTA AGTTATCTAATCTTGGACTAACTGGATCGTTGCCTTATGGATTACAAGTCTTGAC ATCTTTGACCTACGTAGACATGAGTAGCAACAGTCTTGGTGGCAGCATACCGTAC CAACTTCCTCCATATTTGCAGCACTTAAATCTTGCTTATAACAACATCACAGGGA CAGTACCTTATTCGATTTCTAACTTGACTGCTCTTACTGACCTGAATTTTAGTCAC AATCAGCTCCAGCAAGGACTGGGTGTTGACTTTCTTAATCTTTCTACTCTCTCCAC ATTGGATCTCTCTTTCAATTTTCTAACAGGTGACCTCCCTCAGACTATGAGCTCAC TTTCACGCATAACCACCATGTATCTGCAAAATAACCAGTTTACAGGCACTATTGA TGTCCTTGCTAATCTGCCTCTGGATAATCTGAATGTGGAAAATAATAATTTTACT GGATGGATACCAGAACAGTTGAAGAACATAAACCTACAGACCGGTGGTAATGCA TGGAGCTCAGGGCCTGCACCCCCACCTCCTCCTGGGACACCTCCAGCACCTAAA AGCAACCAGCACCACAAGTCTGGTGGTGGAAGCACCACCCCCTCAGATACTGCC ACTGGCAGCAGCTCAATTGACGAGGGAAAAAAATCTGGTACAGGAGGTGGTGCC ATAGCCGGAATTGTGATCTCTGTCATAGTTGTGGGGGCAATAGTAGCATTCTTTC TGGTGAAGAGAAAATCCAAGAAGTCATCTTCTGATTTAGAAAAGCAGGATAATC AGTCCTTTGCTCCACTTCTTTCAAATGAAGTGCATGAAGAAAAGTCCATGCAAAC TTCCTCTGTAACAGACTTGAAGACGTTTGATACTTCTGCCTCAATAAATCTTAAA CCCCCACCTATTGACCGTCATAAATCATTTGATGATGAAGAATTCTCCAAGAGGC CCACAATTGTGAAGAAGACTGTAACAGCTCCTGCAAATGTGAAATCATATTCTAT TGCTGAACTGCAGATTGGTACTGGCAGCTTCAGTGTGGATCACCTTGTTGGCGAG GGATCTTTTGGGCGTGTTTACCGTGCTCAATTTGATGATGGACAGGTTCTTGCAG TGAAGAAGATAGATTCATCTATCCTTCCCAATGATTTGACAGATGATTTTATACA AATAATTTCAAACATCTCCAATTTACATCATCCAAATGTGACAGAGCTTGTAGGT TATTGCTCAGAGTATGGACAACACCTCTTGGTCTATGAGTTTCATAAAAATGGAT CACTGCATGACTTCCTTCACCTATCAGATGAATATAGTAAACCATTGATATGGAA TTCCCGTGTCAAGATTGCTTTGGGGACTGCACGTGCTCTAGAGTACCTACATGAA GTTAGTTCGCCATCAGTTGTTCATAAGAATATTAAGTCAGCCAACATATTACTTG ATACAGAACTTAATCCTCATCTTTCAGATAGTGGATTGGCAAGTTATATTCCAAA TGCCGACCAGATATTGAATCATAATGTTGGATCTGGATATGATGCACCTGAAGTT GCCTTGTCTGGTCAGTATACTTTGAAAAGTGATGTCTACAGCTTTGGAGTCGTCA TGTTGGAACTTCTCAGTGGACGGAACCCTTTTGATAGCTCAAGGCCAAGATCTGA GCAGTCTTTGGTTCGATGGGCAACACCTCAACTCCATGATATTGATGCATTGGCT AAAATGGTTGATCCTGCAATGAAAGGGTTATATCCTGTTAAGTCTCTTTCTCGAT TTGCCGATGTTATTGCTCTTTGCGTTCAGCCGGAGCCAGAATTCCGACCACCGAT GTCAGAAGTGGTTCAAGCACTGGTGCGACTAGTGCAGCGAGCTAACATGAGCAA GCGAACATTTAGTAGTAGTGATCATGGAGGATCCCAACGAGGGAGTGATGAGCC AGTTCTACGAGACATCTAAATCCCAAAGCAAATGTAGTTATATTTTTCTCCCAAG CTAGTTCGGTTATTTGTAATATAATTTCCAATAGTTGCAAATTTGAATTGATGGGT TCCATATTCTGTTGATACCTATGTAAACCTGTCCAAATCAGCTTATTACAATGAC AGTAACGGTTCACTGGCAAAAAAAAAAAAAAAAA (SEQ ID NO: 28) Deduced amino acid sequence of GmPK-4 from Glycine max MPNGATAATDPNDAAAVRFLFQNMNSPPQLGWPPNGDDPCGQSWKGITCSGNRVT EIKLSNLGLTGSLPYGLQVLTSLTYVDMSSNSLGGSIPYQLPPYLQHLNLAYNNITGTV PYSISNLTALTDLNFSHNQLQQGLGVDFLNLSTLSTLDLSFNFLTGDLPQTMSSLSRITT MYLQNNQETGTIDVLANLPLDNLNVENNNFTGWIPEQLKNINLQTGGNAWSSGPAPP PPPGTPPAPKSNQHHKSGGGSTTPSDTATGSSSIDEGKKSGTGGGAIAGIVISVIVVGAI VAFFLVKRKSKKSSSDLEKQDNQSFAPLLSNEVHEEKSMQTSSVTDLKTFDTSASINL KPPPIDRHKSFDDEEFSKRPTIVKKTVTAPANVKSYSIAELQIATGSFSVDHLVGEGSF GRVYRAQFDDGQVLAVKKIDSSILPNDLTDDFIQIISNISNLHHPNVTELVGYCSEYGQ HLLVYEFHKNGSLHDFLHLSDEYSKPLIWNSRVKIALGTARALEYLHEVSSPSVVHK NIKSANILLDTELNPHLSDSGLASYIPNADQILNHNVGSGYDAPEVALSGQYTLKSDV YSFGVVMLELLSGRNPFDSSRPRSEQSLVRWATPQLHDIDALAKMVDPAMKGLYPV KSLSRFADVIALCVQPEPEFRPPMSEVVQALVRLVQRANMSKRTFSSSDHGGSQRGS DEPVLRDI* (SEQ ID NO: 29) Nucleotide sequence of OsPK-1 from Oryza sativa ACCACACAAAAAAGCAAAACAGAGAGAACAACTGTTACTCACACACGCCATGG GTAAATGAATGGTTTTTGAGCAACAGCAGTTAAAAGAGAAAAGGGATTCAGCGA AGATGACATCGGTTGGTGTGGCACCAACTTCGGGTTTGAGAGAAGCCAGTGGGC ATGGAGCAGCAGCTGCGGATAGATTGCCAGAGGAGATGAACGATATGAAAATTA GGGATGATAGAGAAATGGAAGCCACAGTTGTTGATGGCAACGGAACGGAGACA GGACATATCATTGTGACTACCATTGGGGGTAGAAATGGTCAGCCCAAGCAGACT ATAAGCTACATGGCAGAGCGTGTTGTAGGGCATGGATCATTTGGAGTTGTCTTCC AGGCTAAGTGCTTGGAAACCGGTGAAACTGTGGCTATCAAAAAGGTTCTTCAAG ATAAGAGGTACAAGAACCGGGAGCTGCAAACAATGCGCCTTCTTGACCACCCAA ATGTTGTCGCTTTGAAGCACTGTTTCTTTTCAACCACTGAAAAGGATGAACTATA CCTCAATTTGGTACTTGAATATGTTCCTGAAACAGTTAATCGTGTGATCAAACAT TACAACAAGTTAAACCAAAGGATGCCGCTGATATATGTGAAACTCTATACATAC
CAGATCTTTAGGGCGTTATCTTATATTCATCGTTGTATTGGAGTCTGCCATCGGG ATATCAAGCCTCAAAATCTATTGGTCAATCCACACACTCACCAGGTTAAATTATG TGACTTTGGAAGTGCAAAGGTTTTGGTAAAAGGCGAACCAAATATATCATACAT ATGTTCTAGATACTATAGAGCACCTGAGCTCATATTTGGCGCAACTGAATATACT TCAGCCATTGACATCTGGTCTGTTGGATGTGTTTTAGCTGAGCTGCTGCTTGGAC AGCCTCTGTTCCCTGGTGAGAGTGGAGTTGATCAACTTGTTGAGATCATCAAGGT TCTGGGCACTCCAACAAGGGAAGAGATTAAGTGCATGAACCCTAATTATACAGA ATTTAAATTCCCACAGATTAAAGCACATCCATGGCACAAGATCTTCCATAAGCGC ATGCCTCCAGAGGCTGTTGATTTGGTATCAAGACTACTACAATACTCCCCTAACT TGCGGTGCACAGCTTTTGATGCCTTGACGCATCCTTTCTTCGACGAGCTTCGTGAT CCAAATACTCGCTTGCCAAATGGCCGATTCCTTCCACCACTATTTAATTTCAAAT CCCATGAACTGAAAGGAGTCCCATCTGAGATTTTGGTGAAATTGGTTCCAGAGC ATGCAAGGAAGCAATGCCCGTTTCTAGGCTCGTGAAGTGTTGTTTCCATATGAGA ATGCTGCGCTTTCCTTTTCTATTTAATATGATATTTTTGTTGGTATCTTTATTGTAT TCGGTTGCCCTGTAAAAGCAGATTTAGAGATACATGCTACTCATTATCACCCAAC CCCCGATGGTTATGTAGAATACCCTGTTTCCTGTATCACAGCAGATTGTAACATA CAATAGAGGACAAAATGTCTGCAATTATCTAAATGTTGCATCAATATTTGTATTT GTTGAGGCAAAAAAAAAAAAAAAAAA (SEQ ID NO: 30) Deduced amino acid sequence of OsPK-1 from Oryza sativa MVFEQQQLKEKRDSAKMTSVGVAPTSGLREASGHGAAAADRLPEEMNDMKIRDDR EMEATVVDGNGTETGHIIVTTIGGRNGQPKQTISYMAERVVGHGSFGVVFQAKCLET GETVAIKKVLQDKRYKNRELQTMRLLDHPNVVALKHCFFSTTEKDELYLNLVLEYV PETVNRVIKHYNKLNQRMPLIYVKLYTYQIFRALSYIHRCIGVCHRDIKPQNLLVNPH THQVKLCDFGSAKVLVKGEPNISYICSRYYRAPELIFGATEYTSAIDIWSVGCVLAELL LGQPLFPGESGVDQLVEIIKVLGTPTREEIKCMNPNYTEFKFPQIKAHPWHKIFHKRMP PEAVDLVSRLLQYSPNLRCTAFDALTHPFFDELRDPNTRLPNGRFLPPLFNFKSHELK GVPSEILVKLYPEHARKQCPFLGS*
Sequence CWU
1
5111100DNAPhyscomitrella patens 1cggcaccagc atcttcgcga ggcatgtgat
gtgtggtcgg tggagttagc ttctacgggc 60aactggaaat ccagggaatt ctgccagaat
tatacgtact aaagtagaaa tttacgtttc 120ggggacttcg agtcttctat ggcatctgcg
actgcgggta ttatcaacag cacaaacatg 180atcggaggag gaatagctcc aactaaagct
ggctcaagcg gagtagaatt gttaccgaaa 240gaaatgcacg acatgaagct cagggatgac
aaggttgacc acagcgacga caaggaaatt 300gaggcttcaa tagtagatgg aaacggtacc
gaaactggcc acatcatagc tactactatt 360ggagggcgaa atggacaacc taagcagacg
atcagctatt cggcagaacg tgttgttggc 420actggatcat tcgggattgt cttccaggca
aaatgcatcg aaactgggga gacggtggct 480ataaagaaag tgttgcagga caaaagatac
aagaatcgag agctgcagat catgcgactg 540ttggaccacc cgaatattgt agctttgaag
cattgcttct tctcgacgac ggataaagac 600gaattgtact taaacctggt gctggagtat
gtacccgaga cggtgtatcg tattgcaaag 660cactacaatc gcatgaatca gcgaatgccc
cttgtttacg tgaaactgta cacgtatcag 720atatgccgat cactggcata tatccacaat
ggcatcggtg tctgccaccg cgacatcaag 780ccccagaacc tgctggtgga atcctcatac
gcaccagctg aaactgtgtg attttgggaa 840gtgcgaaagt gctggtgaaa ggggagccca
atatctcgta catttgttcg cggtactacc 900gtgctccggg agcttatttt tggagcgacg
gagtacacga ctgccataga tatatggtcg 960atgggttgcg tgatggcaga gcttctacta
ggacagcctt tgtttcctgg agagagtgga 1020gtggatcaat tggtggaaat catcaaggtt
ttggggacac cgactcgtga ggagatcaag 1080tgcatgaatc cgaactacac
110021849DNAPhyscomitrella patens
2ggcaccagca tcttcgcgag gcatgtgatg tgtggtcggt ggagttagct tctacgggca
60actggaaatc cagggaattc tgccagaatt atacgtacta aagtagaaat ttacgtttcg
120gggacttcga gtcttctatg gcatctgcga ctgcgggtat tatcaacagc acaaacatga
180tcggaggagg aatagctcca actaaagctg gctcaagcgg agtagaattg ttaccgaaag
240aaatgcacga catgaagctc agggatgaca aggttgacca cagcgacgac aaggaaattg
300aggcttcaat agtagatgga aacggtaccg aaactggcca catcatagct actactattg
360gagggcgaaa tggacaacct aagcagacga tcagctattc ggcagaacgt gttgttggca
420ctggatcatt cgggattgtc ttccaggcaa aatgcatcga aactggggag acggtggcta
480taaagaaagt gttgcaggac aaaagataca agaatcgaga gctgcagatc atgcgactgt
540tggaccaccc gaatattgta gctttgaagc attgcttctt ctcgacgacg gataaagacg
600aattgtactt aaacctggtg ctggagtatg tacccgagac ggtgtatcgt attgcaaagc
660actacaatcg catgaatcag cgaatgcccc ttgtttacgt gaaactgtac acgtatcaga
720tatgccgatc actggcatat atccacaatg gcatcggtgt ctgccaccgc gacatcaagc
780cccagaacct gctggtgaat cctcatacgc accagctgaa actgtgtgat tttggaagtg
840cgaaagtgct ggtgaaaggg gagcccaata tctcgtacat ttgttcgcgg tactaccgtg
900ctccggagct tatttttgga gcgacggagt acacgactgc catagatata tggtcgatgg
960gttgcgtgat ggcagagctt ctactaggac agcctttgtt tcctggagag agtggagtgg
1020atcaattggt ggaaatcatc aaggttttgg ggacaccgac tcgtgaggag atcaagtgca
1080tgaatccgaa ctacacagag ttcaagtttc cacaaatcaa ggcgcacccg tggcacaaag
1140ttttccacaa acgcatgcca cctgaagcag ttgacttggt gtcaaggctc cttcagtact
1200ctccaaatct gcggtgcaac gctctggaag cgtgtgtgca cccgttcttt gatgagctaa
1260gggatcctaa ctgccggctt ccgaatgggc ggccactgcc ctctctgttc aacttcaaaa
1320cccaagagtt gaagggtgca actcctgata ttctgcagcg tttgataccc gagcacgcga
1380ggaagcagaa tccgatgctg gcgctgtgag gggtgcctgg aaagagatcg gaagagtcta
1440ctgcgtgaaa ggttttcctc tgtttggagg agtggtccgc tttgtggagg gcttcatagg
1500cactctgtat cattgcttaa acacgtaaag tcaaccaatt tgctatggat ccctgctttc
1560gctgtgattg gaggaagact tagtagacga ttagcatgcc acttttagga acggcaattc
1620tcctgtagtg aaggttacga ttctattgta cttcagaacg gtaaaggtat ttaggggttc
1680tcagtgcttc ctgatttggg tacgtgatgt accattggaa aggcttcaaa cgcatgtata
1740tctatgagac tttgacgtta ctttttatcg tcagtactca ggaagctcct ctctggatgg
1800gattatccat tcgtgccgtt cgaatcgcaa taaaaaaaaa aaaaaaaaa
