Patent application title: METHODS AND COMPOSITIONS FOR PLANT PEST CONTROL
Inventors:
John D. Bradley (St. Louis, MO, US)
Catherine C. Baublite (Overland, MO, US)
Michael J. Crawford (St. Louis, MO, US)
Michael J. Crawford (St. Louis, MO, US)
Stanislaw Flasinski (Chesterfield, MO, US)
Deryck J. Williams (University City, MO, US)
Deryck J. Williams (University City, MO, US)
IPC8 Class: AC12N1582FI
USPC Class:
Class name:
Publication date: 2015-07-23
Patent application number: 20150203866
Abstract:
The present invention is directed to controlling nematode infestation.
The invention discloses methods and compositions for use in controlling
nematode infestation by providing recombinant DNA molecules to the cells
of a plant in order to achieve a reduction in nematode infestation. The
invention is also directed to methods for making transgenic plants that
express the recombinant DNA molecule for use in protecting plants from
nematode infestation.Claims:
1. A polynucleotide comprising a sequence encoding a polypeptide
comprising a plant methylketone synthase and a heterologous transit
peptide operably linked to the plant methylketone synthase, wherein the
plant methylketone synthase has at least 90% identity to a polypeptide
sequence selected from the group consisting of SEQ ID NO: 3 and 5
2. (canceled)
3. The polynucleotide of claim 1, wherein the transit peptide is a chloroplast transit peptide.
4. The polynucleotide of claim 3, wherein the chloroplast transit peptide is selected from the group consisting of an EPSPS chloroplast transit peptide, a small subunit ribulose-1,5,-bisphosphate carboxylase chloroplast transit peptide, a ferredoxin chloroplast transit peptide, a ferredoxin oxidoreductase chloroplast transit peptide, a light-harvesting complex protein I and protein II chloroplast transit peptide, and a thioredoxin F chloroplast transit peptide.
5. The polynucleotide of claim 1, wherein the sequence encoding the plant methylketone synthase exhibits at least about 80% percent sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1, 2 and 4.
6. A construct comprising the polynucleotide of claim 1 operably linked to a promoter functional in plants.
7. A plant cell comprising the polynucleotide of claim 1.
8. The plant cell of claim 7, wherein the transit peptide is a chloroplast transit peptide.
9. The plant cell of claim 7, wherein said plant cell is from a seed, root, leaf, shoot, flower, pollen, or ovule.
10. The plant cell of claim 7, wherein said cell produces a methylketone.
11. The plant cell of claim 10, wherein said methylketone is 2-undecanon; 2-tridecanone, or 2-pentadecanone.
12. The plant cell of claim 7, wherein said cell is a crop plant cell.
13. The plant cell of claim 7, wherein said cell is from a plant selected from the group selected from cotton, soybean, canola, corn, wheat, rice, sunflower, sorghum, sugarcane, potato, tomato, and a tree.
14. A plant or a part thereof comprising the polynucleotide of claim 1.
15. The plant or part thereof of claim 14, wherein the part thereof is selected from the group consisting of a seed, pollen, a root, a leaf, a shoot, a flower and an ovule.
16. A processed product comprising a plant tissue comprising the polynucleotide of claim 1.
17. The processed product of claim 16, selected from the group consisting of meal, flour, oil, hay, starch, juice, protein extract, and fiber.
18. A method for controlling a pathogen or pest in a plant comprising expressing in the plant the construct of claim 6.
19. The method for controlling a pathogen or pest in a plant of claim 18, wherein the polynucleotide sequence comprises a sequence that encodes a second heterologous transit peptide operably linked to a sequence that encodes an acyl carrier protein.
20. The method of claim 19, wherein the pathogen or pest is a nematode.
21. The method of claim 20, wherein the nematode is selected from the group consisting of Heterodera species, Globodera species, Meloidogyne species, Rotylenchulus species, Hoplolaimus species, Belonolaimus species, Pratylenchus species, Longidorus species, Paratrichodorus species, Ditylenchus species, Xiphinema species, Dolichodorus species, Helicotylenchus species, Radopholus species, Hirschmanniella species, Tylenchorhynchus species, and Trichodorus species.
22. The method of claim 19, wherein the pathogen or pest is an insect pest.
23. The method of claim 22, wherein the insect pest is selected from the group consisting of Diabrotica, Diaprepes, Pachnaeus, Asynonychus, Lycoriella, Sciara, Stenophlus, and Bradysia.
24. A method of producing seed, comprising crossing the plant of claim 14 with itself or a second plant.
25. The method of producing seed of claim 24, wherein the polynucleotide sequence comprises a sequence that encodes a second heterologous transit peptide operably linked to a sequence that encodes an acyl carrier protein.
26. The polynucleotide of claim 1, further comprising a sequence that encodes a second heterologous transit peptide operably linked to a sequence that encodes an acyl carrier protein.
27. The polynucleotide of claim 26, wherein said sequence that encodes a second heterologous transit peptide operably linked to a sequence that encodes an acyl carrier protein encodes a polypeptide that comprises an amino acid sequence exhibiting at least about 85% identity to a polypeptide selected from the group consisting of SEQ ID NO: 41, 43, 45, 47, 49, 51, 53, 55, and 57.
28. The polynucleotide of claim 26, wherein said second transit peptide is a chloroplast transit peptide.
29. The polynucleotide of claim 26, wherein said second transit peptide is selected from the group consisting of an EPSPS chloroplast transit peptide, a small subunit ribulose-1,5,-bisphosphate carboxylase chloroplast transit peptide, a ferredoxin chloroplast transit peptide, a ferredoxin oxidoreductase chloroplast transit peptide, a light-harvesting complex protein I and protein II chloroplast transit peptide, and a thioredoxin F chloroplast transit peptide.
30. The polynucleotide of claim 26, wherein said sequence that encodes a second heterologous transit peptide operably linked to a sequence that encodes an acyl carrier protein exhibits at least about 80% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 40, 42, 44, 46, 48, 50, 52, 54, and 56.
31. The plant cell of claim 7, wherein the polynucleotide sequence comprises a sequence that encodes a second heterologous transit peptide operably linked to a sequence that encodes an acyl carrier protein.
32. The plant or part thereof of claim 14, wherein the polynucleotide sequence comprises a sequence that encodes a second heterologous transit peptide operably linked to a sequence that encodes an acyl carrier protein.
33. A processed product of a plant, plant part, seed or progeny, wherein the product comprises the plant cell of claim 7.
34. The processed product of claim 33, wherein the processed product is selected from the group consisting of meal, flour, oil, hay, starch, juice, protein extract, and fiber.
35. The polynucleotide of claim 1, wherein the polypeptide comprises an amino acid sequence with at least 95% identity to a polypeptide sequence selected from the group consisting of SEQ ID NO: 3 and 5.
Description:
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority of U.S. Provisional Application Ser. No. 61/027,473, filed Feb. 10, 2008, the entire disclosure of which is incorporated herein by reference.
INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING IN COMPUTER READABLE FORM
[0002] The Sequence Listing, which is a part of the present disclosure, includes a computer readable form 96 KB file entitled "MNDI005WOsequence" comprising nucleotide sequences of the present invention submitted via EFS-Web. The subject matter of the Sequence Listing is incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to methods and compositions for pest or pathogen control in plants. More particularly, it discloses transgenic plant cells, plants and seeds comprising recombinant DNA and methods of making and using such plant cells, plants and seeds that are associated with pest resistance.
[0005] 2. Description of Related Art
[0006] Plants and animals are targets for infection by many nematode pests. Improved methods for protecting plants from nematode infection are therefore desired since they would increase the amount and stability of food production.