18493423PRTPhyscomitrella patens 3Met Ala Ser Ala Thr Ala Gly Ile Ile Asn
Ser Thr Asn Met Ile Gly1 5 10
15Gly Gly Ile Ala Pro Thr Lys Ala Gly Ser Ser Gly Val Glu Leu Leu
20 25 30Pro Lys Glu Met His Asp
Met Lys Leu Arg Asp Asp Lys Val Asp His 35 40
45Ser Asp Asp Lys Glu Ile Glu Ala Ser Ile Val Asp Gly Asn
Gly Thr 50 55 60Glu Thr Gly His Ile
Ile Ala Thr Thr Ile Gly Gly Arg Asn Gly Gln65 70
75 80Pro Lys Gln Thr Ile Ser Tyr Ser Ala Glu
Arg Val Val Gly Thr Gly 85 90
95Ser Phe Gly Ile Val Phe Gln Ala Lys Cys Ile Glu Thr Gly Glu Thr
100 105 110Val Ala Ile Lys Lys
Val Leu Gln Asp Lys Arg Tyr Lys Asn Arg Glu 115
120 125Leu Gln Ile Met Arg Leu Leu Asp His Pro Asn Ile
Val Ala Leu Lys 130 135 140His Cys Phe
Phe Ser Thr Thr Asp Lys Asp Glu Leu Tyr Leu Asn Leu145
150 155 160Val Leu Glu Tyr Val Pro Glu
Thr Val Tyr Arg Ile Ala Lys His Tyr 165
170 175Asn Arg Met Asn Gln Arg Met Pro Leu Val Tyr Val
Lys Leu Tyr Thr 180 185 190Tyr
Gln Ile Cys Arg Ser Leu Ala Tyr Ile His Asn Gly Ile Gly Val 195
200 205Cys His Arg Asp Ile Lys Pro Gln Asn
Leu Leu Val Asn Pro His Thr 210 215
220His Gln Leu Lys Leu Cys Asp Phe Gly Ser Ala Lys Val Leu Val Lys225
230 235 240Gly Glu Pro Asn
Ile Ser Tyr Ile Cys Ser Arg Tyr Tyr Arg Ala Pro 245
250 255Glu Leu Ile Phe Gly Ala Thr Glu Tyr Thr
Thr Ala Ile Asp Ile Trp 260 265
270Ser Met Gly Cys Val Met Ala Glu Leu Leu Leu Gly Gln Pro Leu Phe
275 280 285Pro Gly Glu Ser Gly Val Asp
Gln Leu Val Glu Ile Ile Lys Val Leu 290 295
300Gly Thr Pro Thr Arg Glu Glu Ile Lys Cys Met Asn Pro Asn Tyr
Thr305 310 315 320Glu Phe
Lys Phe Pro Gln Ile Lys Ala His Pro Trp His Lys Val Phe
325 330 335His Lys Arg Met Pro Pro Glu
Ala Val Asp Leu Val Ser Arg Leu Leu 340 345
350Gln Tyr Ser Pro Asn Leu Arg Cys Asn Ala Leu Glu Ala Cys
Val His 355 360 365Pro Phe Phe Asp
Glu Leu Arg Asp Pro Asn Cys Arg Leu Pro Asn Gly 370
375 380Arg Pro Leu Pro Ser Leu Phe Asn Phe Lys Thr Gln
Glu Leu Lys Gly385 390 395
400Ala Thr Pro Asp Ile Leu Gln Arg Leu Ile Pro Glu His Ala Arg Lys
405 410 415Gln Asn Pro Met Leu
Ala Leu 4204420DNAPhyscomitrella patensmodified_base(223)a, t,
c , g, unknown or other 4gcacgaggat cgaccgggtg gagtacgtgc actcgcgagg
tctaattcat cgtgacttga 60aaccagataa tttcctcatg ggctgcggcc ggcaagggaa
ccaagtgttc attattgact 120ttggcttggc aaaagagtac atcgaccccg cgacacgtag
acacattcct taccgagata 180gaaagagctt tacaggaaca gcgcggtatg ctagtaggaa
tcnccacnaa ggaatcgaac 240acagcaggag agatgacata naatcncttg gttacattct
tatgtacttt cttaggggga 300atttaccatg gcaaggtcaa ggggggcaac gtttcaccga
tcagaagcaa catgagtaca 360tgcncaacaa aattaagatg gagactanca tcnaggatct
ctgcgatggg tacccagaca 42051795DNAPhyscomitrella patens 5gcccttatcc
cgggaggcat tgaactacct ggagtgagat ttttttggga atttgaaaga 60gaattacata
tatacaaggt tgaggctcac cgagaacaag tctgctgata gcttcttcac 120tcttgaaata
gatagttcat catggattca ggaggtgacc gcgtgcgagc tcctcagaag 180cagtctcgcg
aggaggatca gtaccgttca ttgaacattg ctacagagca tcgtcagcat 240atacagaagc
accaacaaca ccaacagcag ccggggactg gattggttgt tgaaacgctt 300caaaaaacac
tatgtaacgt gactgtgacc tcacctacaa gcagtccgga ggggggtaga 360ttacgtactg
ttgcgaacaa gtatgcagtg gaaggaatgg tcggcagtgg cgcattttgc 420aaggtgtacc
agggttctga cttaaccaac catgaggttg tgggcatcaa gctcgaggat 480acaagaacag
agcacgcaca attgatgcac gagtcgcgat tatacaacat tttgcggggt 540ggaaagggag
tgcccaacat gagatggttt gggaaagagc aagactacaa tgtgatggtg 600ctagatttgc
tggggcctaa cctactgcac cttttcaagg tgtgtgggca aagattttcg 660ttgaagacgg
tgatcatgtt ggggtaccaa atgatcgacc gggtggagta cgtgcactcg 720cgaggtctag
ttcatcgtga cttgaaacca gataatttcc tcatgggctg cggccggcaa 780gggaaccaag
tgttcattat tgactttggc ttggcaaaag agtacatcga ccccgcgaca 840cgtagacaca
ttccttaccg agatagaaag agctttacag gaacagcgcg gtatgctagt 900aggaatcagc
acaaaggaat cgaacacagc aggagagatg acatagaatc acttggttac 960attcttatgt
actttcttag ggggaattta ccatggcaag gtcaaggggg gcaacgtttc 1020accgatcaga
agcaacatga gtacatgcac aacaaaatta agatggagac taccatcgag 1080gatctctgcg
atgggtaccc cagacaattt gccgactttt tacaccacgc gcgcgagttg 1140ggattctatg
agcagcctga ctactcgtac cttcgcagcc tgttccgtga tcttttcatt 1200cagaagaaat
tccagcttga ccatgtctac gactggacag tgtacactca acctcctcag 1260aatggctctg
cacaaacagt tcgaagcccg gctgccggtc cacagactca cttacaaagt 1320cgcccttcca
atgtatcata ttgtccacct ctgactaaac cagagttccg gcgtgaggta 1380gttgcggcga
attagggttt acacaggaag agatgtggta aagcatctca tcttcttcgt 1440tctggtgcca
aaatggtaca aggtcgtctg ctgtctcttt ctcgcaagcc ctcacatata 1500gatgaaggtt
tgtgaagtta gagatgcaac taccaagcaa aggctaggaa aagagctgta 1560gactttctag
tgtgtagtgt gtaaatcaag gcttctggca tggtatcggc agtcaggtgc 1620atggagcaga
atagaaatta cttcgtgcat gacaagattt tttttcttgc agagctctcg 1680acggttctgc
gatctcactt ctctacacaa ccagcgctcc tttaattgaa aagaggatct 1740ggtacgagta
tgataaagtt aacacagatt actagttcaa cgatatcgca agggc
17956417PRTPhyscomitrella patens 6Met Asp Ser Gly Gly Asp Arg Val Arg Ala
Pro Gln Lys Gln Ser Arg1 5 10
15Glu Glu Asp Gln Tyr Arg Ser Leu Asn Ile Ala Thr Glu His Arg Gln
20 25 30His Ile Gln Lys His Gln
Gln His Gln Gln Gln Pro Gly Thr Gly Leu 35 40
45Val Val Glu Thr Leu Gln Lys Thr Leu Cys Asn Val Thr Val
Thr Ser 50 55 60Pro Thr Ser Ser Pro
Glu Gly Gly Arg Leu Arg Thr Val Ala Asn Lys65 70
75 80Tyr Ala Val Glu Gly Met Val Gly Ser Gly
Ala Phe Cys Lys Val Tyr 85 90
95Gln Gly Ser Asp Leu Thr Asn His Glu Val Val Gly Ile Lys Leu Glu
100 105 110Asp Thr Arg Thr Glu
His Ala Gln Leu Met His Glu Ser Arg Leu Tyr 115
120 125Asn Ile Leu Arg Gly Gly Lys Gly Val Pro Asn Met
Arg Trp Phe Gly 130 135 140Lys Glu Gln
Asp Tyr Asn Val Met Val Leu Asp Leu Leu Gly Pro Asn145
150 155 160Leu Leu His Leu Phe Lys Val
Cys Gly Gln Arg Phe Ser Leu Lys Thr 165
170 175Val Ile Met Leu Gly Tyr Gln Met Ile Asp Arg Val
Glu Tyr Val His 180 185 190Ser
Arg Gly Leu Val His Arg Asp Leu Lys Pro Asp Asn Phe Leu Met 195
200 205Gly Cys Gly Arg Gln Gly Asn Gln Val
Phe Ile Ile Asp Phe Gly Leu 210 215
220Ala Lys Glu Tyr Ile Asp Pro Ala Thr Arg Arg His Ile Pro Tyr Arg225
230 235 240Asp Arg Lys Ser
Phe Thr Gly Thr Ala Arg Tyr Ala Ser Arg Asn Gln 245
250 255His Lys Gly Ile Glu His Ser Arg Arg Asp
Asp Ile Glu Ser Leu Gly 260 265
270Tyr Ile Leu Met Tyr Phe Leu Arg Gly Asn Leu Pro Trp Gln Gly Gln
275 280 285Gly Gly Gln Arg Phe Thr Asp
Gln Lys Gln His Glu Tyr Met His Asn 290 295
300Lys Ile Lys Met Glu Thr Thr Ile Glu Asp Leu Cys Asp Gly Tyr
Pro305 310 315 320Arg Gln
Phe Ala Asp Phe Leu His His Ala Arg Glu Leu Gly Phe Tyr
325 330 335Glu Gln Pro Asp Tyr Ser Tyr
Leu Arg Ser Leu Phe Arg Asp Leu Phe 340 345
350Ile Gln Lys Lys Phe Gln Leu Asp His Val Tyr Asp Trp Thr
Val Tyr 355 360 365Thr Gln Pro Pro
Gln Asn Gly Ser Ala Gln Thr Val Arg Ser Pro Ala 370
375 380Ala Gly Pro Gln Thr His Leu Gln Ser Arg Pro Ser
Asn Val Ser Tyr385 390 395
400Cys Pro Pro Leu Thr Lys Pro Glu Phe Arg Arg Glu Val Val Ala Ala
405 410
415Asn7539DNAPhyscomitrella patens 7gcacgagcgc acttggtttc tgccacttat
tccagctggt aaagaaaaac cacctaaaat 60gaaagtgttt gaagcagata catttgagaa
ggaagtggaa gaaccgaaga tcaaggcctt 120acctccattg aagtcactta aagtacctcc
agctttgaag gttgaggaag ctacctacaa 180ggttgaaagt gaagggaagg tgaacaagag
caacattaca gcaagagagt tttccgtcgc 240agaacttcag gcggctacgg acagtttctc
agaggataat ttacttggcg aaggttcgct 300tggttgtgtt taccgcgcgg agttccccga
cggtgaggtt ctagctgtca gaaacttgat 360acaacagcct ccatggttcg gaatgaagat
gatttcttga gcgttgtcga tggcttggcc 420cggctacaat accaattcta atgaactcgt
aggctactgt gccgagcatg ggcaacgact 480tctggtctac aagttcatca gtcgagggac
actccatgaa ctgcttcatg gctcagccg 53981468DNAPhyscomitrella patens
8tttctggaat agctcagaag cgttgcaaaa tttatcagga ggtttgcaga catggtgatg
60aggaaagtgg gcaagtatga agtggggcga actattggtg agggaacctt cgccaaggtg
120aaatttgccc agaacaccga gacaggggag agcgtggcca tgaaggtgct agatcgtcag
180acggtgctca agcacaagat ggtagagcag atcaggcgag aaatatccat aatgaagctg
240gttaggcatc ctaatgttgt ccgattgcac gaggttctgg caagtcgttg caagatttac
300atcattttgg agtttgtaac gggcggggag ctttttgaca aaattgtgca tcaaggaagg
360cttaatgaga acgactctcg caaatatttt cagcagctca tggatggagt tgattattgc
420cacagcaagg gcgtctcaca tcgagatttg aagcctgaaa atctccttct tgattcactg
480gacaatctca aaatatcaga ttttggtctg agtgctcttc ctcagcaagt gagggaagat
540ggacttttgc acaccacttg tggtactccc aattatgttg cacctgaggt tcttaatgat
600aagggctacg atggtgcagt ggctgatatc tggtcttgcg gtgtcatctt gtttgtatta
660atggctggat ttctcccatt tgatgaggct gacttgaata ctctttacag caagatacga
720gaggcagatt ttacttgtcc accttggttt tcctccggcg ccaaaacact gattactaat
780attctggatc ccaatcccct aacacgtatc aggatgagag gaattcggga tgacgaatgg
840ttcaaaaaga actatgttcc tgttcgtatg tatgacgatg aagatattaa tcttgatgat
900gtggagactg cttttgatga ttctaaggaa caatttgtga aagagcagag ggaggtgaaa
960gacgtgggtc cgtcgttgat gaatgccttt gaactcataa gcctatctca aggactaaac
1020ctctctgcgt tgtttgatag acgtcaggac catgtaaagc gccaaactcg tttcacttca
1080aagaaaccag ctcgagatat aattaataga atggaaaccg ctgcgaagtc gatgggcttt