[0007] There are numerous plant-parasitic nematode species, including various cyst nematodes (e.g. Heterodera spp.), root knot nematodes (e.g. Meloidogyne spp.), lesion nematodes (e.g. Pratylenchus spp.), dagger nematodes (e.g. Xiphinema spp.) and stem and bulb nematodes (e.g. Ditylenchus spp.), among others. Tylenchid nematodes (members of the order Tylenchida), including the families Heteroderidae, Meloidogynidae, and Pratylenchidae, are the largest and most economically important group of plant-parasitic nematodes. Other important plant-parasitic nematodes include Dorylaimid nematodes (e.g. Xiphinenza spp.), among others. Nematode species grow through a series of lifecycle stages and molts. Typically, there are five stages and four molts: egg stage; J1 (i.e. first juvenile stage); M1 (i.e. first molt); J2 (second juvenile stage; sometimes hatch from egg); M2; J3; M3; J4; M4; A (adult). Juvenile ("J") stages are also sometimes referred to as larval ("L") stages. Gene expression may be specific to one or more lifecycle stages. Nematodes have evolved as very successful parasites of both plants and animals and are responsible for significant economic losses in agriculture and livestock and for morbidity and mortality in humans. Nematode parasites of plants can inhabit all parts of plants, including roots, developing flower buds, leaves, and stems. Plant parasites are classified on the basis of their feeding habits into the broad categories migratory ectoparasites, migratory endoparasites, and sedentary endoparasites. Sedentary endoparasites, which include the root knot nematodes (Meloidogyne species, RKN), cyst nematodes (Globodera and Heterodera species) and reniform nematodes (Rotylenchulus species) induce feeding sites and establish long-term infections within roots that are often very damaging to crops. Nematode infection is a significant problem in the farming of many agriculturally significant crops. For example, soybean cyst nematode (Heteodera glycines, SCN) is believed to be responsible for yield losses in soybeans estimated to be in excess of $1 billion per year in North America. Such damage is the result of the stunting of the soybean plant caused by the cyst nematode. The stunted plants have smaller root systems, show symptoms of mineral deficiencies in their leaves, and wilt easily. It is estimated that parasitic nematodes cost the horticulture and agriculture industries in excess of $78 billion worldwide a year, based on an estimated average 12 percent annual loss spread across all major crops.
[0008] Traditional approaches for control of plant diseases have been the use of chemical treatment and the construction of interspecific hybrids between resistant crops and their wild-type relatives as sources of resistant germplasm. Chemical nematode control agents are not effective in eradicating nematode infestations. Because of the lack of selectivity, the chemical nematode control agents exert their effects on non-target fauna as well, often effectively sterilizing a field for a period of time following the application of nematode control agents. Nematicides such as Aldicarb and its environmental breakdown products are known to be highly toxic to mammals. As a result, government restrictions have been imposed on the use of these chemicals. The most widely used nematicide, methyl bromide, is scheduled to be soon retired from use, and at present, there is no promising candidate to replace this treatment.
[0009] Methods employing plant biotechnology have provided effective means to control insect infestations, for instance through plant expression of an insect control agent. Biotechnologically-related nematode control agents have generally been reported to be nucleotides expressed by a plant that are selectively toxic to the target nematode when ingested by the nematode. However, there are few examples of effectively applied biotechnology methods to control nematode infection.
SUMMARY OF THE INVENTION
[0010] In one aspect, the invention provides agents effective as a plant nematode control agent. The effective compounds are, in one embodiment, methylketones not previously known to be toxic to plant parasitic nematodes. Additionally, the inventors have developed compositions and methods to express methylketones, such as 2-undecanone, 2-tridecanone and 2-pentadecanone, in the roots of plants that nematodes infect, to reduce or inhibit nematode growth, development, or the plant disease caused by nematode infection. In particular embodiments the method comprises production of transgenic plants containing one or more transgenes that provide for the production of 2-undecanone, 2-tridecanone and/or 2-pentadecanone in plant tissues susceptible to nematode infection.
[0011] In another aspect, the invention provides methods for construction and use of a transgene expression cassette comprising a methylketone synthase coding region and expression of the synthase in a plant cell, particularly the root cells of a plant. The invention provides for a transgenic plant comprising the transgene wherein the roots of the transgenic plant produce a methylketonc. The methylketone synthase transgene, in certain embodiments, additionally comprises a sequence region comprising a heterologous plastid transit peptide molecule in operable linkage to the methylketone synthase coding region. By "heterologous" it is meant that a given sequence is not in its native context with respect to any other referenced sequence. Thus one sequence may be heterologous with respect to second, operably linked, sequence where both sequences can be isolated from the same species, but will be not be in their native orientation. A heterologous transit peptide operably linked to a given methylketone synthase coding region is therefore not a transit peptide normally found in nature in an unmodified state in operable linkage to the methylketone synthase coding region.
[0012] In yet another aspect of the invention, modified DNA coding sequences comprising SEQ ID NO: 1 or 2 are provided that encode a methylketone synthase of SEQ ID NO: 3; SEQ ID NO: 4 is provided encoding the methylketone synthase of SEQ ID NO: 5; and SEQ ID NO: 6 is provided encoding the methylketone synthase SEQ ID NO: 7. In certain embodiments, the DNA coding sequence encoding a polypeptide with methylketone synthase activity shares at least about 80%, 85%, 90%, 95%, 98%, or 99% percent sequence identity to any one or more of said SEQ ID NOs.
[0013] In still yet another aspect of the invention, a heterologous fusion protein is provided that comprises a plastid transit peptide molecule (such as SEQ ID NO: 9 or 11) and a methylketone synthase molecule (such as SEQ ID NO: 13, 15, 17, 19, 21, 23, 25 or 27) or methylketone synthase molecule variant (such as SEQ ID NO: 29, 31, 33, 35, 37, or 39) with methylketone synthase activity, or a methylketone synthase molecule having at least about 80%, 85%, 90%, 95%, 98%, or 99% percent sequence identity to any one or more of said SEQ ID NOs.
[0014] In still yet another aspect of the invention, a transgene expression cassette is provided comprising a heterologous acyl carrier protein coding region that encodes for an acyl carrier protein (such as SEQ ID NO: 41, 43, 45, 47, 49, 51, 53, 55, or 57) that is expressed in plant tissues with the transgene comprising the methylketone synthase coding region.
[0015] In still yet another aspect of the invention, a transgenic seed is provided comprising a heterologous plastid transit peptide molecule in operable linkage to the methylketone synthase coding region. The transgenic seed may additionally comprise a transgene expression cassette comprising a heterologous acyl carrier protein coding region.
[0016] Other aspects of the invention are specifically directed to transgenic plant cells, and transgenic plants comprising a plurality of the plant cells, nuclei and organelles, and progeny transgenic seed, embryo, ovule and transgenic pollen from such plants. A plant cell and parts thereof is selected from a population of transgenic plant cells transformed with a heterologous methylketone synthase coding region and may additionally comprise a heterologous acyl carrier protein coding region by selecting the transgenic plant cell from any population comprising the heterologous coding region as compared to a cell that does not have the heterologous coding region.
[0017] This invention also provides methods for manufacturing non-natural, transgenic seed that can be used to produce a crop of transgenic plants with pest resistance resulting from expression of a heterologous methylketone synthase coding region and in certain embodiments the co-expression of a heterologous acyl carrier protein coding region in the nucleus or organelle or cytoplasm of the plant cells making up the transgenic plants. The various aspects of this invention are especially useful for transgenic plants having nematode resistance activity that include, without limitation, cereals including corn, wheat, barley, rye, and rice; vegetables; tomatoes; potatoes; clovers; legumes including beans, soybeans, peas and alfalfa; sugar cane; sugar beets; tobacco; cotton; rapeseed (canola); sunflower; safflower; and sorghum.
[0018] The present invention provides for a transgenic soybean plant comprising within its genome a heterologous methylketone synthase coding region and may additionally comprise a heterologous acyl carrier protein coding region, wherein the plant is resistant to nematode infection or displays reduced disease symptoms caused by nematode infection.
[0019] The present invention further provides a method of increasing the yield of a nematode tolerant crop plant. The method comprises growing a crop plant comprising a heterologous methylketone synthase coding region which may additionally comprise a heterologous acyl carrier protein coding region in the presence of nematodes.
[0020] Another aspect of the invention provides a method of producing a hybrid seed comprising acquiring hybrid seed from a nematode tolerant plant which also has a stably-integrated heterologous nucleotide sequence encoding a methylketone synthase and may also have integrated a heterologous nucleotide sequence encoding an acyl carrier protein. The method further comprises producing a crop from plants grown from the hybrid seed, wherein a fraction of the plants produced from said hybrid seed are homozygous for the heterologous methylketone synthase coding sequence and if present, the heterologous acyl carrier protein coding sequence, a fraction of the plants produced from said hybrid seed are hemizygous for the heterologous methylketone synthase coding sequence and if present, the heterologous acyl carrier protein coding sequence, and a fraction of the plants produced from the hybrid have no heterologous methylketone synthase coding sequence or heterologous acyl carrier protein coding sequence; selecting plants which are homozygous and hemizygous; collecting seed from the selected plants, and planting the seed to produce further progeny plants; repeating the selecting and collecting steps at least once from these progeny plants to produce an inbred line; and crossing the inbred line with a second line to produce hybrid seed. The plants of the invention are selected, without limitation, from the group of corn (maize), soybean, cotton, canola (rape), wheat, sunflower, sorghum, alfalfa, barley, millet, rice, tobacco, tomato, potato, fruit and vegetable crops, turfgrass, sugar cane, sugar beets, and safflower.
[0021] In a further aspect of the invention, control of agronomically important soil inhabiting insects is contemplated, which include, but are not limited to Diabrotica, Diaprepes, Pachnaeus, Asynonychus, Lycoriella, Sciara, Stenophlus, and Bradysia among others. Broader acaricidal, insecticidal, and pest repellent properties are also contemplated.
DETAILED DESCRIPTION OF THE INVENTION
[0022] The invention relates to methods and compositions for pest control in plants, in particular nematode control. In one aspect, the invention relates to controlling, preventing or treating nematode infection in transgenic plants. The method comprises, in one embodiment, generation of transgenic plant containing a recombinant construct and expression of such construct to impart nematode resistance to plants. The recombinant construct may comprise a nucleotide sequence encoding one or more proteins, wherein the sequence is operably linked to a heterologous promoter functional in a plant cell, and to cells transformed with the recombinant construct. Cells comprising (meaning including but not limited to) the recombinant construct may be prokaryotic or eukaryotic. In particular, they may be plant cells. Plants and seeds derived from such transformed plant cells are also contemplated. The transgenic plants or parts thereof of the present invention, in one embodiment, produce one or more fatty acid compounds for which at least one is 2-tridecanone. 2-tridecanone is the major methylketone (76% of total volatile content) produced in Lycopersicon hirsutum (Solanum habrochaites) (compared to 21% 2-undecanone, and 3% 2-pentadecanone; Fridman, et al., Plant Cell 17:1252-67, 2005). Higher plants synthesize fatty acids via a metabolic pathway involving an acyl carrier protein co-factor (ACP) and a fatty acid synthase (FAS) enzyme complex. The FAS complex consists of about eight separate enzymes that catalyze thirty or more individual reaction steps, all of which, in plants, are located in the plastids.
[0023] The present invention provides heterologous molecules that are directed into the plastid of a plant to provide production of a methylketone, especially 2-tridecanone, from the FAS complex, including, but not limited to, nucleotides that encode polypeptides having methylketone synthase activity such as SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or the amino acid sequence given in GenBank Accession AY701574. In certain embodiments, the polypeptide having methylketone synthase activity (e.g. allowing for production of methylketones such as 2-undecanone, 2-tridecanone, and 2-pentadecanone) may share at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity, to any one or more amino acid sequence(s) set forth in SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, 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, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:39. The function of the encoded polypeptide may also be determined by measuring the efficacy of the presence of the transgene that encodes it in reducing nematode infection, growth, reproduction, or symptomatology. For instance, a reduction in root galls, cysts, or worm number of 20% or more, 25% or more, 50% or more, 80% or more, or 95% or more, in a transgenic plant comprising a heterologous nucleotide construct encoding methylketone synthase activity, relative to a control plant, for instance an otherwise isogenic plant not comprising the heterologous molecule, under similar conditions, indicates the presence of a functional molecule.
[0024] In certain embodiments, a heterologous molecule provided by the present invention that is directed into the plastid of a plant to provide production of a methylketone may share at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity at the nucleotide level with one or more sequence(s) as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, 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, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:38; or any of SEQ ID NOs:58-61. Thus, in particular embodiments, the heterologous molecule may comprise a sequence encoding a heterologous chloroplast transit peptide, for instance, without limitation, as shown in SEQ ID NO:9 or SEQ ID NO:11.
[0025] Likewise, in certain embodiments, a nucleotide of the present invention may further comprise a sequence that encodes an acyl carrier protein (e.g. ACP1), as set forth in any of SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, or SEQ ID NO:57, or may comprise a sequence that encodes an acyl carrier protein with at least about 85%, 90%, 95%, 98%, or 99% sequence similarity to any of these sequences.
[0026] Yet another aspect of the invention provides methods for production and for use of one or more methylketone(s), such as 2-tridecanone, to control nematode infestation. Thus, methods for production of a methylketone, for instance in a plant cell, are provided. The methylketone may then be applied to soil prior to, during, or subsequent to planting of a crop, in order to control or reduce nematode infestation or symptomatology of crop plants grown in that soil.
[0027] Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. Definitions of common terms in molecular biology may also be found in Rieger et al., Glossary of Genetics: Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991; and Lewin, Genes V, Oxford University Press: New York, 1994. The nomenclature for DNA bases as set forth at Title 37 of the United States Code of Federal Regulations, Part 1, section 1.822.
[0028] As used herein, a "transgenic plant" is any plant in which one or more, or all, of the cells of the plant include a transgene. A transgene may be integrated within a nuclear genome or organelle genome, or it may be extra-chromosomally replicating DNA. The term "transgene" means a nucleic acid that is partly or entirely heterologous, foreign, to a transgenic microbe, plant, animal, or cell into which it is introduced. Cells that make up various cell and tissue types of plants include but are not limited to seed, root, leaf, shoot, flower, pollen and ovule.
[0029] "Recombinant DNA" is a polynucleotide having a genetically engineered modification introduced through combination of endogenous and/or exogenous molecules in a transcription unit, manipulation via mutagenesis, restriction enzymes, and the like or simply by inserting multiple copies of a native transcription unit. Recombinant DNA may comprise DNA segments obtained from different sources, or DNA segments obtained from the same source, but which have been manipulated to join DNA segments which do not naturally exist in the joined form. An isolated recombinant polynucleotide may exist, for example as a purified molecule, or integrated into a genome, such as a plant cell, or organelle genome or a microbe plasmid or genome. The polynucleotide comprises linked regulatory molecules that cause transcription of an RNA in a plant cell.
[0030] As used herein, "percent identity" means the extent to which two optimally aligned DNA or protein segments are invariant throughout a window of alignment of components, for example nucleotide sequence or amino acid sequence. An "identity fraction" for aligned segments of a test sequence and a reference sequence is the number of identical components that are shared by sequences of the two aligned segments divided by the total number of sequence components in the reference segment over a window of alignment which is the smaller of the full test sequence or the full reference sequence. "Percent identity" ("% identity") is the identity fraction times 100.
[0031] "Expression" means transcription of DNA to produce RNA. The resulting RNA may be without limitation mRNA encoding a protein, antisense RNA, or a double-stranded RNA for use in RNAi technology. Expression also may refer to translation of RNA, i.e. the production of encoded protein from an mRNA.
[0032] As used herein, "promoter" means regulatory DNA molecules for initializing transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells whether or not its origin is a plant cell. For example it is well known that certain Agrobacterium promoters are functional in plant cells. Thus, plant promoters include promoter DNA obtained from plants, plant viruses (in particular, double stranded DNA viruses) and bacteria such as Agrobacterium and Bradyrhizobium bacteria. Constitutive promoters generally provide transcription in most or all of the cells of a plant. In particular, promoters such as the FMV promoter (FMV, U.S. Pat. No. 6,051,753), the enhanced 35S promoter (E35S, U.S. Pat. No. 5,359,142), rice actin promoter (U.S. Pat. No. 5,641,876), and various chimeric promoters (U.S. Pat. No. 6,660,911) are useful in the present invention. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, or seeds. Such promoters are referred to as "tissue-preferred". Promoters that initiate transcription only in certain tissues are referred to as "tissue specific."
[0033] A number of root-specific or root-enhanced promoters or fragments of such that provide enhanced expression in root tissues relative to other plant tissues have been identified and are known in the art (e.g. U.S. Pat. Nos. 5,110,732, 5,837,848, 5,837,876; 5,633,363; 5,459,252; 5,401,836; 7,196,247; 7,232,940; 7,119,254; and 7,078,589). Examples include root-enhanced or root-specific promoters such as the CaMV-derived as-1 promoter or the wheat PDX1 promoter (U.S. Pat. No. 5,023,179), the acid chitinase gene promoter (Samac et al., Plant Mol. Biol. 25:587-596 (1994); the root specific subdomains of the CaMV35S promoter (Lam et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:7890-7894 (1989); the root-enhanced ORF13 promoter from Agrobacterium rhizogenes (Hansen et al., Mol. Gen. Genet. 254:337-343 (1997); the promoter for the tobacco root-specific gene RB7 (U.S. Pat. No. 5,750,386); and the root cell-specific promoters reported by Conkling et al. (Plant Physiol. 93:1203-1211 (1990). Additional examples include RCc2 and RCc3, promoters that direct root-specific gene transcription in rice (Xu et al., Plant Mol. Biol. 27:237, 1995); soybean root-specific glutamine synthetase promoter (Hire et al., Plant Mol. Biol. 20:207-218, 1992); root-specific control element in the GRP 1.8 gene of French bean (Keller and Baumgartner, Plant Cell 3:1051-1061, 1991.); a root-specific promoter of the mannopine synthase (MAS) gene of Agrobacterium tumefaciens (Sanger et al., Plant Mol. Biol. 14:433-443, 1990); and full-length cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in roots and root nodules of soybean (Miao et al., Plant Cell 3:11-22, 1991). See also Bogusz et al., Plant Cell 2:633-641, 1990, where two root-specific promoters isolated from hemoglobin genes from the nitrogen-fixing non-legume Parasponia andersonii and the related non-nitrogen-fixing non-legume Trema tomentosa are described. Leach and Aoyagi (1991) describe their analysis of the promoters of the highly expressed rolC and rolD root-inducing genes of Agrobacterium rhizogenes (see Plant Science (Limerick) 79:69-76). Additional root-preferred promoters include the VfENOD-GRP3 gene promoter (Kuster et al., Plant Mol. Biol. 29(4):759-772, 1995); and rolB promoter (Capana et al., Plant Mol. Biol. 25:681-691, 1994). Examples of nematode-induced promoters include, for instance, the TobRB7 promoter (Opperman et al., Science 263:221-223, 1994), and promoters described in U.S. Pat. Nos. 6,262,344, and 7,193,136.