1140ggtgttggaa cgcgtaacta caagatgaga ctcgaggcag ctagtgagtg cagaatatca
1200cagcacttgg ctgtggctat cgaagtgtac gaggtggctc cttctttatt catgattgaa
1260gtgcggaagg ctgcgggtga tactttggaa tatcacaagt tctataaaag cttttgtacc
1320cggttgaaag atatcatatg gacaacggca gttgataagg acgaagttaa gacattgacg
1380ccatctgtag ttaagaataa ataattctgc tccagcatta acttggatga ggagcaagga
1440tataccgctg catcgagctc cgaagggc
14689450PRTPhyscomitrella patens 9Met Val Met Arg Lys Val Gly Lys Tyr Glu
Val Gly Arg Thr Ile Gly1 5 10
15Glu Gly Thr Phe Ala Lys Val Lys Phe Ala Gln Asn Thr Glu Thr Gly
20 25 30Glu Ser Val Ala Met Lys
Val Leu Asp Arg Gln Thr Val Leu Lys His 35 40
45Lys Met Val Glu Gln Ile Arg Arg Glu Ile Ser Ile Met Lys
Leu Val 50 55 60Arg His Pro Asn Val
Val Arg Leu His Glu Val Leu Ala Ser Arg Cys65 70
75 80Lys Ile Tyr Ile Ile Leu Glu Phe Val Thr
Gly Gly Glu Leu Phe Asp 85 90
95Lys Ile Val His Gln Gly Arg Leu Asn Glu Asn Asp Ser Arg Lys Tyr
100 105 110Phe Gln Gln Leu Met
Asp Gly Val Asp Tyr Cys His Ser Lys Gly Val 115
120 125Ser His Arg Asp Leu Lys Pro Glu Asn Leu Leu Leu
Asp Ser Leu Asp 130 135 140Asn Leu Lys
Ile Ser Asp Phe Gly Leu Ser Ala Leu Pro Gln Gln Val145
150 155 160Arg Glu Asp Gly Leu Leu His
Thr Thr Cys Gly Thr Pro Asn Tyr Val 165
170 175Ala Pro Glu Val Leu Asn Asp Lys Gly Tyr Asp Gly
Ala Val Ala Asp 180 185 190Ile
Trp Ser Cys Gly Val Ile Leu Phe Val Leu Met Ala Gly Phe Leu 195
200 205Pro Phe Asp Glu Ala Asp Leu Asn Thr
Leu Tyr Ser Lys Ile Arg Glu 210 215
220Ala Asp Phe Thr Cys Pro Pro Trp Phe Ser Ser Gly Ala Lys Thr Leu225
230 235 240Ile Thr Asn Ile
Leu Asp Pro Asn Pro Leu Thr Arg Ile Arg Met Arg 245
250 255Gly Ile Arg Asp Asp Glu Trp Phe Lys Lys
Asn Tyr Val Pro Val Arg 260 265
270Met Tyr Asp Asp Glu Asp Ile Asn Leu Asp Asp Val Glu Thr Ala Phe
275 280 285Asp Asp Ser Lys Glu Gln Phe
Val Lys Glu Gln Arg Glu Val Lys Asp 290 295
300Val Gly Pro Ser Leu Met Asn Ala Phe Glu Leu Ile Ser Leu Ser
Gln305 310 315 320Gly Leu
Asn Leu Ser Ala Leu Phe Asp Arg Arg Gln Asp His Val Lys
325 330 335Arg Gln Thr Arg Phe Thr Ser
Lys Lys Pro Ala Arg Asp Ile Ile Asn 340 345
350Arg Met Glu Thr Ala Ala Lys Ser Met Gly Phe Gly Val Gly
Thr Arg 355 360 365Asn Tyr Lys Met
Arg Leu Glu Ala Ala Ser Glu Cys Arg Ile Ser Gln 370
375 380His Leu Ala Val Ala Ile Glu Val Tyr Glu Val Ala
Pro Ser Leu Phe385 390 395
400Met Ile Glu Val Arg Lys Ala Ala Gly Asp Thr Leu Glu Tyr His Lys
405 410 415Phe Tyr Lys Ser Phe
Cys Thr Arg Leu Lys Asp Ile Ile Trp Thr Thr 420
425 430Ala Val Asp Lys Asp Glu Val Lys Thr Leu Thr Pro
Ser Val Val Lys 435 440 445Asn Lys
45010606DNAPhyscomitrella patens 10ggcacgagat ttggttgcaa aataggtaac
tacaacttaa gaagaaaaac aatctctctc 60tttccccaca caagatacaa cttcgctttt
tccatcactt acaccagaaa gcccaaagta 120gggtagattg tcacacatcg ctatgatccc
aattaagcat ctactacttt tcatcagatc 180agcaaactac caatcataga aactaggtga
tgaatattac gatactttca ggttcaatgc 240gaaatccaag gttaacagta atgaatgtat
tcaagctctg tacatgcatt aattttatgc 300taccagtaga aaacttcatt tgacgatgca
gcggtatatc cttgctcctc atccaagtta 360atgctggagc agaattattt attcttaact
acagatggcg tcaatgtctt aacttcgtcc 420ttatcaactg ccgttgtcca tatgatatct
ttcaaccggg tacaaaagct tttatagaac 480ttgtgatatt ccaaagtatc acccgcagcc
ttccgcactt caatcatgaa taaagaagga 540gccacctcgt acacttcgat agcccagcca
agtgctgtga tattctgcct cactactgcc 600tcgagc
606113025DNAPhyscomitrella patens
11atcccgggtg tcggaattcg gtcacaatga gctagtgtgt tgtttgattg tggcctcagc
60tggagaggct ttggtatcgt tagcagcgag tgacgctgtt gaaggattgt atccatccac
120aagcgagaag ccttgcctaa tttttgggag ggaaaggtgg ttctcacatg agaggagcag
180ttgtcgatgc cccaatgaag ggtgacagga gagcatgcat tttgggagga atgggaagac
240ctaatggtgg aaccatcttg tacgtgttgg tgatttcatt cattgctttg gtgaatggag
300ccaccgatcc gaacgatgtg tctgctttga atactatgtt cactggcttc aacagcgatc
360ctaagctcac gaactgggtg caaaacgcgg gtgatccctg cggaaccaac tggctgggcg
420ttacttgtga tgggaccttc gtcacctcaa tcaagctatc caacatggga ctgaatggga
480aggtggaggg atgggtgttg cagaagtttc aacacctctc tgtgcttgac cttagccata
540ataatcttgc tagcggaatt cctgagatgt ttcctcccaa gttgactgaa ctagatttgt
600cttacaacca gctcacgggt agttttcctt atttgataat caacatccct actttgacaa
660gcataaaact gaataacaac aagctgagtg gaacgctcga tgggcaggtt ttcagtaaac
720tcacaaactt aatcaccctc gatatttcca acaacgcaat tacagggccg attcccgagg
780gcatgggtga catggtcagc ctaagatttt tgaacatgca aaataataag ctgactggac
840caatcccaga cacattggct aatattccat ctctagaaac attggacgta tctaacaacg
900cgcttactgg ctttctccca ccaaacctga acccaaagaa tttcagatat ggaggcaatc
960cactcaacac ccaagcccct cctccaccac cgtttacacc accgccacct tcaaagaatc
1020caaagcctat tcctcctcca ccccaccctg gtagccgaac accagatact gctcctaagg
1080ctgaaggcgg catcgtatca ggcgcagcaa ttgctgggat tgtcgtggga gcaattttgg
1140tgcttgcagc aattttcata gctgtatggt tctttgtcgt ccgtaaaaga tctgagctta
1200ccaaaccttt ggatttagag gctaatcaca gcagccgacg cacttggttt ctgccactta
1260ttccagctgg taaagaaaaa ccacctaaaa tgaaagtgtt tgaagcagat acatttgaga
1320aggaagtgga agagccgaag atcaaggcct tacctccatt gaagtcactt aaagtacctc
1380cagcattgaa ggttgaggaa gctacctaca aggttgaaag tgaagggaag gtgaacaaga
1440gcaacattac agcaagagag ttttccgtcg cagaacttca ggcggctacg gacagtttct
1500cagaggataa tttacttggc gaaggttcgc ttggttgtgt ttaccgcgcg gagttccccg
1560acggtgaggt tctagctgtc aagaaacttg atacaacagc ctccatggtt cggaatgaag
1620atgatttctt gagcgttgtc gatggcttgg cccggctaca acataccaat tctaatgaac
1680tcgtaggcta ctgtgccgag catgggcaac gacttctggt ctacaagttc atcagtcgag
1740ggacactcca tgaactgctt catggctcag ccgatagccc caaggagttg tcatggaatg
1800tccgtgtgaa gattgcactt ggttgtgcgc gggctcttga gtatttccat gaaatcgttt
1860cgcagccggt tgtgcaccgc aactttagat cctcaaacat tcttttggat gatgagctga
1920acccacatgt gtcggattgt ggtttggctg cttttacccc atccagtgct gaacggcagg
1980tctctgccca agtgttggga tcttttggac acagtccccc tgaattcagc acatctggaa
2040tgtatgatgt gaaaagcgac gtttatagct ttggtgttgt gatgcttgag cttatgacag
2100gacgcaagcc tttagacagc tcaagaccaa gatccgagca aaacctggtg cgatgggcaa
2160caccacaact gcatgatatt gatgcactcg caagaatggt ggatccagcg ttagagggtg
2220cttaccctgc caagtccctc tcccggttcg ccgacatcgt tgccttgtgt gtccagcccg
2280aacccgaatt ccgacctcct atatctgaag tagtgcagtc cctggtaagg cttatgcagc
2340gtgcagcttt aagtaaacgc cggcatgagt acaacgcagg cgttcctcag actgatatgg
2400aggaccctag tgattacttg tgacagaagt aagtatcctg gtcgatactt cccaatttca
2460agcatagaga acctcccgcg cgtctactcc cacttgattt tcaaagctgg cgaaaagtgg
2520ccaaatttgt ggatttgtga caccttgcaa ctaaatcggg gagatattca gcttctttgc
2580aattccagac catgatggca cagactttgg cttgcatcct cctcattatt actgaagctt
2640ttgcttctaa tggcggatta ctgattatgg atgactatcc cgtttccagg cagacgtgaa
2700gagaagtgtt ggcttccgaa gttgttaaat tgtatcgacg gctgaaagct tttttaagag
2760cttacttctg ggtcctagtt agtgatatta aggtccctgt gccttaagag taatgtgcaa
2820ttcctgttgt gttgcaaact cgggtaacgc tttgtcttgt agttttggca cattacaagg
2880ttagttcgac agtgaactca caatttgaac agattagtta gggagtgtaa ctctagcaaa
2940agttgattcc ttgtggttac ccaatttttt gaatgtgaac tcccactcat tggtgtgatg
3000gagtacatga ttcgcacgag ctcgc
302512751PRTPhyscomitrella patens 12Met Arg Gly Ala Val Val Asp Ala Pro
Met Lys Gly Asp Arg Arg Ala1 5 10
15Cys Ile Leu Gly Gly Met Gly Arg Pro Asn Gly Gly Thr Ile Leu
Tyr 20 25 30Val Leu Val Ile
Ser Phe Ile Ala Leu Val Asn Gly Ala Thr Asp Pro 35
40 45Asn Asp Val Ser Ala Leu Asn Thr Met Phe Thr Gly
Phe Asn Ser Asp 50 55 60Pro Lys Leu
Thr Asn Trp Val Gln Asn Ala Gly Asp Pro Cys Gly Thr65 70
75 80Asn Trp Leu Gly Val Thr Cys Asp
Gly Thr Phe Val Thr Ser Ile Lys 85 90
95Leu Ser Asn Met Gly Leu Asn Gly Lys Val Glu Gly Trp Val
Leu Gln 100 105 110Lys Phe Gln
His Leu Ser Val Leu Asp Leu Ser His Asn Asn Leu Ala 115
120 125Ser Gly Ile Pro Glu Met Phe Pro Pro Lys Leu
Thr Glu Leu Asp Leu 130 135 140Ser Tyr
Asn Gln Leu Thr Gly Ser Phe Pro Tyr Leu Ile Ile Asn Ile145
150 155 160Pro Thr Leu Thr Ser Ile Lys
Leu Asn Asn Asn Lys Leu Ser Gly Thr 165
170 175Leu Asp Gly Gln Val Phe Ser Lys Leu Thr Asn Leu
Ile Thr Leu Asp 180 185 190Ile
Ser Asn Asn Ala Ile Thr Gly Pro Ile Pro Glu Gly Met Gly Asp 195
200 205Met Val Ser Leu Arg Phe Leu Asn Met
Gln Asn Asn Lys Leu Thr Gly 210 215
220Pro Ile Pro Asp Thr Leu Ala Asn Ile Pro Ser Leu Glu Thr Leu Asp225
230 235 240Val Ser Asn Asn
Ala Leu Thr Gly Phe Leu Pro Pro Asn Leu Asn Pro 245
250 255Lys Asn Phe Arg Tyr Gly Gly Asn Pro Leu
Asn Thr Gln Ala Pro Pro 260 265
270Pro Pro Pro Phe Thr Pro Pro Pro Pro Ser Lys Asn Pro Lys Pro Ile
275 280 285Pro Pro Pro Pro His Pro Gly
Ser Arg Thr Pro Asp Thr Ala Pro Lys 290 295
300Ala Glu Gly Gly Ile Val Ser Gly Ala Ala Ile Ala Gly Ile Val
Val305 310 315 320Gly Ala
Ile Leu Val Leu Ala Ala Ile Phe Ile Ala Val Trp Phe Phe
325 330 335Val Val Arg Lys Arg Ser Glu
Leu Thr Lys Pro Leu Asp Leu Glu Ala 340 345
350Asn His Ser Ser Arg Arg Thr Trp Phe Leu Pro Leu Ile Pro
Ala Gly 355 360 365Lys Glu Lys Pro
Pro Lys Met Lys Val Phe Glu Ala Asp Thr Phe Glu 370
375 380Lys Glu Val Glu Glu Pro Lys Ile Lys Ala Leu Pro