[0034] The term "resistance," or "tolerance" when used in the context of comparing the effectiveness of a transgene in a transgenic plant, refers to the ability of the transgenic plant to maintain a desirable phenotype when exposed to nematode infestation pressures relative to the phenotype presented by a nematode sensitive non-transgenic plant under similar conditions. The level of resistance can be determined by comparing the physical characteristics of the transgenic plant to non-transgenic plants that either have or have not been exposed to nematode infection. Exemplary physical characteristics to observe include plant height, an increase in population of plants that have ability to survive nematode challenge (that is, plants that come in contact with a parasitic nematode may have enhanced root growth, enhanced fruit or grain yield, and reproduction nematode infection or population increase rate). The product of expression of the recombinant DNA may be directly toxic to the nematode (nematicidal) or may affect the mobility, host finding, feeding site establishment, fecundity or have other nematistatic effects.
[0035] "Transformed seed" is the seed which has been generated from the transformed plant. A transformed plant contains transformed cells. A transformed cell is a cell that has been altered by the introduction of an exogenous DNA molecule or in the present invention comprises a heterologous methylketone synthase or a heterologous acyl carrier protein or a combination of both.
[0036] Nematodes include but are not limited to plant parasitic species, for example, Heterodera species, Globodera species, Meloidogyne species, Rotylenchulus species, Hoplolalaimus species, Belonolaimus species, Pratylenchus species, Longidorus species, Paratrichodorus species, Ditylenchus species, Xiphinema species, Dolichodorus species, Helicotylenchus species, Radopholus species, Hirschmanniella species, Tylenchorhynchus species, and Trichodorus species, and the like.
[0037] The present invention provides recombinant DNA constructs comprising a polynucleotide that, when incorporated in a plant cell, imparts to the plant resistance to nematode infection or plant disease caused by the nematode infection. Such constructs also typically comprise a promoter operatively linked to said polynucleotide to provide for expression in the plant cells. Other construct components may include additional regulatory molecules, such as 5' leader regions or 3' untranslated regions (such as polyadenylation sites), intron regions, and transit or signal peptides. Such recombinant DNA constructs can be assembled using methods known to those of ordinary skill in the art.
[0038] Recombinant constructs prepared in accordance with the present invention also generally include a 3' untranslated DNA region (UTR) that typically contains a polyadenylation sequence following the polynucleotide coding region. Examples of useful 3' UTRs include but arc not limited to those from the nopalinc synthase gene of Agrobacterium tumefaciens (nos), a gene encoding the small subunit of a ribulose-1,5-bisphosphate carboxylase-oxygenase (rbcS), and the T7 transcript of Agrobacterium tumefaciens.
[0039] Constructs and vectors may also include a transit peptide for targeting of a protein product, particularly to a chloroplast, leucoplast or other plastid organelle, mitochondria, peroxisome, or vacuole or an extracellular location. For descriptions of the use of chloroplast transit peptides, see U.S. Pat. No. 5,188,642 and U.S. Pat. No. 5,728,925. Many chloroplast-localized proteins are expressed from nuclear genes as precursors and are targeted to the chloroplast by a chloroplast transit peptide (CTP). Examples of other such isolated chloroplast proteins include, but are not limited to those associated with the small subunit (SSU) of ribulose-1,5,-bisphosphate carboxylase, ferredoxin, ferredoxin oxidoreductase, the light-harvesting complex protein I and protein II, thioredoxin F, enolpyruvyl shikimate phosphate synthase (EPSPS) and transit peptides described in U.S. Pat. No. 7,193,133. It has been demonstrated in vivo and in vitro that non-chloroplast proteins may be targeted to the chloroplast by use of protein fusions with a heterologous CTP and that the CTP is sufficient to target a protein to the chloroplast. Incorporation of a suitable chloroplast transit peptide, such as, the Arabidopsis thaliana EPSPS CTP (CTP2, Klee et al., Mol. Gen. Genet. 210:437-442, 1987), and the Petunia hybrida EPSPS CTP (CTP4, della-Cioppa et al., Proc. Natl. Acad. Sci. USA 83:6873-6877, 1986) has been show to target heterologous EPSPS protein sequences to chloroplasts in transgenic plants. The production of glyphosate tolerant plants by expression of a fusion protein comprising an amino-terminal CTP with a glyphosate resistant EPSPS enzyme is well known by those skilled in the art, (U.S. Pat. No. 5,627,061, U.S. Pat. No. 5,633,435, U.S. Pat. No. 5,312,910, EP 0218571, EP 189707, EP 508909, and EP 924299). Those skilled in the art will recognize that various chimeric constructs can be made that utilize the functionality of a CTP to import various methylketone synthases or acyl carrier proteins into the plant cell plastid.
[0040] Stable methods for plant transformation include virtually any method by which DNA can be introduced into a cell, such as by direct delivery of DNA (for example, by PEG-mediated transformation of protoplasts, by electroporation, by agitation with silicon carbide fibers, and by acceleration of DNA coated particles), by Agrobacterium-mediated transformation, by viral or other vectors. One preferred method of plant transformation is microprojectile bombardment, for example, as illustrated in U.S. Pat. No. 5,015,580 (soy), U.S. Pat. No. 5,550,318 (maize), U.S. Pat. No. 5,538,880 (maize), U.S. Pat. No. 6,153,812 (wheat), U.S. Pat. No. 6,160,208 (maize), U.S. Pat. No. 6,288,312 (rice) and U.S. Pat. No. 6,399,861 (maize), and U.S. Pat. No. 6,403,865 (maize).
[0041] Detailed procedures for Agrobacterium-mediated transformation of plants, especially crop plants, include, for example, procedures disclosed in U.S. Pat. Nos. 5,004,863, 5,159,135, 5,518,908, 5,846,797, and 6,624,344 (cotton); U.S. Pat. Nos. 5,416,011, 5,569,834, 5,824,877, 5,914,451 6,384,301, and 7,002,058 (soy); U.S. Pat. Nos. 5,591,616 5,981,840, and 7,060,876 (maize); U.S. Pat. Nos. 5,463,174 and 5,750,871 (Brassica species, including rapeseed and canola), and in U.S. Patent Application Publications 2004/0244075 (maize), 2004/0087030 (cotton) and 2005/0005321 (soybean). Additional procedures for Agrobacterium-mediated transformation are disclosed in W09506722 (maize). Similar methods have been reported for many plant species, both dicots and monocots, including, among others, peanut (Cheng et al., Plant Cell Rep., 15:653, 1996); asparagus (Bytebier et al., Proc. Natl. Acad. Sci. U.S.A., 84:5345, 1987); barley (Wan and Lemaux, Plant Physiol., 104:37, 1994); rice (Toriyama et al., Bio/Technology, 6:10, 1988; Zhang et al., Plant Cell Rep., 7:379, 1988; wheat (Vasil et al., Bio/Technology,10:667, 1992; Becker et al., Plant J. , 5:299, 1994), alfalfa (Masoud et al., Transgen. Res., 5:313, 1996); Brassica species (Radke et al., Plant Cell Rep., 11:499-505, 1992); and tomato (Sun et al., Plant Cell Physiol., 47:426-431, 2006). Transgenic plant cells and transgenic plants can also be obtained by transformation with other vectors, such as but not limited to viral vectors (for example, tobacco etch virus (TEV), barley stripe mosaic virus (BSMV), and the viruses referenced in Edwardson and Christie, "The Potyvirus Group: Monograph No. 16 ", 1991, Agric. Exp. Station, Univ. of Florida), plasmids, cosmids, YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes) or any other suitable cloning vector, when used with an appropriate transformation protocol such as but not limited to bacterial infection (for example, with Agrobacterium as described above), binary bacterial artificial chromosome constructs, direct delivery of DNA (for example, via PEG-mediated transformation, desiccation/inhibition-mediated DNA uptake, electroporation, agitation with silicon carbide fibers, and microprojectile bombardment). It would be clear to one of ordinary skill in the art that various transformation methodologies can be used and modified for production of stable transgenic plants from any number of plant species of interest. For example the construction of stably inherited recombinant DNA constructs and mini-chromosomes can be used as vectors for the construction of transgenic plants (U.S. Pat. No. 7,235,716).
[0042] Plants of the present invention include, but are not limited to, Acacia, alfalfa, aneth, apple, apricot, artichoke, arugula, asparagus, avocado, banana, barley, beans, beet, blackberry, blueberry, broccoli, brussels sprouts, cabbage, canola, cantaloupe, carrot, cassava, cauliflower, celery, cherry, cilantro, citrus, clementine, coffee, corn, cotton, cucumber, Douglas fir, eggplant, endive, escarole, eucalyptus, fennel, figs, forest trees, gourd, grape, grapefruit, honey dew, jicama, kiwifruit, lettuce, leeks, lemon, lime, loblolly pine, mango, melon, mushroom, nut, oat, okra, onion, orange, an ornamental plant, papaya, parsley, pea, peach, peanut, pear, pepper, persimmon, pine, pineapple, plantain, plum, pomegranate, poplar, potato, pumpkin, quince, radiata pine, radicchio, radish, rapeseed, raspberry, rice, rye, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugarbeet, sugarcane, sunflower, sweet potato, sweetgum, tangerine, tea, tobacco, tomato, turf, a vine, watermelon, wheat, yams, and zucchini. Crop plants are defined as plants which are cultivated to produce one or more commercial products. Examples of such crops or crop plants include but are not limited to soybean, canola, rape, cotton (cottonseeds), peanut, sunflower, pigeon pea, chickpea, and the like, and grains such as corn, wheat, rice, oat, millet, and rye, and the like. Rape, rapeseed and canola are used synonymously in the present disclosure.
[0043] Transformation methods to provide transgenic plant cells and transgenic plants containing stably integrated recombinant DNA are preferably practiced in tissue culture on media and in a controlled environment. Recipient cell targets include but are not limited to meristem cells, callus, immature embryos or parts of embryos, gametic cells such as microspores, pollen, sperm, and egg cells. Any cell from which a fertile plant can be regenerated is contemplated as a useful recipient cell for practice of the invention. Callus can be initiated from various tissue sources, including, but not limited to, immature embryos or parts of embryos, seedling apical meristems, microspores, and the like. Those cells which are capable of proliferating as callus can serve as recipient cells for genetic transformation. Practical transformation methods and materials for making transgenic plants of this invention (for example, various media and recipient target cells, transformation of immature embryos, and subsequent regeneration of fertile transgenic plants) are disclosed, for example, in U.S. Pat. Nos. 6,194,636 and 6,232,526 and U.S. Patent Application Publication 2004/0216189.
[0044] In general transformation practice, DNA is introduced into only a small percentage of target cells in any one transformation experiment. Marker genes are generally used to provide an efficient system for identification of those cells that are transformed by a transgenic DNA construct. Preferred marker genes provide selective markers which confer resistance to a selective agent, such as an antibiotic or herbicide. Any of the antibiotics or herbicides to which a plant cell may be resistant can be a useful agent for selection. Potentially transformed cells are exposed to the selective agent. In the population of surviving cells will be those cells where, generally, the resistance-conferring gene is expressed at sufficient levels to permit cell survival in the presence of the selective agent. Cells can be tested further to confirm integration of the recombinant DNA. Commonly used selective marker genes include those conferring resistance to antibiotics such as kanamycin or paromomycin (nptII), hygromycin B (aph IV), gentamycin (aac3 and aacC4) and glufosinate (bar or pat), glyphosate (EPSPS), and dicamba (dicamba monooxygenase). Examples of useful selective marker genes and selection agents are illustrated in U.S. Pat. Nos. 5,550,318, 5,633,435, 5,780,708, and 6,118,047. Screenable markers or reporters, such as markers that provide an ability to visually identify transformants can also be employed. Non-limiting examples of useful screenable markers include, for example, a gene expressing a protein that produces a detectable color by acting on a chromogenic substrate (for example, beta-glucuronidase, GUS, uidA, or luciferase, luc) or that itself is detectable, such as green fluorescent protein (GFP, gfp) or an immunogenic molecule. Those of skill in the art will recognize that many other useful markers or reporters are available for use.
Trait Stacking and Breeding:
[0045] The recombinant DNA constructs of the invention can be stacked with other recombinant DNA for imparting additional agronomic traits (such as in the case of transformed plants, traits including but not limited to herbicide resistance, insect resistance, cold germination tolerance, water deficit tolerance, enhanced yield, enhanced quality, fungal, viral, and bacterial disease resistance) for example, by expressing other transgenes. The recombinant DNA constructs of the present invention can also be transformed into plant varieties that carry natural pest or pathogen resistance genes to enhance the efficacy of the resistance phenotype. Constructs for coordinated decrease and/or increase of gene expression are disclosed in U.S. Patent Application Publication 2004/0126845 A1. Seeds of transgenic, fertile plants can be harvested and used to grow progeny generations, including hybrid generations, of transgenic plants of this invention that include the recombinant DNA construct in their genome. Thus, in addition to direct transformation of a plant with a recombinant DNA construct of this invention, transgenic plants of the invention can be prepared by crossing a first plant having the recombinant DNA with a second plant lacking the construct. For example, the recombinant DNA can be introduced into a plant line that is amenable to transformation to produce a transgenic plant, which can be crossed with a second plant line to introgress the recombinant DNA into the resulting progeny. A transgenic plant of the invention can be crossed with a plant line having other recombinant DNA or naturally occurring genetic regions that confers one or more additional trait(s) (such as, but not limited to, herbicide resistance, pest or disease resistance, environmental stress resistance, modified nutrient content, and yield improvement) to produce progeny plants having recombinant DNA that confers both the desired target sequence expression behavior and the additional trait(s). Typically, in such breeding for combining traits the transgenic plant donating the additional trait is a male line and the transgenic plant carrying the base traits is the female line. The progeny of this cross segregate such that some of the plant will carry the DNA for both parental traits and some will carry DNA for one parental trait; such plants can be identified by markers associated with parental recombinant DNA. Progeny plants carrying DNA for both parental traits can be crossed back into the female parent line multiple times, for example, usually 6 to 8 generations, to produce a progeny plant with substantially the same genotype as one original transgenic parental line but for the recombinant DNA of the other transgenic parental line.
[0046] The transgenic plant, plant part, seed or progeny plants of the present invention can be processed into products useful in commerce. These products include but are not limited to meal, flour, oil, hay, starch, juice, protein extract, and fiber.
EXAMPLES
[0047] The following examples are included to illustrate embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
Example 1
[0048] The example illustrates the surprising nematicidal efficacy of various methylketones. Methylketones of various chain lengths were tested in vitro against C. elegans L1 and L4 larvae and M. incognita pre-parasitic J2 larvae, the dispersal larval stage found in the soil. As illustrated in Table 1, nematicidal activity was observed for the medium-length methylketones (10-14 carbon chain lengths). The table shows the in vitro IC30 values (in parts per million) of various methylketones effective against C. elegans L1 and L4 larvae and M. incognita J2 larvae. IC30 is defined as the concentration of the methylketone at which 30 percent of the nematodes are killed after an exposure of 4 hours for C. elegans and 24 hours for M. incognita.
TABLE-US-00001 TABLE 1 In vitro efficacy of various methylketones on nematodes. C. elegans M. incognita Compound vs.L1 vs.L4 vs.J2 2-heptanone >400 >400 400 2-nonanone 12.5 >400 200 2-decanone 6.3 >400 25 2-undecanone 6.3 25 50 2-dodecanone 3.2 25 50 2-tridecanone 3.2 12.5 50 2-tetradecanone 3.2 >100 25 2-pentadecanone 12.5 >100 400
[0049] Whole plant assays were used to determine the efficacy of the methylketones on the infection of soybean plants and tomato plants by nematodes, H. glycines and M. incognita, respectively. The seeds were planted in 100 percent sand in two-inch square plastic pots and grown to a sufficient size for treatment. Methylketone chemical treatment was applied when the soybean plants showed the first trifoliate beginning to emerge and when the tomato plants reached the 2-3 leaf stage. Following methylketone treatment, nematodes were inoculated into each pot and for soybeans are then incubated for 28 days before harvest and for tomatoes incubated 21 days before harvest.