Pro Leu Lys Ser385 390 395
400Leu Lys Val Pro Pro Ala Leu Lys Val Glu Glu Ala Thr Tyr Lys Val
405 410 415Glu Ser Glu Gly Lys
Val Asn Lys Ser Asn Ile Thr Ala Arg Glu Phe 420
425 430Ser Val Ala Glu Leu Gln Ala Ala Thr Asp Ser Phe
Ser Glu Asp Asn 435 440 445Leu Leu
Gly Glu Gly Ser Leu Gly Cys Val Tyr Arg Ala Glu Phe Pro 450
455 460Asp Gly Glu Val Leu Ala Val Lys Lys Leu Asp
Thr Thr Ala Ser Met465 470 475
480Val Arg Asn Glu Asp Asp Phe Leu Ser Val Val Asp Gly Leu Ala Arg
485 490 495Leu Gln His Thr
Asn Ser Asn Glu Leu Val Gly Tyr Cys Ala Glu His 500
505 510Gly Gln Arg Leu Leu Val Tyr Lys Phe Ile Ser
Arg Gly Thr Leu His 515 520 525Glu
Leu Leu His Gly Ser Ala Asp Ser Pro Lys Glu Leu Ser Trp Asn 530
535 540Val Arg Val Lys Ile Ala Leu Gly Cys Ala
Arg Ala Leu Glu Tyr Phe545 550 555
560His Glu Ile Val Ser Gln Pro Val Val His Arg Asn Phe Arg Ser
Ser 565 570 575Asn Ile Leu
Leu Asp Asp Glu Leu Asn Pro His Val Ser Asp Cys Gly 580
585 590Leu Ala Ala Phe Thr Pro Ser Ser Ala Glu
Arg Gln Val Ser Ala Gln 595 600
605Val Leu Gly Ser Phe Gly His Ser Pro Pro Glu Phe Ser Thr Ser Gly 610
615 620Met Tyr Asp Val Lys Ser Asp Val
Tyr Ser Phe Gly Val Val Met Leu625 630
635 640Glu Leu Met Thr Gly Arg Lys Pro Leu Asp Ser Ser
Arg Pro Arg Ser 645 650
655Glu Gln Asn Leu Val Arg Trp Ala Thr Pro Gln Leu His Asp Ile Asp
660 665 670Ala Leu Ala Arg Met Val
Asp Pro Ala Leu Glu Gly Ala Tyr Pro Ala 675 680
685Lys Ser Leu Ser Arg Phe Ala Asp Ile Val Ala Leu Cys Val
Gln Pro 690 695 700Glu Pro Glu Phe Arg
Pro Pro Ile Ser Glu Val Val Gln Ser Leu Val705 710
715 720Arg Leu Met Gln Arg Ala Ala Leu Ser Lys
Arg Arg His Glu Tyr Asn 725 730
735Ala Gly Val Pro Gln Thr Asp Met Glu Asp Pro Ser Asp Tyr Leu
740 745 750131757DNABrassica napus
13aacaaaaaaa aatctaaggt ttatcttttt cttcttctat ctgatcatca atcatcgaga
60gagaaaaaag tatacttttt tagatgtgaa gaagctcatc aatcgaagaa gacaatcatc
120aaatgcttca ctttggttcc ctttcttcat cagaaaactc gaggtagatc agttctttga
180tgggatggga caccaaatcg ctaagtgtta tgataccagc aactactagt tacgtgctat
240ctccagagca aataccatgg cttcaaacgg agtaggcagt tcgagatctt ccaaaggtgt
300gaaggcctct tctagctcag tcgattggtt gaccagagat ttggttgaga tgaggataag
360ggacaaggtc gagactgatg atgagaggga tagtgaacca gatattattg atggcgctgg
420cactgaacct ggccatgtga ttagaaccac agtccgtgga cgcaatggtc aatcaagaca
480gacagtcagt tacatatcag agcatgtagt tggtactggt tcctttggca tggtttttca
540agccaagtgt agggaaactg gggagattgt tgcaatcaag aaggttctac aagacaagcg
600ttacaagaac agggagctac aaattatgca gatgctagac caccccaatg tcgttgctct
660aaagcatagc ttctacacga gagctgataa cgaagaggtt tatttgaatc ttgtccttga
720gtttgtgcct gagaccgtca atagggctgc aagaagttac actaggacga accagctaat
780gcctttaata tacgttaaac tctacaccta tcagatttgc agggcgcttg cttacatcca
840taattgcttt ggtctttgtc accgtgatat taagcctcaa aacttgctag tgaacccaca
900tacgcatcag ctgaaaatct gtgacttcgg gagtgcaaaa gtgttggtga aaggagaacc
960caatgtttct tacatctgtt ctagatacta tcgtgctcca gaactcattt ttggcgccag
1020cgaatacaca cctgcaattg atatatggtc aactggttgt gtgatggctg aattgcttct
1080tggacagcct ctgttccctg gtgaaagcgg agtcgatcag cttgttgaaa tcattaaggt
1140tttaggtaca ccaacgaggg aggaaatcaa gtgcatgaat ccaaactata cagaatttaa
1200attcccccag ataaaacctc acccatggca caaggtcttc caaaaacgtt taccgccaga
1260agcggttgat cttctatgta ggttcttcca atattcccct aatctgagat gcacagcttt
1320ggaagcgtgt attcatccgt tatttgatga gctaagggac ccgaacactc gtcttcccaa
1380tggccggcca cttcctccgc ttttcaactt caaacctcaa gagctatctg gcatcccttc
1440tgaaatcgtg aacaggcttg taccagaaca tgcccgtaag cagaacttct tcatggcgtt
1500ggatgcctaa gcgcttatcc tgtttctttt ctttttcttg cttatgtata aactctctag
1560atatcgggta tttggagcag ccagaaggca ttacacgccc tctttggctt ttttttatca
1620gtgagttgtt tggttatcgg gacacgatga tgcatgaata caaacagtac ttgaggtcgc
1680tgctggctta taagaccact tgtttgtttc acaaccagtt cttatatata ttattataca
1740aaaaaaaaaa aaaaaaa
175714417PRTBrassica napus 14Met Ala Ser Asn Gly Val Gly Ser Ser Arg Ser
Ser Lys Gly Val Lys1 5 10
15Ala Ser Ser Ser Ser Val Asp Trp Leu Thr Arg Asp Leu Val Glu Met
20 25 30Arg Ile Arg Asp Lys Val Glu
Thr Asp Asp Glu Arg Asp Ser Glu Pro 35 40
45Asp Ile Ile Asp Gly Ala Gly Thr Glu Pro Gly His Val Ile Arg
Thr 50 55 60Thr Val Arg Gly Arg Asn
Gly Gln Ser Arg Gln Thr Val Ser Tyr Ile65 70
75 80Ser Glu His Val Val Gly Thr Gly Ser Phe Gly
Met Val Phe Gln Ala 85 90
95Lys Cys Arg Glu Thr Gly Glu Ile Val Ala Ile Lys Lys Val Leu Gln
100 105 110Asp Lys Arg Tyr Lys Asn
Arg Glu Leu Gln Ile Met Gln Met Leu Asp 115 120
125His Pro Asn Val Val Ala Leu Lys His Ser Phe Tyr Thr Arg
Ala Asp 130 135 140Asn Glu Glu Val Tyr
Leu Asn Leu Val Leu Glu Phe Val Pro Glu Thr145 150
155 160Val Asn Arg Ala Ala Arg Ser Tyr Thr Arg
Thr Asn Gln Leu Met Pro 165 170
175Leu Ile Tyr Val Lys Leu Tyr Thr Tyr Gln Ile Cys Arg Ala Leu Ala
180 185 190Tyr Ile His Asn Cys
Phe Gly Leu Cys His Arg Asp Ile Lys Pro Gln 195
200 205Asn Leu Leu Val Asn Pro His Thr His Gln Leu Lys
Ile Cys Asp Phe 210 215 220Gly Ser Ala
Lys Val Leu Val Lys Gly Glu Pro Asn Val Ser Tyr Ile225
230 235 240Cys Ser Arg Tyr Tyr Arg Ala
Pro Glu Leu Ile Phe Gly Ala Ser Glu 245
250 255Tyr Thr Pro Ala Ile Asp Ile Trp Ser Thr Gly Cys
Val Met Ala Glu 260 265 270Leu
Leu Leu Gly Gln Pro Leu Phe Pro Gly Glu Ser Gly Val Asp Gln 275
280 285Leu Val Glu Ile Ile Lys Val Leu Gly
Thr Pro Thr Arg Glu Glu Ile 290 295
300Lys Cys Met Asn Pro Asn Tyr Thr Glu Phe Lys Phe Pro Gln Ile Lys305
310 315 320Pro His Pro Trp
His Lys Val Phe Gln Lys Arg Leu Pro Pro Glu Ala 325
330 335Val Asp Leu Leu Cys Arg Phe Phe Gln Tyr
Ser Pro Asn Leu Arg Cys 340 345
350Thr Ala Leu Glu Ala Cys Ile His Pro Leu Phe Asp Glu Leu Arg Asp
355 360 365Pro Asn Thr Arg Leu Pro Asn
Gly Arg Pro Leu Pro Pro Leu Phe Asn 370 375
380Phe Lys Pro Gln Glu Leu Ser Gly Ile Pro Ser Glu Ile Val Asn
Arg385 390 395 400Leu Val
Pro Glu His Ala Arg Lys Gln Asn Phe Phe Met Ala Leu Asp
405 410 415Ala151621DNABrassica napus
15ttttctctct ctctctctct ctccacattt gatgatcatt accaaccaaa ctaattgaaa
60tccatttgtt ctctctctct ctctctctct ctcacactct cttctctgct cttctctgcg
120cctctaacgt catggctgac gatagggaga tgccgccggc tgctgtagtt gatggacatg
180accaagtcac tggccacata atctccacca ccatcggtgg taaaaacgga gaaccaaaac
240agacaataag ttacatggcg gagcgagttg tcggtacagg ctccttcggg atagtgttcc
300aggcgaagtg tctggagact ggagaaaccg tggcgataaa gaaggttttg caagacagga
360ggtacaagaa ccgagagctt cagctgatgc gtgtgatgga ccatccgaat gttgtttgtt
420tgaagcattg cttcttctcg accacgagca aagacgagct gtttctgaac ttggttatgg
480agtatgtccc tgagagcttg taccgagttc tgaaacatta cagcactgct aaccagagga
540tgccgcttgt ttatgttaaa ctctatatgt accagatctt cagaggactt gcttacattc
600acaatgttgc tggagtttgt cacagagatc taaagcctca aaatcttctg gttgatcctc
660tgactcatca agtgaagatc tgtgattttg gcagtgcgaa acagcttgtt aaaggtgaag
720ccaacatctc ttacatatgt tcaagattct accgtgcacc tgaacttata ttcggtgcca
780ctgagtacac aacttccatt gatatttggt ctgctggttg tgttctcgct gagcttcttc
840ttggtcagcc actattccct ggagaaaatg ctgtgggtca gctcgttgaa atcatcaaag
900ttcttggtac accaactcga gaagagatcc gttgtatgaa tccacactac acagacttta
960ggttcccgca gataaaggca catccttggc acaagatttt ccacaaaagg atgcctccag
1020aagccattga ttttgcatca aggctgcttc agtactctcc aagtcttaga tgcacagcgc
1080ttgaagcttg tgcacatccg ttctttgatg agcttagaga accaaatgct cgtttaccaa
1140acggacggcc tttcccgccg ctcttcaact tcaaacaaga ggtagctgga gcttcacctg
1200agctggtcaa caagttgatt ccagaccata tcaagacgca gttgggtcta agcttcttga
1260atcagtctgg aacttaaaca aacgatcaaa aagacaagaa cttttttata tataattgta
1320ccattactca gaaccagaag aaggttagtt gaaggcacgt ggaggacaca gttagaggtt
1380ttgcctcctc aaaactcgtt ccaggaatga aggtcaaaaa agacaagctt ctctacaacc
1440tgacttcccc caagcctgca agaaaagcta ctcagttgta tcttcttctt cttcttttgt
1500ccttttttaa aaatgtttgg ttaaagcaaa gaacaaaatc ttctcttttt gctttattct
1560tactgcatct gtaaatgagt ttagtcagag atttttatat agtaaaaaaa aaaaaaaaaa
1620a
162116381PRTBrassica napus 16Met Ala Asp Asp Arg Glu Met Pro Pro Ala Ala
Val Val Asp Gly His1 5 10
15Asp Gln Val Thr Gly His Ile Ile Ser Thr Thr Ile Gly Gly Lys Asn
20 25 30Gly Glu Pro Lys Gln Thr Ile
Ser Tyr Met Ala Glu Arg Val Val Gly 35 40
45Thr Gly Ser Phe Gly Ile Val Phe Gln Ala Lys Cys Leu Glu Thr
Gly 50 55 60Glu Thr Val Ala Ile Lys
Lys Val Leu Gln Asp Arg Arg Tyr Lys Asn65 70
75 80Arg Glu Leu Gln Leu Met Arg Val Met Asp His
Pro Asn Val Val Cys 85 90
95Leu Lys His Cys Phe Phe Ser Thr Thr Ser Lys Asp Glu Leu Phe Leu
100 105 110Asn Leu Val Met Glu Tyr
Val Pro Glu Ser Leu Tyr Arg Val Leu Lys 115 120
125His Tyr Ser Thr Ala Asn Gln Arg Met Pro Leu Val Tyr Val
Lys Leu 130 135 140Tyr Met Tyr Gln Ile
Phe Arg Gly Leu Ala Tyr Ile His Asn Val Ala145 150
155 160Gly Val Cys His Arg Asp Leu Lys Pro Gln
Asn Leu Leu Val Asp Pro 165 170
175Leu Thr His Gln Val Lys Ile Cys Asp Phe Gly Ser Ala Lys Gln Leu
180 185 190Val Lys Gly Glu Ala
Asn Ile Ser Tyr Ile Cys Ser Arg Phe Tyr Arg 195
200 205Ala Pro Glu Leu Ile Phe Gly Ala Thr Glu Tyr Thr