[0050] To each of four pots, five milliliters of the appropriate chemical solution is applied to the surface making sure to avoid contact with the base of the plant. Immediately following the chemical application, the pot surface is wetted sufficiently to water in the chemical. One milligram of chemical per four pots is approximately equivalent to one kilogram per hectare of chemical. A standard test uses four replications. For rates above 2 kg/ha, the desired amount of chemical is weighed into a 30 ml vial (example: 8 kg/ha rate=8 mg chemical in 30 ml vial). The chemical is dissolved in 2 ml of an appropriate solvent, generally acetone. For rates below 2 kg/ha, 2 milligrams of chemistry is weighed into the vial and dissolved in 2 ml of the solvent. The appropriate amount of chemical concentrate is then applied into a separate 30 ml vial and solvent is added to bring the volume to 2 ml (example 0.5 kg/ha-0.5 ml of concentrate+1.5 ml solvent). Each dissolved concentrate is then brought to a total of 20 milliliters using 0.05% Triton X-100 surfactant solution.
[0051] Nematode eggs, either SCN or RKN, are added to distilled water to create a concentration of 1000 vermiform eggs per liter of water. At least four hours after chemical treatment the eggs are applied to the treated pots plus non-treated check plants. A small hole about 1 cm deep is punched into the pot surface. One milliliter of the nematode egg slurry is applied into the hole. Immediately afterwards the hole is gently covered. Watering of the test plants is then restricted to a minimum volume needed to prevent wilting for a period of 24 hours. After the 24 hour restricted watering, normal sub-irrigation watering is done for the duration of the test.
[0052] The 2-undecanone, 2-tridecanone, and 2-pentadecanone are tested in greenhouse studies against M. incognita infection of tomato roots in sand. The tomato plants are commercial varieties sensitive to nematode infection (e.g., Mountain Spring) and do not accumulate the methylketones that are found in the leaf trichomes of some wild tomato species. Shown in Table 2 is a high level of nematicidal activity of various methylketones observed against root knot nematode (M. incognita) inoculated into treated pots containing tomato plants. 2-tridecanone is highly effective at controlling nematode-induced galling at both 40 kilograms per hectare (kg/ha) (100% control) and 8 kg/ha (97% control), while 2-undecanone and 2-pentadecanone also demonstrated nematode control. The listed kilograms/hectare (kg/ha) rating is based upon the surface area of the test pots; 1 kg/ha equates to about 1.65 mg compound per kilogram of soil in these assays.
TABLE-US-00002 TABLE 2 Activity of various methylketones on root knot nematode disease. % Galled Compound Rate (kg/ha) Roots % Control 2-undecanone 40 21 65% 2-undecanone 8 39 35% 2-tridecanone 40 0 100% 2-tridecanone 8 2 97% 2-pentadecanone 40 38 37% 2-pentadecanone 8 45 25% No Compound added -- 60 NA
[0053] 2-tridecanone was also assayed in the greenhouse for control of soybean cyst nematode (H. glycines) in soybeans (Table 3). Nematode control (#cysts/plant) is observed at 40 kg/ha (96% control relative to the non-treated) and 8 kg/ha (80% control relative to the non-treated) when 2-tridecanone was applied as a soil drench prior to nematode inoculation.
TABLE-US-00003 TABLE 3 Efficacy of methylketone on cyst nematode infection of soybean Rate Compound (kg/ha) #cysts/plant % Control 2-tridecanone 40 2 96% 2-tridecanone 8 10 80% No Compound added -- 49 NA
Example 2
[0054] This example provides descriptions of compositions in use or contemplated for use in controlling plant parasitic nematodes singularly or in any combination. Table 4 provides a list of the compositions. A crop transformation base vector comprising selection expression cassettes and elements necessary for the maintenance of the plasmid in a bacterial cell is used to assemble DNA segments (promoters, leaders, introns, 3'UTR) that provide regulatory activity when operably linked to DNA segments that provide functionality in the present invention. The assembly of these DNA segments can be accomplished using methods known in the art of recombinant DNA technology. DNA coding sequences of the present invention such as any one or more of the DNA molecules identified as SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 59, 60, and 61 are cloned and inserted into an expression cassette or inserted into operable linkage with another coding sequence or genetic element of an expression cassette. Other genetic elements can be selected and tested by those skilled in the art that provide functional expression of a methylketone in plant tissues.
TABLE-US-00004 TABLE 4 Descriptions of genetic elements. SEQ ID NO: Name Description SEQ ID NO: 1 MKS1a A codon-optimized polynucleotide sequence variant for L. hirsutum methylketone synthase SEQ ID NO: 2 MKS1b A codon-optimized polynucleotide sequence variant for L. hirsutum methylketone synthase SEQ ID NO: 3 MKS1 Amino acid sequence of the methylketone synthase protein from L. hirsutum SEQ ID NO: 4 LhMKS1 Polynucleotide sequence for a codon- optimized L. hirsutum methylketone synthase SEQ ID NO: 5 LhMKS1 protein Amino acid sequence variant of the variant methylketone synthase protein from L. hirsutum SEQ ID NO: 6 LsMKS1 Polynucleotide sequence for a codon- optimized Lycopersicon esculentum (Solanum lycopersicum) methylketone synthase SEQ ID NO: 7 LsMKS1 protein Amino acid sequence of the methylketone synthase protein from L. esculentum SEQ ID NO: 8 AtCTP2 A polynucleotide sequence encoding a chloroplast transit peptide from A. thaliana EPSPS protein SEQ ID NO: 9 AtCTP2 protein Amino acid sequence of the chloroplast transit peptide from A. thaliana EPSPS protein SEQ ID NO: 10 PhCTP4 A polynucleotide sequence encoding a chloroplast transit peptide from Petunia hybrida EPSPS protein SEQ ID NO: 11 PhCTP4 protein Amino acid sequence of the chloroplast transit peptide from Petunia hybrida EPSPS protein SEQ ID NO: 12 CTP2-MKS1a Polynucleotide sequence of the AtCTP2 chloroplast transit peptide fused to the MKS1a codon optimized sequence encoding methylketone synthase SEQ ID NO: 13 CTP2-MKS1a Amino acid sequence of the protein heterologous AtCTP2-MKS1 fusion protein SEQ ID NO: 14 CTP4-MKS1a Polynucleotide sequence of the PhCTP4 chloroplast transit peptide fused to the MKS1a codon optimized sequence encoding methylketone synthase SEQ ID NO: 15 CTP4-MKS1a Amino acid sequence of the protein heterologous PhCTP4-MKS1a fusion protein SEQ ID NO: 16 CTP2-MKS1b Polynucleotide sequence of the CTP2 chloroplast transit peptide fused to the MKS1b codon optimized sequence encoding methylketone synthase SEQ ID NO: 17 CTP2-MKS1b Amino acid sequence of the protein heterologous AtCTP2-MKS1b fusion protein SEQ ID NO: 18 CTP4-MKS1b Polynucleotide sequence of the PhCTP4 chloroplast transit peptide fused to the MKS1b codon optimized sequence encoding methylketone synthase SEQ ID NO: 19 CTP4-MKS1b Amino acid sequence of the protein heterologous PhCTP4-MKS1b fusion protein SEQ ID NO: 20 CTP2-LhMKS1 Polynucleotide sequence of the AtCTP2 chloroplast transit peptide fused to the LhMKS1 sequence encoding methylketone synthase SEQ ID NO: 21 CTP2-LhMKS1 Amino acid sequence of the protein heterologous AtCTP2-LhMKS1 fusion protein SEQ ID NO: 22 CTP4-LhMKS1 Polynucleotide sequence of the PhCTP4 chloroplast transit peptide fused to the LhMKS1 sequence encoding methylketone synthase SEQ ID NO: 23 CTP4-LhMKS1 Amino acid sequence of the protein heterologous PhCTP4-LhMKS1 fusion protein SEQ ID NO: 24 CTP2-LsMKS1 Polynucleotide sequence of the AtCTP2 chloroplast transit peptide fused to the LsMKS1 sequence encoding methylketone synthase SEQ ID NO: 25 CTP2-LsMKS1 Amino acid sequence of the protein heterologous AtCTP2-LsMKS1 fusion protein SEQ ID NO: 26 CTP4-LsMKS1 Polynucleotide sequence of the PhCTP4 chloroplast transit peptide fused