Thr Ser Ile Asp 210 215 220Ile Trp Ser
Ala Gly Cys Val Leu Ala Glu Leu Leu Leu Gly Gln Pro225
230 235 240Leu Phe Pro Gly Glu Asn Ala
Val Gly Gln Leu Val Glu Ile Ile Lys 245
250 255Val Leu Gly Thr Pro Thr Arg Glu Glu Ile Arg Cys
Met Asn Pro His 260 265 270Tyr
Thr Asp Phe Arg Phe Pro Gln Ile Lys Ala His Pro Trp His Lys 275
280 285Ile Phe His Lys Arg Met Pro Pro Glu
Ala Ile Asp Phe Ala Ser Arg 290 295
300Leu Leu Gln Tyr Ser Pro Ser Leu Arg Cys Thr Ala Leu Glu Ala Cys305
310 315 320Ala His Pro Phe
Phe Asp Glu Leu Arg Glu Pro Asn Ala Arg Leu Pro 325
330 335Asn Gly Arg Pro Phe Pro Pro Leu Phe Asn
Phe Lys Gln Glu Val Ala 340 345
350Gly Ala Ser Pro Glu Leu Val Asn Lys Leu Ile Pro Asp His Ile Lys
355 360 365Thr Gln Leu Gly Leu Ser Phe
Leu Asn Gln Ser Gly Thr 370 375
380171654DNABrassica napus 17cgtcgtcgtc tctctctctt tctttctctt ctccgtgaat
catcatcatc atcatcatct 60tcgtgttttc tcgttaagcc cattttgttt tttttttttc
tctggggaaa aactcggctc 120aaaacgatga atgtgatgcg tagattgacg agtatcgctt
ctggacgcgg tttcgtctct 180tctgataacg taggagagac cgagacgccg agatcgaagc
ctaaccaaat ttgtgaagag 240atagaagaga ctacacgaga agactctgtt tctaaaacag
aggattctga ttcattacca 300aaagagatgg gaatcggtga tgacgacaag gataaggacg
gtgggattat caagggtaat 360gggacagagt ctggtcggat cattaccacc acaaagaagg
gtctgaacga tcaaagagac 420aagacaatct cgtacagagc tgaacatgtg attggcactg
gctcattcgg tgttgtcttt 480caggctaagt gcttagagac agaagaaaaa gtagctatca
agaaagtgtt gcaagacaag 540agatacaaga acagagagct tcagatcatg cggatgcttg
atcatcctaa tgttgttgac 600ctcaagcatt ctttcttctc caccactgag aaagatgagc
tttatcttaa ccttgttctt 660gagtatgtac ctgagactat ataccgttct tcaagatctt
acaccaagat gaatcaacac 720atgcccttga tctatattca gctctataca tatcagattt
gccgcgcaat gaactatcta 780catagagttg ttggagtgtg tcaccgtgac attaaacctc
agaatctatt ggtcaataat 840gttacacatg aggtgaaggt atgcgatttt gggagcgcca
agatgctgat tccgggagaa 900cccaatatat cttacatatg ctcaaggtat tacagagctc
ctgaactcat atttggggta 960actgagtaca caaccgccat cgatatgtgg tctgttggct
gtgtcatggc tgaacttttt 1020cttggacatc ctctgttccc tggagagact agtgttgatc
aattggttga gatcattaag 1080attttgggaa caccagcaag agaagagatc agaaacatga
atcctcgtta caatgatttt 1140aagttccctc agatcaaagc tcagccatgg cacaagattt
tccggagaca ggtatctcca 1200gaagcaatgg atcttgcctc tagactcctc cagtactcac
caaacctgag atgttcagcg 1260cttgaagcat gtgcacaccc cttcttcgat gatctgagag
acccgagagc atccttgcct 1320aatggaagag cacttcctcc actgtttgat ttcacagctc
aagaactggc tggtgcatct 1380gttgaattgc gtcatcgctt aatccctgaa catgcaagga
aataacttac tttgtctaac 1440gagaccgctt cttctctaca cagatgttga tatctaaatt
cctttttttt tggcattgtt 1500ctggttatga acaccctcat tgacctctgc aaccaccttg
cactagcagt tccaaaagtg 1560tatgatttgt taagtttgta actttgtaga ctccattgtt
gcagacagaa aatgcagaat 1620tttccgagtt tgtctcaaaa aaaaaaaaaa aaaa
165418432PRTBrassica napus 18Met Asn Val Met Arg
Arg Leu Thr Ser Ile Ala Ser Gly Arg Gly Phe1 5
10 15Val Ser Ser Asp Asn Val Gly Glu Thr Glu Thr
Pro Arg Ser Lys Pro 20 25
30Asn Gln Ile Cys Glu Glu Ile Glu Glu Thr Thr Arg Glu Asp Ser Val
35 40 45Ser Lys Thr Glu Asp Ser Asp Ser
Leu Pro Lys Glu Met Gly Ile Gly 50 55
60Asp Asp Asp Lys Asp Lys Asp Gly Gly Ile Ile Lys Gly Asn Gly Thr65
70 75 80Glu Ser Gly Arg Ile
Ile Thr Thr Thr Lys Lys Gly Leu Asn Asp Gln 85
90 95Arg Asp Lys Thr Ile Ser Tyr Arg Ala Glu His
Val Ile Gly Thr Gly 100 105
110Ser Phe Gly Val Val Phe Gln Ala Lys Cys Leu Glu Thr Glu Glu Lys
115 120 125Val Ala Ile Lys Lys Val Leu
Gln Asp Lys Arg Tyr Lys Asn Arg Glu 130 135
140Leu Gln Ile Met Arg Met Leu Asp His Pro Asn Val Val Asp Leu
Lys145 150 155 160His Ser
Phe Phe Ser Thr Thr Glu Lys Asp Glu Leu Tyr Leu Asn Leu
165 170 175Val Leu Glu Tyr Val Pro Glu
Thr Ile Tyr Arg Ser Ser Arg Ser Tyr 180 185
190Thr Lys Met Asn Gln His Met Pro Leu Ile Tyr Ile Gln Leu
Tyr Thr 195 200 205Tyr Gln Ile Cys
Arg Ala Met Asn Tyr Leu His Arg Val Val Gly Val 210
215 220Cys His Arg Asp Ile Lys Pro Gln Asn Leu Leu Val
Asn Asn Val Thr225 230 235
240His Glu Val Lys Val Cys Asp Phe Gly Ser Ala Lys Met Leu Ile Pro
245 250 255Gly Glu Pro Asn Ile
Ser Tyr Ile Cys Ser Arg Tyr Tyr Arg Ala Pro 260
265 270Glu Leu Ile Phe Gly Val Thr Glu Tyr Thr Thr Ala
Ile Asp Met Trp 275 280 285Ser Val
Gly Cys Val Met Ala Glu Leu Phe Leu Gly His Pro Leu Phe 290
295 300Pro Gly Glu Thr Ser Val Asp Gln Leu Val Glu
Ile Ile Lys Ile Leu305 310 315
320Gly Thr Pro Ala Arg Glu Glu Ile Arg Asn Met Asn Pro Arg Tyr Asn
325 330 335Asp Phe Lys Phe
Pro Gln Ile Lys Ala Gln Pro Trp His Lys Ile Phe 340
345 350Arg Arg Gln Val Ser Pro Glu Ala Met Asp Leu
Ala Ser Arg Leu Leu 355 360 365Gln
Tyr Ser Pro Asn Leu Arg Cys Ser Ala Leu Glu Ala Cys Ala His 370
375 380Pro Phe Phe Asp Asp Leu Arg Asp Pro Arg
Ala Ser Leu Pro Asn Gly385 390 395
400Arg Ala Leu Pro Pro Leu Phe Asp Phe Thr Ala Gln Glu Leu Ala
Gly 405 410 415Ala Ser Val
Glu Leu Arg His Arg Leu Ile Pro Glu His Ala Arg Lys 420
425 430192400DNABrassica napus 19gttttggcat
ctggagaggg agagagagag agagaaaggg gaataagatg atggagaatc 60gagtggtggt
ggtggctgct ctgtttgcgg tctgcattgt aggatttgag tttagcttca 120tccatggagc
cactgatgca tcagacactt cagcattgaa catgttgttc accagtatgc 180attcaccagg
acagttaaca caatggactg catcaggtgg ggatccttgt gttcagaact 240ggagaggcgt
tacttgctcc aaatcacgaa ttactcaatt aaagttatca ggtcttgagc 300tctctggaac
acttgggtac atgcttgata aattgacttc tcttacagag cttgatctaa 360gcagcaataa
tcttggaggt gatttaccat atcagcttcc tccaaatctg caacggttga 420atcttgcaaa
caaccaattc actggagctg ctcaatactc catttctaat atggcatcac 480ttaagtatct
taatcttggt cacaaccagt ttaaggggca agtagctgtg gacttctcca 540agctcacctc
tcttacaacc ttggacttct ctttcaactc tttcacatcg tctctaccgg 600gaacttttac
ttctcttaca agtttaaagt ccctatacct tcagaacaat cagttctcag 660gaacactcaa
tgtattagcc ggtcttcctc ttgagaccct gaacattgca aacaatgact 720tcaccggctg
gatccccagt accttaaagg gtactaattt aataaaagat ggtaactcgt 780tcaataatgg
acctgcacca ccaccaccac ctggtacacc tccaatccac cgctcaccga 840gccataaatc
cggaggaggt tcaaaccgtg attctaccag caatggagat tccaagaaat 900caggaattgg
agctggtgct atagcaggta taatcatttc attactagta gttacagctc 960ttgtggcttt
cttcttagtc aaaagaagaa gaagatcaaa gagatcatca tctatggaca 1020ttgagaaaac
tgacaaccag cctttcactc ttcctccaag cgactttcac gaaaacaatt 1080ctattcagag
ttcttcatca attgagacaa agaaacttga tacttccttg tctattaatc 1140tccgtcctcc
accagctgat cgatcatttg atgatgatga ggattctacg agaaagccta 1200tagttgtcaa
gaaatccacc gtggctgttc cctcgaatgt gagagtttac tcagttgctg 1260atcttcagat
tgccactgcc agtttcagtg ttgataatct tcttggagaa ggcacttttg 1320gaagagtata
cagagctgag tttaacaatg gaaaggttct tgctgtgaag aagattgatt 1380catctgctct
tccacatagc atgactgatg atttcaccga aatagtatcg aaaatagccg 1440ttttggatca
tccaaatgtg acaaagcttg ttggctactg tgctgaacac ggacaacatc 1500tcctggtcta
tgagttccac agcaaaggat cgttacatga cttcctacac ttatcagaag 1560aagaaagcaa
agcattggtg tggaactcgc gagtcaaggt cgcacttggg actgcacggg 1620caatagagta
cttgcatgaa gtttgttcac cgtctatagt tgacaagaac atcaaatcag 1680ccaatatttt
gcttgattcg gagatgaatc ctcacttatc agacacaggt ctcgcaagct 1740tcctccccac
agcaaatgag ttactaaacc aaaccgatga aggttatagc gcaccggaag 1800tatcaatgtc
aggtcaatac tctttgaaga gtgatgttta cagttttgga gtagtgatgc 1860ttgaactttt
aaccgggagg aaaccattcg acagcacaag gtcaagatct gagcagtcat 1920tggttagatg
ggcgacacca cagcttcatg acattgatgc tttaggcaaa atggttgatc 1980cagctcttga
aggactttat ccggttaaat ctctttctcg gtttgcagat gttattgctc 2040tctgcgtcca
gccagagcca gagtttagac caccaatgtc tgaagttgtg cagtcactgg 2100ttgtgttagt
gcagagagct aacatgagca agagaactgt tggagttgat ccatcacagc 2160gttctggtag
tgctgagcca agcaacgatt acatgtaaac ccattaccac agagagagaa 2220aaaaagaaca
ctttgctccc tatgggatga agtcattgtt tttattgtaa tatgtttgat 2280aaaccttcac
acagtatatt atccccattg tattttgttg taatgtgttt ccaaatttgt 2340agcttttaga
tcattgaaat gaacaaatat tctttcttgt gtaaaaaaaa aaaaaaaaaa
240020716PRTBrassica napus 20Met Met Glu Asn Arg Val Val Val Val Ala Ala
Leu Phe Ala Val Cys1 5 10
15Ile Val Gly Phe Glu Phe Ser Phe Ile His Gly Ala Thr Asp Ala Ser
20 25 30Asp Thr Ser Ala Leu Asn Met
Leu Phe Thr Ser Met His Ser Pro Gly 35 40
45Gln Leu Thr Gln Trp Thr Ala Ser Gly Gly Asp Pro Cys Val Gln
Asn 50 55 60Trp Arg Gly Val Thr Cys
Ser Lys Ser Arg Ile Thr Gln Leu Lys Leu65 70
75 80Ser Gly Leu Glu Leu Ser Gly Thr Leu Gly Tyr
Met Leu Asp Lys Leu 85 90
95Thr Ser Leu Thr Glu Leu Asp Leu Ser Ser Asn Asn Leu Gly Gly Asp
100 105 110Leu Pro Tyr Gln Leu Pro
Pro Asn Leu Gln Arg Leu Asn Leu Ala Asn 115 120
125Asn Gln Phe Thr Gly Ala Ala Gln Tyr Ser Ile Ser Asn Met
Ala Ser 130 135 140Leu Lys Tyr Leu Asn
Leu Gly His Asn Gln Phe Lys Gly Gln Val Ala145 150
155 160Val Asp Phe Ser Lys Leu Thr Ser Leu Thr
Thr Leu Asp Phe Ser Phe 165 170
175Asn Ser Phe Thr Ser Ser Leu Pro Gly Thr Phe Thr Ser Leu Thr Ser
180 185 190Leu Lys Ser Leu Tyr
Leu Gln Asn Asn Gln Phe Ser Gly Thr Leu Asn 195
200 205Val Leu Ala Gly Leu Pro Leu Glu Thr Leu Asn Ile
Ala Asn Asn Asp 210 215 220Phe Thr Gly
Trp Ile Pro Ser Thr Leu Lys Gly Thr Asn Leu Ile Lys225
230 235 240Asp Gly Asn Ser Phe Asn Asn
Gly Pro Ala Pro Pro Pro Pro Pro Gly 245
250 255Thr Pro Pro Ile His Arg Ser Pro Ser His Lys Ser
Gly Gly Gly Ser 260 265 270Asn
Arg Asp Ser Thr Ser Asn Gly Asp Ser Lys Lys Ser Gly Ile Gly 275
280 285Ala Gly Ala Ile Ala Gly Ile Ile Ile
Ser Leu Leu Val Val Thr Ala 290 295
300Leu Val Ala Phe Phe Leu Val Lys Arg Arg Arg Arg Ser Lys Arg Ser305
310 315 320Ser Ser Met Asp
Ile Glu Lys Thr Asp Asn Gln Pro Phe Thr Leu Pro 325
330 335Pro Ser Asp Phe His Glu Asn Asn Ser Ile
Gln Ser Ser Ser Ser Ile 340 345
350Glu Thr Lys Lys Leu Asp Thr Ser Leu Ser Ile Asn Leu Arg Pro Pro
355 360 365Pro Ala Asp Arg Ser Phe Asp
Asp Asp Glu Asp Ser Thr Arg Lys Pro 370 375
380Ile Val Val Lys Lys Ser Thr Val Ala Val Pro Ser Asn Val Arg
Val385 390 395 400Tyr Ser
Val Ala Asp Leu Gln Ile Ala Thr Ala Ser Phe Ser Val Asp
405 410 415Asn Leu Leu Gly Glu Gly Thr
Phe Gly Arg Val Tyr Arg Ala Glu Phe 420 425
430Asn Asn Gly Lys Val Leu Ala Val Lys Lys Ile Asp Ser Ser
Ala Leu 435 440 445Pro His Ser Met
Thr Asp Asp Phe Thr Glu Ile Val Ser Lys Ile Ala 450
455 460Val Leu Asp His Pro Asn Val Thr Lys Leu Val Gly
Tyr Cys Ala Glu465 470 475
480His Gly Gln His Leu Leu Val Tyr Glu Phe His Ser Lys Gly Ser Leu
485 490 495His Asp Phe Leu His
Leu Ser Glu Glu Glu Ser Lys Ala Leu Val Trp 500
505 510Asn Ser Arg Val Lys Val Ala Leu Gly Thr Ala Arg
Ala Ile Glu Tyr 515 520 525Leu His
Glu Val Cys Ser Pro Ser Ile Val Asp Lys Asn Ile Lys Ser 530
535 540Ala Asn Ile Leu Leu Asp Ser Glu Met Asn Pro
His Leu Ser Asp Thr545 550 555
560Gly Leu Ala Ser Phe Leu Pro Thr Ala Asn Glu Leu Leu Asn Gln Thr
565 570 575Asp Glu Gly Tyr
Ser Ala Pro Glu Val Ser Met Ser Gly Gln Tyr Ser 580
585 590Leu Lys Ser Asp Val Tyr Ser Phe Gly Val Val
Met Leu Glu Leu Leu 595 600 605Thr
Gly Arg Lys Pro Phe Asp Ser Thr Arg Ser Arg Ser Glu Gln Ser 610
615 620Leu Val Arg Trp Ala Thr Pro Gln Leu His
Asp Ile Asp Ala Leu Gly625 630 635
640Lys Met Val Asp Pro Ala Leu Glu Gly Leu Tyr Pro Val Lys Ser
Leu 645 650 655Ser Arg Phe
Ala Asp Val Ile Ala Leu Cys Val Gln Pro Glu Pro Glu 660
665 670Phe Arg Pro Pro Met Ser Glu Val Val Gln
Ser Leu Val Val Leu Val 675 680
685Gln Arg Ala Asn Met Ser Lys Arg Thr Val Gly Val Asp Pro Ser Gln 690
695 700Arg Ser Gly Ser Ala Glu Pro Ser
Asn Asp Tyr Met705 710
715211499DNAGlycine max 21tttagagaga gaaagagtgt gagtgttgtg ttgagtgcag
tttctttctc acatggcctc 60tatgccgttg gggccgcagc aacagcttcc accgccgccg
ccgcaacaac cgccgccagc 120ggagaatgac gcgatgaaag tggactctcg cggcggctcc
gacgccggca ccgaaaagga 180aatgtcagct cctgtcgcag atggtaatga tgcactcact
ggtcacataa tctcaaccac 240aattgcaggc aaaaatggcg aacctaaaca aaccatcagt
tacatggccg aacgtgttgt 300tggcactgga tcatttggca ttgttttcca ggcgaagtgc
ttggagactg gcgaggcagt 360ggctataaag aaggtcttgc aggacaggcg atacaaaaat
cgtgaactgc agttaatgcg 420cgtgatggat cacccaaata taatttcctt gagtaactat
ttcttctcta caacaagtag 480agatgaactt tttctgaact tggtgatgga atatgtccct
gagacgatct tccgtgttat 540aaagcactac agtagcatga aacagagaat gcccctaatc
tatgtgaaat tatatacata 600tcaaatcttt aggggactgg cgtatatcca tactgtacca
ggaatctgcc atagggattt 660gaagcctcaa aatcttttgg ttgatcgact cacacaccaa
gtcaagctct gtgattttgg 720gagtgcaaaa gttctggtgg agggtgaatc aaacatttca
tacatatgtt cacggtacta 780tcgtgcccca gagctaatat ttggtgcggc agaatacaca
acttctgttg atatttggtc 840cgctggttgt gtccttgcgg aacttcttct aggccagcct
ttgttcccag gagaaaatca 900ggttgaccaa ctcgtggaaa ttatcaagat tcttggcact
cctactcgag aagaaattcg 960atgcatgaat cctaattata cagatttcag attcccccat
atcaaagctc atccttggca 1020taaggttttt cacaagcgaa tgcctcctga agcaattgac
cttgcatcaa ggcttctcca 1080atattcccca aaacttcgtt acagtgcagt ggaagcaatg
gcacatcctt tctttgacga 1140gcttcgcgag cccaatgccc ggctacctaa tggtcgtcca
ctgcctccac ttttcaactt 1200taaacaggaa ttagatggag cgccccctga actgcttcct
aagctcatcc cagagcatgt 1260caggcggcaa acccaaatgt aaagagatag taaaacatag
agtgaactgt tctagtggat 1320tagtgtgaaa tacatgagag cttgcttgtg gtcaatagaa
caggggttag gcccaaatat 1380gcagtttttc tcccccttgt gaagatgtat acatgtgctg
gaaaactcag tgtaacccgg 1440aaatgtagat tatgtctaat gtctaatatt tcattctagt
taaaaaaaaa aaaaaaaaa 149922409PRTGlycine max 22Met Ala Ser Met Pro Leu
Gly Pro Gln Gln Gln Leu Pro Pro Pro Pro1 5
10 15Pro Gln Gln Pro Pro Pro Ala Glu Asn Asp Ala Met
Lys Val Asp Ser 20 25 30Arg
Gly Gly Ser Asp Ala Gly Thr Glu Lys Glu Met Ser Ala Pro Val 35
40 45Ala Asp Gly Asn Asp Ala Leu Thr Gly
His Ile Ile Ser Thr Thr Ile 50 55
60Ala Gly Lys Asn Gly Glu Pro Lys Gln Thr Ile Ser Tyr Met Ala Glu65
70 75 80Arg Val Val Gly Thr
Gly Ser Phe Gly Ile Val Phe Gln Ala Lys Cys 85
90 95Leu Glu Thr Gly Glu Ala Val Ala Ile Lys Lys
Val Leu Gln Asp Arg 100 105
110Arg Tyr Lys Asn Arg Glu Leu Gln Leu Met Arg Val Met Asp His Pro
115 120 125Asn Ile Ile Ser Leu Ser Asn
Tyr Phe Phe Ser Thr Thr Ser Arg Asp 130 135
140Glu Leu Phe Leu Asn Leu Val Met Glu Tyr Val Pro Glu Thr Ile
Phe145 150 155 160Arg Val
Ile Lys His Tyr Ser Ser Met Lys Gln Arg Met Pro Leu Ile
165 170 175Tyr Val Lys Leu Tyr Thr Tyr
Gln Ile Phe Arg Gly Leu Ala Tyr Ile 180 185
190His Thr Val Pro Gly Ile Cys His Arg Asp Leu Lys Pro Gln
Asn Leu 195 200 205Leu Val Asp Arg
Leu Thr His Gln Val Lys Leu Cys Asp Phe Gly Ser 210
215 220Ala Lys Val Leu Val Glu Gly Glu Ser Asn Ile Ser
Tyr Ile Cys Ser225 230 235
240Arg Tyr Tyr Arg Ala Pro Glu Leu Ile Phe Gly Ala Ala Glu Tyr Thr
245 250 255Thr Ser Val Asp Ile
Trp Ser Ala Gly Cys Val Leu Ala Glu Leu Leu 260
265 270Leu Gly Gln Pro Leu Phe Pro Gly Glu Asn Gln Val
Asp Gln Leu Val 275 280 285Glu Ile
Ile Lys Ile Leu Gly Thr Pro Thr Arg Glu Glu Ile Arg Cys 290
295 300Met Asn Pro Asn Tyr Thr Asp Phe Arg Phe Pro
His Ile Lys Ala His305 310 315
320Pro Trp His Lys Val Phe His Lys Arg Met Pro Pro Glu Ala Ile Asp
325 330 335Leu Ala Ser Arg
Leu Leu Gln Tyr Ser Pro Lys Leu Arg Tyr Ser Ala 340
345 350Val Glu Ala Met Ala His Pro Phe Phe Asp Glu
Leu Arg Glu Pro Asn 355 360 365Ala
Arg Leu Pro Asn Gly Arg Pro Leu Pro Pro Leu Phe Asn Phe Lys 370
375 380Gln Glu Leu Asp Gly Ala Pro Pro Glu Leu
Leu Pro Lys Leu Ile Pro385 390 395
400Glu His Val Arg Arg Gln Thr Gln Met
405231523DNAGlycine max 23agacaccaca aagtgtaact tgagtgatta tatctgatga
gtgcagaaag aagggaggat 60tgttggtgat cgatcatcga tcatcgatca tcgatcatcg
atggcgtctg ctagccttgg 120aagtggtggg gtgggcagtt ccaggtctgt taatggtggc
ttcaggggtt cttccagttc 180cgtcgattgg cttggcagag agatgcttga gatgtctttg
agagaccacg aggacgatag 240agatagtgag cctgacatca ttgatggttt gggtgctgag
actggtcacg tgataagaac 300cagcgttggt ggccgaaatg gtcaatctaa gcagaatgtt
agttatattt ctgagcatgt 360tgtgggaaca ggctcttttg gtgttgtttt tcaagccaaa
tgtagagaaa cgggagaaat 420tgtggccatc aagaaagttc tccaggacaa gcgctacaag
aatagagagt tacaaattat 480gcaaatgctg gatcatccaa atattgttgc ccttaggcat
tgtttctatt caacgactga 540caaagaagaa gtttacttga atcttgtact tgaatatgtt
cctgaaactg tgaatcgcat 600cgccaggagc tatagcagga ttaaccagcg aatgccttta
atatatgtaa agctttatac 660ctaccagatt tgcagggccc ttgcttatat acataactgc
attggtatat gtcatcgtga 720catcaaacct cagaacctac ttgtgaaccc gcacactcat
cagctgaaac tatgtgattt 780tgggagtgca aaagtgttgg tgaaaggaga acctaatgtt
tcttacatct gttcaagata 840ctaccgtgct ccggaactta tatttggggc cactgaatat
acaactgcca tagatatatg 900gtcaactggt tgtgtaatgg ctgaattact tcttggacag
cccttgtttc ctggagagag 960tggagttgat cagctagttg aaatcatcaa ggttttggga
actccaacca gggaggagat 1020aaagtgcatg aacccaaatt atactgaatt taagtttcca
cagataaaac ctcatccatg 1080gcacaaggtt tttcagaaac gtttaccccc agaagcagtg
gaccttgtct gtaggttctt 1140tcagtactct cccaatttga gatgcactgc attggaagct
tgcattcatc cattttttga 1200tgaattgagg gacccaaaca cccgccttcc taatggtcga
ccacttcctc cactgtttaa 1260ttttaaacct caggaacttt ctggtgtacc ccctgatgtc
atcaatcggc ttattccaga 1320gcatgcgcgt aaacagaact tatttatggc tttgcacacc
tagcaattcc cgtaccctcc 1380taagttgtcg tcacttacta gcaggttgta aattatccgg
tttatccgag aaaaactcca 1440cagaaagagt tactaggatt atattattat tatataatat
gaaaagtttc ttttttcttt 1500tttggaaaaa aaaaaaaaaa aaa
152324420PRTGlycine max 24Met Ala Ser Ala Ser Leu
Gly Ser Gly Gly Val Gly Ser Ser Arg Ser1 5
10 15Val Asn Gly Gly Phe Arg Gly Ser Ser Ser Ser Val
Asp Trp Leu Gly 20 25 30Arg
Glu Met Leu Glu Met Ser Leu Arg Asp His Glu Asp Asp Arg Asp 35
40 45Ser Glu Pro Asp Ile Ile Asp Gly Leu
Gly Ala Glu Thr Gly His Val 50 55
60Ile Arg Thr Ser Val Gly Gly Arg Asn Gly Gln Ser Lys Gln Asn Val65
70 75 80Ser Tyr Ile Ser Glu
His Val Val Gly Thr Gly Ser Phe Gly Val Val 85
90 95Phe Gln Ala Lys Cys Arg Glu Thr Gly Glu Ile
Val Ala Ile Lys Lys 100 105
110Val Leu Gln Asp Lys Arg Tyr Lys Asn Arg Glu Leu Gln Ile Met Gln
115 120 125Met Leu Asp His Pro Asn Ile
Val Ala Leu Arg His Cys Phe Tyr Ser 130 135
140Thr Thr Asp Lys Glu Glu Val Tyr Leu Asn Leu Val Leu Glu Tyr
Val145 150 155 160Pro Glu
Thr Val Asn Arg Ile Ala Arg Ser Tyr Ser Arg Ile Asn Gln
165 170 175Arg Met Pro Leu Ile Tyr Val
Lys Leu Tyr Thr Tyr Gln Ile Cys Arg 180 185