to the LsMKS1 sequence encoding methylketone synthase SEQ ID NO: 27 CTP4-LsMKS1 Amino acid sequence of the protein heterologous PhCTP4-LsMKS1 fusion protein SEQ ID NO: 28 CTP2-MKS1a_sN Polynucleotide sequence of the AtCTP2 chloroplast transit peptide fused to the MKS1s sequence encoding an Alanine to Serine active site variant of methylketone synthase SEQ ID NO: 29 CTP2-MKS1a_sN Amino acid sequence of the AtCTP2 protein chloroplast transit peptide fused to the MKS1s sequence having an Alanine to Serine active site variant of methylketone synthase SEQ ID NO: 30 CTP2-MKS1a_Ad Polynucleotide sequence of the AtCTP2 chloroplast transit peptide fused to the MKS1s sequence encoding an Asparagine to Aspartic acid active site variant of methylketone synthase SEQ ID NO: 31 CTP2-MKS1a_Ad Amino acid sequence of the AtCTP2 protein chloroplast transit peptide fused to the MKS1s sequence having an Asparagine to Aspartic acid active site variant of methylketone synthase SEQ ID NO: 32 CTP2-MKS1a_sd Polynucleotide sequence of the AtCTP2 chloroplast transit peptide fused to the MKS1s sequence encoding a double variant Alanine to Serine and Asparagine to Aspartic acid active site variant of methylketone synthase SEQ ID NO: 33 CTP2-MKS1a_sd Amino acid sequence of the AtCTP2 protein chloroplast transit peptide fused to the MKS1s sequence having a double variant Alaninc to Serine and Asparagine to Aspartic acid active site variant of methylketone synthase SEQ ID NO: 34 CTP2-LsMKS1_sN Polynucleotide sequence of the AtCTP2 chloroplast transit peptide fused to the LsMKS1s sequence encoding an Alanine to Serine active site variant of methylketone synthase SEQ ID NO: 35 CTP2-LsMKS1_sN Amino acid sequence of the AtCTP2 protein chloroplast transit peptide fused to the LsMKS1s sequence encoding an Alanine to Serine active site variant of methylketone synthase SEQ ID NO: 36 CTP2-LsMKS1_Ad Polynucleotide sequence of the AtCTP2 chloroplast transit peptide fused to the LsMKS1s sequence encoding an Asparagine to Aspartic acid active site variant of methylketone synthase SEQ ID NO: 37 CTP2-LsMKS1_Ad Amino acid sequence of the AtCTP2 protein chloroplast transit peptide fused to the LsMKS1s sequence encoding an Asparagine to Aspartic acid active site variant of methylketone synthase SEQ ID NO: 38 CTP2-LsMKS1_sd Polynucleotide sequence of the AtCTP2 chloroplast transit peptide fused to the LsMKS1s sequence encoding a double variant Alanine to Serine and Asparagine to Aspartic acid active site variant of methylketone synthase SEQ ID NO: 39 CTP2-LsMKS1_sd Amino acid sequence of the AtCTP2 protein chloroplast transit peptide fused to the LsMKS1s sequence having a double variant Alanine to Serine and Asparagine to Aspartic acid active site variant of methylketone synthase SEQ ID NO: 40 LhACP1-PI126449 Polynucleotide sequence of an Acyl carrier protein ACP1 coding sequence from PI126449 SEQ ID NO: 41 LhACP1-PI126449 Amino acid sequence of an Acyl carrier protein protein ACP1 from PI126449 SEQ ID NO: 42 LhACP2-PI126449 Polynucleotide sequence of an Acyl carrier protein ACP2 coding sequence from PI126449 SEQ ID NO: 43 LhACP2-PI126449 Amino acid sequence of an Acyl carrier protein protein ACP2 from PI126449 SEQ ID NO: 44 LhACP1-LA1777 Polynucleotide sequence of an Acyl carrier protein ACP1 coding sequence from LA1777 SEQ ID NO: 45 LhACP1-LA1777 Amino acid sequence of an Acyl carrier protein protein ACP1 from LA1777 SEQ ID NO: 46 LeACP2 Polynucleotide sequence of an Acyl carrier protein ACP2 from L. esculentum SEQ ID NO: 47 LeACP2 protein Amino acid sequence of an Acyl carrier protein ACP2 from L. esculentum SEQ ID NO: 48 StACP2 Polynucleotide sequence of an Acyl carrier protein ACP2 from Solanum tuberosum SEQ ID NO: 49 StACP2 protein Amino acid sequence of an Acyl carrier protein ACP2 from Solanum tuberosum SEQ ID NO: 50 ScACP2 Polynucleotide sequence of an Acyl carrier protein ACP2 from Solanum chacoense SEQ ID NO: 51 ScACP2 protein Amino acid sequence of an Acyl carrier protein ACP2 from Solanum chacoense SEQ ID NO: 52 NtACP2 Polynucleotide sequence of an Acyl carrier protein ACP2 from Nicotiana tabacum SEQ ID NO: 53 NtACP2 protein Amino acid sequence of an Acyl carrier protein ACP2 from Nicotiana tabacum SEQ ID NO: 54 PhACP2 Polynucleotide sequence of an Acyl carrier protein ACP2 from Petunia hybrida SEQ ID NO: 55 PhACP2 protein Amino acid sequence of an Acyl carrier protein ACP2 from Petunia hybrida SEQ ID NO: 56 CaACP2 Polynucleotide sequence of an Acyl carrier protein ACP2 from Capsicum annum SEQ ID NO: 57 CaACP2 protein Amino acid sequence of an Acyl carrier protein ACP2 from Capsicum annum SEQ ID NO: 58 LeMKS1 homolog Polynucleotide sequence of an MKS1 homolog from L. esculentum SEQ ID NO: 59 StMKS1 homolog Polynucleotide sequence of an MKS1 homolog from S. tuberosum SEQ ID NO: 60 NtMKS1 homolog Polynucleotide sequence of an MKS1 homolog from N. tabacum SEQ ID NO: 61 CaMKS1 homolog Polynucleotide sequence of an MKS1 homolog from C. annum SEQ ID NO: 62 Act7 intron 558 nucleotide actin 7 intron sequence from A. thaliana
Example 3
[0055] This example describes generation of tomato or soybean transgenic hairy roots expressing MKS and the nematode infection assay. Hairy root cultures allow the rapid growth of root tissue on a large scale which can be used for testing the effectiveness of the gene of interest as set forth herein, for controlling plant parasitic nematode infestation of a crop plant. Hairy roots are characterized by fast growth, frequent branching, plagiotropism, and the ability to synthesize the same compounds as the roots of the intact plant (David et al., Biotechnology 2:73-76, 1984). Transfer and integration of the genes located on the root-inducing plasmid Ri of Agrobacterium rhizogenes into the plant genome and their expression therein (White and Nester, J Bacteriol., 141:1134-1141, 1980). These types of roots continue to grow in vitro on hormone-free medium and also exhibit a high degree of genetic stability (Aird et al., Plant Cell Tiss. Org. Cult. 15: 47-57, 1988). The natural ability of the soil bacterium A. rhizogenes to transform genes into a host plant genome results in roots being formed at the site of infection. Infection of the plant with A. rhizogenes, leads to the integration and expression of T-DNA in the plant genome, which causes development of a hairy root. Hairy root cultures grow rapidly, show plagiotropic root growth and are highly branched on hormone-free medium.
[0056] For soybean hairy roots, A. rhizogenes strain K599 is grown and maintained on LB, minimal A, or yeast extract and peptone (YEP) media. Methods for generation of transgenic tomato hairy root cultures for evaluating lesion or root knot nematodes are not significantly different other than the use of A. rhizogenes D1 strain. Soybean seeds are surface-sterilized by setting in chlorine gas under controlled conditions for 12-16 hours, and then aerating in a clean air hood for at least 30 minutes. Seeds are germinated in Petri dishes containing 1/4 MS.
[0057] The hypocotyl or cotyledons of 6-days-old seedlings are wounded using a scalpel. The wounded cotyledons are then immersed in freshly grown A. rhizogenes containing the construct and subsequently vacuum infiltrated. Cotyledons are cultured under the same conditions used for seed germination with the exception that the antibiotic cefotaxime is added to the 1/4 MS agar plates to prevent the A. rhizogenes from subsequent growth. Adventitious roots are excised from hypocotyls or cotyledons inoculated with A. rhizogenes. The putative transformed roots are cultured on Gamborg's B-5 agar containing 3% sucrose plus 3 g/1 Gelrite, BASTA, and cefotaxime). Roots passing selection are transferred to fresh media and maintained. Cultured roots are maintained in an incubator, without light, set at 24-30° C. Roots are maintained on Gamborg's B-5 agar. A piece of root tip is excised and transferred to fresh medium every 2-4 weeks.