190Ala Leu Ala Tyr Ile His Asn Cys Ile Gly Ile Cys His Arg
Asp Ile 195 200 205Lys Pro Gln Asn
Leu Leu Val Asn Pro His Thr His Gln Leu Lys Leu 210
215 220Cys Asp Phe Gly Ser Ala Lys Val Leu Val Lys Gly
Glu Pro Asn Val225 230 235
240Ser Tyr Ile Cys Ser Arg Tyr Tyr Arg Ala Pro Glu Leu Ile Phe Gly
245 250 255Ala Thr Glu Tyr Thr
Thr Ala Ile Asp Ile Trp Ser Thr Gly Cys Val 260
265 270Met Ala Glu Leu Leu Leu Gly Gln Pro Leu Phe Pro
Gly Glu Ser Gly 275 280 285Val Asp
Gln Leu Val Glu Ile Ile Lys Val Leu Gly Thr Pro Thr Arg 290
295 300Glu Glu Ile Lys Cys Met Asn Pro Asn Tyr Thr
Glu Phe Lys Phe Pro305 310 315
320Gln Ile Lys Pro His Pro Trp His Lys Val Phe Gln Lys Arg Leu Pro
325 330 335Pro Glu Ala Val
Asp Leu Val Cys Arg Phe Phe Gln Tyr Ser Pro Asn 340
345 350Leu Arg Cys Thr Ala Leu Glu Ala Cys Ile His
Pro Phe Phe Asp Glu 355 360 365Leu
Arg Asp Pro Asn Thr Arg Leu Pro Asn Gly Arg Pro Leu Pro Pro 370
375 380Leu Phe Asn Phe Lys Pro Gln Glu Leu Ser
Gly Val Pro Pro Asp Val385 390 395
400Ile Asn Arg Leu Ile Pro Glu His Ala Arg Lys Gln Asn Leu Phe
Met 405 410 415Ala Leu His
Thr 420251744DNAGlycine max 25agagagagaa acgaagaaga agagtgtttc
tcacatcaca tggcctcctt gcccttgggg 60caccaccacc accaccacaa accggcggcg
gcggctatac atccgtcgca accgccgcag 120tctcagccgc aacccgaagt tcctcgccgg
agctccgatg tggagaccga taaggatatg 180tcagctactg tcattgaggg gaatgatgct
gtcactggcc acataatctc caccacaatt 240ggaggcaaaa atggggaacc taaagagacc
atcagttaca tggcagaacg tgttgttggc 300actggatcat ttggagttgt ttttcaggca
aagtgcttgg agactggaga agcagtggct 360attaaaaagg tcttgcaaga caggcggtac
aaaaatcgtg aattgcagtt aatgcgctta 420atggatcacc ctaatgtaat ttccctgaag
cactgtttct tctccacaac aagcagagat 480gaactttttc taaacttggt aatggaatat
gttcccgaat caatgtaccg agttataaag 540cactacacta ctatgaacca gagaatgcct
ctcatctatg tgaaactgta tacatatcaa 600atctttaggg gattagcata tatccatacc
gcactgggag tttgccatag ggatgtgaag 660cctcaaaatc ttttggttca tcctcttact
caccaagtta agctatgtga ttttgggagt 720gccaaagttc tggtcaaggg tgaatcaaac
atttcataca tatgttcacg ttactatcgg 780gctccagaac taatatttgg tgcaacagaa
tacacagctt ctattgatat ctggtcagct 840ggttgtgttc ttgctgaact tcttctagga
cagccattat ttcctggaga aaaccaagtg 900gaccaacttg tggaaattat caaggttctt
ggtactccaa cacgcgagga aatccgttgt 960atgaacccaa attatacaga gtttagattc
cctcagatta aagctcatcc ttggcacaag 1020gttttccaca agcgaatgcc tcctgaagca
attgaccttg catcaaggct tctccaatat 1080tcacctagtc tccgctgcac tgcgctggaa
gcatgtgcac atcctttctt tgatgagctt 1140cgcgaaccaa atgcccggct acctaatggc
cgtccactgc ccccactttt caacttcaaa 1200caggagttag ctggagcatc acctgaactg
atcaataggc tcatcccaga gcatattagg 1260cggcagatgg gtctcagctt cccgcattct
gccggtacat agatgtaaag ggataatgaa 1320acgatgagtc aacctacata gtgatcgatg
tgaatcaaca gaagggctgt ttgaggccta 1380tgtataactg ggagtcccaa cataatatgc
agtttttcct cccccttgtg aagatgtata 1440catgtgttgg ttgctcggta aagcttgaaa
gttggtgatt ctgtgtagta tttcattcaa 1500gttaaagcat acttatccct gcatctgtat
attgttttgg tcagatttca gaaagctagg 1560agtataaaat gatagcaatc atgtcttcat
aggtagaggg gcccagctga attgaggggc 1620ccctatagta gtttggcttg ctttttatga
gattaaattc aggatgtcgt ttatattatg 1680tttataacaa tctcttgatt caaaacaaga
aattttctcg ttgttgaaaa aaaaaaaaaa 1740aaaa
174426420PRTGlycine max 26Met Ala Ser
Leu Pro Leu Gly His His His His His His Lys Pro Ala1 5
10 15Ala Ala Ala Ile His Pro Ser Gln Pro
Pro Gln Ser Gln Pro Gln Pro 20 25
30Glu Val Pro Arg Arg Ser Ser Asp Val Glu Thr Asp Lys Asp Met Ser
35 40 45Ala Thr Val Ile Glu Gly Asn
Asp Ala Val Thr Gly His Ile Ile Ser 50 55
60Thr Thr Ile Gly Gly Lys Asn Gly Glu Pro Lys Glu Thr Ile Ser Tyr65
70 75 80Met Ala Glu Arg
Val Val Gly Thr Gly Ser Phe Gly Val Val Phe Gln 85
90 95Ala Lys Cys Leu Glu Thr Gly Glu Ala Val
Ala Ile Lys Lys Val Leu 100 105
110Gln Asp Arg Arg Tyr Lys Asn Arg Glu Leu Gln Leu Met Arg Leu Met
115 120 125Asp His Pro Asn Val Ile Ser
Leu Lys His Cys Phe Phe Ser Thr Thr 130 135
140Ser Arg Asp Glu Leu Phe Leu Asn Leu Val Met Glu Tyr Val Pro
Glu145 150 155 160Ser Met
Tyr Arg Val Ile Lys His Tyr Thr Thr Met Asn Gln Arg Met
165 170 175Pro Leu Ile Tyr Val Lys Leu
Tyr Thr Tyr Gln Ile Phe Arg Gly Leu 180 185
190Ala Tyr Ile His Thr Ala Leu Gly Val Cys His Arg Asp Val
Lys Pro 195 200 205Gln Asn Leu Leu
Val His Pro Leu Thr His Gln Val Lys Leu Cys Asp 210
215 220Phe Gly Ser Ala Lys Val Leu Val Lys Gly Glu Ser
Asn Ile Ser Tyr225 230 235
240Ile Cys Ser Arg Tyr Tyr Arg Ala Pro Glu Leu Ile Phe Gly Ala Thr
245 250 255Glu Tyr Thr Ala Ser
Ile Asp Ile Trp Ser Ala Gly Cys Val Leu Ala 260
265 270Glu Leu Leu Leu Gly Gln Pro Leu Phe Pro Gly Glu
Asn Gln Val Asp 275 280 285Gln Leu
Val Glu Ile Ile Lys Val Leu Gly Thr Pro Thr Arg Glu Glu 290
295 300Ile Arg Cys Met Asn Pro Asn Tyr Thr Glu Phe
Arg Phe Pro Gln Ile305 310 315
320Lys Ala His Pro Trp His Lys Val Phe His Lys Arg Met Pro Pro Glu
325 330 335Ala Ile Asp Leu
Ala Ser Arg Leu Leu Gln Tyr Ser Pro Ser Leu Arg 340
345 350Cys Thr Ala Leu Glu Ala Cys Ala His Pro Phe
Phe Asp Glu Leu Arg 355 360 365Glu
Pro Asn Ala Arg Leu Pro Asn Gly Arg Pro Leu Pro Pro Leu Phe 370
375 380Asn Phe Lys Gln Glu Leu Ala Gly Ala Ser
Pro Glu Leu Ile Asn Arg385 390 395
400Leu Ile Pro Glu His Ile Arg Arg Gln Met Gly Leu Ser Phe Pro
His 405 410 415Ser Ala Gly
Thr 420272342DNAGlycine max 27gagtttcaaa ggttgttggt gtgcatcacc
acctgcattc tatgttggat gcccaatggt 60gccactgccg ccacagatcc aaatgatgct
gctgctgtga gatttttgtt tcaaaatatg 120aactcaccac cccagctagg ttggcctcct
aatggtgatg atccatgtgg acaatcttgg 180aaaggcatta cttgctctgg caatcgtgtt
acagagatta agttatctaa tcttggacta 240actggatcgt tgccttatgg attacaagtc
ttgacatctt tgacctacgt agacatgagt 300agcaacagtc ttggtggcag cataccgtac
caacttcctc catatttgca gcacttaaat 360cttgcttata acaacatcac agggacagta
ccttattcga tttctaactt gactgctctt 420actgacctga attttagtca caatcagctc
cagcaaggac tgggtgttga ctttcttaat 480ctttctactc tctccacatt ggatctctct
ttcaattttc taacaggtga cctccctcag 540actatgagct cactttcacg cataaccacc
atgtatctgc aaaataacca gtttacaggc 600actattgatg tccttgctaa tctgcctctg
gataatctga atgtggaaaa taataatttt 660actggatgga taccagaaca gttgaagaac
ataaacctac agaccggtgg taatgcatgg 720agctcagggc ctgcaccccc acctcctcct
gggacacctc cagcacctaa aagcaaccag 780caccacaagt ctggtggtgg aagcaccacc
ccctcagata ctgccactgg cagcagctca 840attgacgagg gaaaaaaatc tggtacagga
ggtggtgcca tagccggaat tgtgatctct 900gtcatagttg tgggggcaat agtagcattc
tttctggtga agagaaaatc caagaagtca 960tcttctgatt tagaaaagca ggataatcag
tcctttgctc cacttctttc aaatgaagtg 1020catgaagaaa agtccatgca aacttcctct
gtaacagact tgaagacgtt tgatacttct 1080gcctcaataa atcttaaacc cccacctatt
gaccgtcata aatcatttga tgatgaagaa 1140ttctccaaga ggcccacaat tgtgaagaag
actgtaacag ctcctgcaaa tgtgaaatca 1200tattctattg ctgaactgca gattgctact
ggcagcttca gtgtggatca ccttgttggc 1260gagggatctt ttgggcgtgt ttaccgtgct
caatttgatg atggacaggt tcttgcagtg 1320aagaagatag attcatctat ccttcccaat
gatttgacag atgattttat acaaataatt 1380tcaaacatct ccaatttaca tcatccaaat
gtgacagagc ttgtaggtta ttgctcagag 1440tatggacaac acctcttggt ctatgagttt
cataaaaatg gatcactgca tgacttcctt 1500cacctatcag atgaatatag taaaccattg
atatggaatt cccgtgtcaa gattgctttg 1560gggactgcac gtgctctaga gtacctacat
gaagttagtt cgccatcagt tgttcataag 1620aatattaagt cagccaacat attacttgat
acagaactta atcctcatct ttcagatagt 1680ggattggcaa gttatattcc aaatgccgac
cagatattga atcataatgt tggatctgga 1740tatgatgcac ctgaagttgc cttgtctggt
cagtatactt tgaaaagtga tgtctacagc 1800tttggagtcg tcatgttgga acttctcagt
ggacggaacc cttttgatag ctcaaggcca 1860agatctgagc agtctttggt tcgatgggca
acacctcaac tccatgatat tgatgcattg 1920gctaaaatgg ttgatcctgc aatgaaaggg
ttatatcctg ttaagtctct ttctcgattt 1980gccgatgtta ttgctctttg cgttcagccg
gagccagaat tccgaccacc gatgtcagaa 2040gtggttcaag cactggtgcg actagtgcag
cgagctaaca tgagcaagcg aacatttagt 2100agtagtgatc atggaggatc ccaacgaggg
agtgatgagc cagttctacg agacatctaa 2160atcccaaagc aaatgtagtt atatttttct
cccaagctag ttcggttatt tgtaatataa 2220tttccaatag ttgcaaattt gaattgatgg
gttccatatt ctgttgatac ctatgtaaac 2280ctgtccaaat cagcttatta caatgacagt
aacggttgca ctggcaaaaa aaaaaaaaaa 2340aa
234228703PRTGlycine max 28Met Pro Asn
Gly Ala Thr Ala Ala Thr Asp Pro Asn Asp Ala Ala Ala1 5
10 15Val Arg Phe Leu Phe Gln Asn Met Asn
Ser Pro Pro Gln Leu Gly Trp 20 25
30Pro Pro Asn Gly Asp Asp Pro Cys Gly Gln Ser Trp Lys Gly Ile Thr
35 40 45Cys Ser Gly Asn Arg Val Thr
Glu Ile Lys Leu Ser Asn Leu Gly Leu 50 55
60Thr Gly Ser Leu Pro Tyr Gly Leu Gln Val Leu Thr Ser Leu Thr Tyr65
70 75 80Val Asp Met Ser
Ser Asn Ser Leu Gly Gly Ser Ile Pro Tyr Gln Leu 85
90 95Pro Pro Tyr Leu Gln His Leu Asn Leu Ala
Tyr Asn Asn Ile Thr Gly 100 105
110Thr Val Pro Tyr Ser Ile Ser Asn Leu Thr Ala Leu Thr Asp Leu Asn
115 120 125Phe Ser His Asn Gln Leu Gln
Gln Gly Leu Gly Val Asp Phe Leu Asn 130 135
140Leu Ser Thr Leu Ser Thr Leu Asp Leu Ser Phe Asn Phe Leu Thr
Gly145 150 155 160Asp Leu
Pro Gln Thr Met Ser Ser Leu Ser Arg Ile Thr Thr Met Tyr
165 170 175Leu Gln Asn Asn Gln Phe Thr
Gly Thr Ile Asp Val Leu Ala Asn Leu 180 185
190Pro Leu Asp Asn Leu Asn Val Glu Asn Asn Asn Phe Thr Gly
Trp Ile 195 200 205Pro Glu Gln Leu
Lys Asn Ile Asn Leu Gln Thr Gly Gly Asn Ala Trp 210
215 220Ser Ser Gly Pro Ala Pro Pro Pro Pro Pro Gly Thr
Pro Pro Ala Pro225 230 235
240Lys Ser Asn Gln His His Lys Ser Gly Gly Gly Ser Thr Thr Pro Ser
245 250 255Asp Thr Ala Thr Gly
Ser Ser Ser Ile Asp Glu Gly Lys Lys Ser Gly 260
265 270Thr Gly Gly Gly Ala Ile Ala Gly Ile Val Ile Ser
Val Ile Val Val 275 280 285Gly Ala
Ile Val Ala Phe Phe Leu Val Lys Arg Lys Ser Lys Lys Ser 290
295 300Ser Ser Asp Leu Glu Lys Gln Asp Asn Gln Ser
Phe Ala Pro Leu Leu305 310 315
320Ser Asn Glu Val His Glu Glu Lys Ser Met Gln Thr Ser Ser Val Thr
325 330 335Asp Leu Lys Thr
Phe Asp Thr Ser Ala Ser Ile Asn Leu Lys Pro Pro 340
345 350Pro Ile Asp Arg His Lys Ser Phe Asp Asp Glu
Glu Phe Ser Lys Arg 355 360 365Pro
Thr Ile Val Lys Lys Thr Val Thr Ala Pro Ala Asn Val Lys Ser 370
375 380Tyr Ser Ile Ala Glu Leu Gln Ile Ala Thr
Gly Ser Phe Ser Val Asp385 390 395
400His Leu Val Gly Glu Gly Ser Phe Gly Arg Val Tyr Arg Ala Gln
Phe 405 410 415Asp Asp Gly
Gln Val Leu Ala Val Lys Lys Ile Asp Ser Ser Ile Leu 420
425 430Pro Asn Asp Leu Thr Asp Asp Phe Ile Gln
Ile Ile Ser Asn Ile Ser 435 440
445Asn Leu His His Pro Asn Val Thr Glu Leu Val Gly Tyr Cys Ser Glu 450
455 460Tyr Gly Gln His Leu Leu Val Tyr
Glu Phe His Lys Asn Gly Ser Leu465 470
475 480His Asp Phe Leu His Leu Ser Asp Glu Tyr Ser Lys
Pro Leu Ile Trp 485 490
495Asn Ser Arg Val Lys Ile Ala Leu Gly Thr Ala Arg Ala Leu Glu Tyr
500 505 510Leu His Glu Val Ser Ser
Pro Ser Val Val His Lys Asn Ile Lys Ser 515 520
525Ala Asn Ile Leu Leu Asp Thr Glu Leu Asn Pro His Leu Ser
Asp Ser 530 535 540Gly Leu Ala Ser Tyr
Ile Pro Asn Ala Asp Gln Ile Leu Asn His Asn545 550
555 560Val Gly Ser Gly Tyr Asp Ala Pro Glu Val
Ala Leu Ser Gly Gln Tyr 565 570
575Thr Leu Lys Ser Asp Val Tyr Ser Phe Gly Val Val Met Leu Glu Leu
580 585 590Leu Ser Gly Arg Asn
Pro Phe Asp Ser Ser Arg Pro Arg Ser Glu Gln 595
600 605Ser Leu Val Arg Trp Ala Thr Pro Gln Leu His Asp
Ile Asp Ala Leu 610 615 620Ala Lys Met
Val Asp Pro Ala Met Lys Gly Leu Tyr Pro Val Lys Ser625
630 635 640Leu Ser Arg Phe Ala Asp Val
Ile Ala Leu Cys Val Gln Pro Glu Pro 645
650 655Glu Phe Arg Pro Pro Met Ser Glu Val Val Gln Ala
Leu Val Arg Leu 660 665 670Val
Gln Arg Ala Asn Met Ser Lys Arg Thr Phe Ser Ser Ser Asp His 675
680 685Gly Gly Ser Gln Arg Gly Ser Asp Glu
Pro Val Leu Arg Asp Ile 690 695
700291610DNAOryza sativa 29accacacaaa aaagcaaaac agagagaaca actgttactc
acacacgcca tgggtaaatg 60aatggttttt gagcaacagc agttaaaaga gaaaagggat
tcagcgaaga tgacatcggt 120tggtgtggca ccaacttcgg gtttgagaga agccagtggg
catggagcag cagctgcgga 180tagattgcca gaggagatga acgatatgaa aattagggat
gatagagaaa tggaagccac 240agttgttgat ggcaacggaa cggagacagg acatatcatt
gtgactacca ttgggggtag 300aaatggtcag cccaagcaga ctataagcta catggcagag
cgtgttgtag ggcatggatc 360atttggagtt gtcttccagg ctaagtgctt ggaaaccggt
gaaactgtgg ctatcaaaaa 420ggttcttcaa gataagaggt acaagaaccg ggagctgcaa
acaatgcgcc ttcttgacca 480cccaaatgtt gtcgctttga agcactgttt cttttcaacc
actgaaaagg atgaactata 540cctcaatttg gtacttgaat atgttcctga aacagttaat
cgtgtgatca aacattacaa 600caagttaaac caaaggatgc cgctgatata tgtgaaactc
tatacatacc agatctttag 660ggcgttatct tatattcatc gttgtattgg agtctgccat
cgggatatca agcctcaaaa 720tctattggtc aatccacaca ctcaccaggt taaattatgt
gactttggaa gtgcaaaggt 780tttggtaaaa ggcgaaccaa atatatcata catatgttct
agatactata gagcacctga 840gctcatattt ggcgcaactg aatatacttc agccattgac
atctggtctg ttggatgtgt 900tttagctgag ctgctgcttg gacagcctct gttccctggt
gagagtggag ttgatcaact 960tgttgagatc atcaaggttc tgggcactcc aacaagggaa
gagattaagt gcatgaaccc 1020taattataca gaatttaaat tcccacagat taaagcacat
ccatggcaca agatcttcca 1080taagcgcatg cctccagagg ctgttgattt ggtatcaaga
ctactacaat actcccctaa 1140cttgcggtgc acagcttttg atgccttgac gcatcctttc
ttcgacgagc ttcgtgatcc 1200aaatactcgc ttgccaaatg gccgattcct tccaccacta
tttaatttca aatcccatga 1260actgaaagga gtcccatctg agattttggt gaaattggtt
ccagagcatg caaggaagca 1320atgcccgttt ctaggctcgt gaagtgttgt ttccatatga
gaatgctgcg ctttcctttt 1380ctatttaata tgatattttt gttggtatct ttattgtatt
cggttgccct gtaaaagcag 1440atttagagat acatgctact cattatcacc caacccccga
tggttatgta gaataccctg 1500tttcctgtat cacagcagat tgtaacatac aatagaggac
aaaatgtctg caattatcta 1560aatgttgcat caatatttgt atttgttgag gcaaaaaaaa
aaaaaaaaaa 161030426PRTOryza sativa 30Met Val Phe Glu Gln
Gln Gln Leu Lys Glu Lys Arg Asp Ser Ala Lys1 5
10 15Met Thr Ser Val Gly Val Ala Pro Thr Ser Gly
Leu Arg Glu Ala Ser 20 25
30Gly His Gly Ala Ala Ala Ala Asp Arg Leu Pro Glu Glu Met Asn Asp
35 40 45Met Lys Ile Arg Asp Asp Arg Glu
Met Glu Ala Thr Val Val Asp Gly 50 55
60Asn Gly Thr Glu Thr Gly His Ile Ile Val Thr Thr Ile Gly Gly Arg65
70 75 80Asn Gly Gln Pro Lys
Gln Thr Ile Ser Tyr Met Ala Glu Arg Val Val 85
90 95Gly His Gly Ser Phe Gly Val Val Phe Gln Ala
Lys Cys Leu Glu Thr 100 105
110Gly Glu Thr Val Ala Ile Lys Lys Val Leu Gln Asp Lys Arg Tyr Lys
115 120 125Asn Arg Glu Leu Gln Thr Met
Arg Leu Leu Asp His Pro Asn Val Val 130 135
140Ala Leu Lys His Cys Phe Phe Ser Thr Thr Glu Lys Asp Glu Leu
Tyr145 150 155 160Leu Asn
Leu Val Leu Glu Tyr Val Pro Glu Thr Val Asn Arg Val Ile
165 170 175Lys His Tyr Asn Lys Leu Asn
Gln Arg Met Pro Leu Ile Tyr Val Lys 180 185
190Leu Tyr Thr Tyr Gln Ile Phe Arg Ala Leu Ser Tyr Ile His
Arg Cys 195 200 205Ile Gly Val Cys
His Arg Asp Ile Lys Pro Gln Asn Leu Leu Val Asn 210
215 220Pro His Thr His Gln Val Lys Leu Cys Asp Phe Gly
Ser Ala Lys Val225 230 235
240Leu Val Lys Gly Glu Pro Asn Ile Ser Tyr Ile Cys Ser Arg Tyr Tyr
245 250 255Arg Ala Pro Glu Leu
Ile Phe Gly Ala Thr Glu Tyr Thr Ser Ala Ile 260
265 270Asp Ile Trp Ser Val Gly Cys Val Leu Ala Glu Leu
Leu Leu Gly Gln 275 280 285Pro Leu
Phe Pro Gly Glu Ser Gly Val Asp Gln Leu Val Glu Ile Ile 290
295 300Lys Val Leu Gly Thr Pro Thr Arg Glu Glu Ile
Lys Cys Met Asn Pro305 310 315
320Asn Tyr Thr Glu Phe Lys Phe Pro Gln Ile Lys Ala His Pro Trp His
325 330 335Lys Ile Phe His
Lys Arg Met Pro Pro Glu Ala Val Asp Leu Val Ser 340
345 350Arg Leu Leu Gln Tyr Ser Pro Asn Leu Arg Cys
Thr Ala Phe Asp Ala 355 360 365Leu
Thr His Pro Phe Phe Asp Glu Leu Arg Asp Pro Asn Thr Arg Leu 370
375 380Pro Asn Gly Arg Phe Leu Pro Pro Leu Phe
Asn Phe Lys Ser His Glu385 390 395
400Leu Lys Gly Val Pro Ser Glu Ile Leu Val Lys Leu Val Pro Glu
His 405 410 415Ala Arg Lys
Gln Cys Pro Phe Leu Gly Ser 420
4253118DNAArtificial SequenceDescription of Artificial Sequence Primer
31caggaaacag ctatgacc
183219DNAArtificial SequenceDescription of Artificial Sequence Primer
32ctaaagggaa caaaagctg
193318DNAArtificial SequenceDescription of Artificial Sequence Primer
33tgtaaaacga cggccagt
183433DNAArtificial SequenceDescription of Artificial Sequence Primer
34atcccgggcg agtcttctat ggcatctgcg act
333533DNAArtificial SequenceDescription of Artificial Sequence Primer
35atgagctcaa tatcaggagt tgcacccttc aac
333625DNAArtificial SequenceDescription of Artificial Sequence Primer
36tgtgtctacg tgtcgcgggg tcgat
253732DNAArtificial SequenceDescription of Artificial Sequence Primer
37atcccgggag gcattgaact acctggagtg ag
323834DNAArtificial SequenceDescription of Artificial Sequence Primer
38gcgatatcgt tgaactagta atctgtgtta actt
343925DNAArtificial SequenceDescription of Artificial Sequence Primer
39ctgcgacgga aaactctctt gctgt
254034DNAArtificial SequenceDescription of Artificial Sequence Primer
40gcgagctcgt gcgaatcatg tactcccatc acac
344125DNAArtificial SequenceDescription of Artificial Sequence Primer
41gcaacgactt gccagaacct cgtgc
254233DNAArtificial SequenceDescription of Artificial Sequence Primer
42cggagctcga tgcagcggta tatccttgct cct
334338DNAArtificial SequenceDescription of Artificial Sequence Primer
43gcgatatcgt tgaactagta atctgtgtta actttatc
384426DNAArtificial SequenceDescription of Artificial Sequence Primer
44gcagcggtat atccttgctc ctcatc
264526DNAArtificial SequenceDescription of Artificial Sequence Primer
45cgatgtgaga cgcccttgct gtggca
264633DNAArtificial SequenceDescription of Artificial Sequence Primer
46atcccgggtg tcggaattcg gtcacaatga gct
334731DNAArtificial SequenceDescription of Artificial Sequence Primer
47atcccgggtt tctggaatag ctcagaagcg t
314830DNAArtificial SequenceDescription of Artificial Sequence Primer
48gcgctgcaga tttcatttgg agaggacacg
304935DNAArtificial SequenceDescription of Artificial Sequence Primer
49cgcggccggc ctcagaagaa ctcgtcaaga aggcg
355027DNAArtificial SequenceDescription of Artificial Sequence Primer
50cgagagctgc agatcatgcg actgttg
275126DNAArtificial SequenceDescription of Artificial Sequence Primer
51gctctgccat cacgcaaccc atcgac
26
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