[0058] Following hairy root line selection, roots for the plant nematode bioassay are transferred to fresh plates containing Gamborg's B-5 medium and allowed to grow for approximately two weeks to provide sufficient tissue for nematode infection before inoculation with a mixed population of root lesion nematodes or second-stage juveniles of soybean cyst nematode (SCN) or root knot nematode (RKN). Individual hairy root tips are placed on infection plates. 20 plates are used for testing transformed roots for reaction to lesion, SCN or RKN. Each plate contains a transformed root from a separate integration. An additional 20 plates containing a transformed lesion susceptible, SCN-susceptible or RKN-susceptible control and an additional 20 plates containing a transformed SCN-resistant or RKN-resistant control are also tested. Transformed controls are empty vectors. Plates are then inoculated with approximately 400 axenic lesion worms or 1000 sterile H. glycines J2s or 450 sterile M. incognita J2s and incubated at 26-28 ° C. (SCN or RKN) or 25 ° C. or 30 ° C. (lesion nematode).
[0059] Approximately six weeks after inoculation with M. incognita or five weeks after inoculation with H. glycines, infected tomato or soybean hairy roots are removed from the agar plates and the number of galls or cysts counted. For SCN hairy root plates cysts are counted directly, whereas for RKN gall numbers may be estimated. Gall scores are weighted estimates based on size. A scale is created at the beginning of scoring process. The smallest galls are given a score of 1 and as the galled areas become larger the gall score increases. The scale is then used to rate each gall on each plate in the experiment. Egg numbers are also scored at 42 days for RKN infections in tomato hairy roots. At 42 days post-infection, plates are microwaved and sieved to collect the roots. The roots are weighed, then blended in a 10% bleach solution and poured over a series of sieves to remove the root debris and collect the eggs. Eggs are removed from each plate and are counted. For lesion nematodes, plates are harvested after approximately 56 days by placing roots in glass bowls filled with sterilized water containing 50 mg/L carbenicillin and 50 mg/L kanamycin. After 9-10 days to allow the worms to exit the roots, the worms are counted under a microscope. To determine weights, root bowls are then microwaved to melt the agar and roots are collected with a sieve. The extra water is absorbed with a paper towel and the root weights recorded.
[0060] Axenic lesion, SCN and RKN larvae are prepared for use with the hairy root culture system. Axenic SCN J2s are produced as follows. Clean soybean cyst nematode eggs (i.e., eggs with soil and other debris removed) are collected and placed in a 50 ml centrifuge vial containing 30 ml of a 10% bleach solution. The bleach solution is mildly agitated and then left to settle for 2-3 minutes. The vial is mildly agitated again to re-suspend the eggs and then centrifuged for 1 minute at 1000 rpm. Under a sterile hood, the bleach solution is removed into a receptacle and 25 ml of sterile water is added into the vial of eggs. The vial is recapped under the sterile hood, mildly agitated to re-suspend the eggs and centrifuged for 1 minute at 1000 rpm. Under the sterile hood, this liquid is poured off and 25 ml of sterile water is again placed in the vial. The vial is recapped under the sterile hood and the process of agitation and centrifugation repeated. This process of washing the eggs with sterile water is repeated approximately 4 times to thoroughly rinse the bleach from the eggs. Following the last rinse under the sterile hood the liquid is removed leaving about 1-2 ml of egg concentrate. Axenic eggs are hatched by incubating them on the surface of moist filter paper resting in a solution of 5 mM zinc sulfate just deep enough to cover the surface of the filter paper. After 2-3 days J2 larvae are collected in the solution underneath the filter paper. J2s are centrifuged and further cleaned using chlorhexidine (Atkinson et al., J. Nematol. 28:209-215, 1996).
[0061] Axenic RKN larvae are prepared by collecting eggs by placing chopped RKN infected roots into a blender with a sufficient quantity of 10% bleach solution. The blender is pulsed on/off for 5 second intervals. This process is repeated 5-6 times. The root slurry is the passed through a series of sieves where the eggs and small debris are collected in a 500 micron sieve. Any remaining bleach solution is thoroughly rinsed from this egg/debris. Twenty milliliters of the egg/debris is added to a 50 ml conical tube and 20 ml of a 40% sucrose solution is added into the bottom of the tube, bringing the total volume to 40 milliliters. This solution is then centrifuged at 3750 rpm for 5 minutes to separate the eggs from the debris. After centrifugation, the eggs are removed and thoroughly rinsed to remove any remaining sucrose solution. Eggs are then placed into a hatch bowl containing filter paper moistened with just enough aerated tap water to cover the eggs. After 1-2 days J2 larvae are collected in the solution underneath the filter paper. J2 larvae are centrifuged and further cleaned using chlorhexidine (Atkinson et al. (1996, see above).
[0062] Axenic lesion larvae are prepared from lesion nematodes grown on corn explant plates. The nematodes are harvested by placing roots with medium onto filter paper supported by a wire sieve in a sterilized glass bowl which has been filled with sterilized water containing 50 mg/L carbenicillin and 50 mg/L kanamycin. The amount of the water is sufficient to submerge the agar, and the bowls are stored at room temperature (25° C.) for two days. The sieve is removed and the solution poured into a 50 ml conical tube, which was then centrifuged for 5 minutes at 3500 x g at room temperature. After the worms settle to the bottom of the tube (further 15 minute incubation), the supernatant is decanted. Sterilized water is then added to the worm pellet containing 12 mg/L of the antifungal compound Imazilil and 50 mg/L kanamycin.
[0063] The following are results found after the transgenic expression of various combinations of promoters, transit peptides and methylketone synthase coding sequences for control of plant parasitic nematode infections in hairy roots. SCN cysts in the transgenic soybean hairy root inoculated plates are counted and the average number of cysts per replication (Rep 1 and Rep 2) tabulated. The results shown in Table 5 demonstrate that transgenic soybean roots containing the chimeric CTP-methylketone synthase coding region provides resistance to SCN infection, where all treatments having a heterologous CTP fused to a methylketone synthase show a reduction in the average cyst counts compared to the transgenic empty vector control, 4211. The constructs that lack a heterologous CTP (FMV-LsMKS1 and FMV-LhMKS1) do not show a reduction in cyst counts.
TABLE-US-00005 TABLE 5 Transgenic soybean roots expressing MKS reduce SCN infection (cysts). control Test constructs Test constructs D4211 E35S-ctp2/mks1 E35S-ctp4/mks1 Rep 1 31.4 12.6 21.9 Rep 2 18.3 9.1 -- 4211 E35Sp-ctp2/mks1 E35Sp-ctp4/mks1 Rep 1 19.4 17.6 13 Rep 2 18.4 11.2 13.7 4211 FMV-ctp2/LsMKS1 E35Sp-ctp2/LsMKS1 Rep 1 33 16.5 19.3 Rep 2 25.4 15 22.9 4211 FMV-LsMKS1 (no CTP) FMV-LhMKS1 (no CTP) Rep 1 21 27 23 Rep 2 18 17 18
[0064] As can be seen in table 6 below, the expression of MKS constructs containing CTP leaders either with or without certain targeted active site mutations leads to reduction in the ability of root knot nematode to infect plants roots. In addition to the elements listed in the table above, the constructs shown contain a ˜540 nucleotide actin 7 intron incorporated into the 5' untranslated region (UTR) of the fused methylketone synthase transcript and a visual fluorescent DsRED marker (driven by the FMV promoter) co-expressed in the T-DNA, downstream of the MKS open reading frame.
TABLE-US-00006 TABLE 6 Transgenic tomato roots expressing MKS reduce root knot nematode infection (eggs). control Test construct Test construct Test construct 8221 E35sp-ctp2/LsMKS1 E35sp-ctp2/ E35sp-ctp2/ LsMKS1_sN LsMKS1_sd 1884.5 968.5 -- 1333.4 3059.8 1927.4 -- 1558 716.7 -- 283.3 298.1
[0065] As can be seen in table 7 below, the expression of MKS constructs containing CTP leaders either with or without certain targeted active site mutations leads to reduction in the ability of root lesion nematodes to infect plants roots. In addition to the elements listed in the table above, the constructs shown contain a ˜540 nucleotide actin 7 intron incorporated into the 5' untranslated region (UTR) of the fused methylketone synthase transcript and a visual fluorescent DsRED marker (driven by the FMV promoter) co-expressed in the T-DNA, downstream of the MKS open reading frame.
TABLE-US-00007 TABLE 7 Transgenic tomato roots expressing MKS reduce lesion nematode infection (larvae). control Test construct Test construct Test construct 8221 E35sp-ctp2/LsMKS1 E35sp-ctp2/ E35sp-ctp2/ LsMKS1_sN LsMKS1_sd 5857.8 4842.4 3926.2 4897.2
Example 4
[0066] This example describes a plant transformation method useful in producing transgenic soybean plants and transgenic seed. Other methods are known in the art of plant cell transformation that can be applied using the DNA constructs of the present invention.
[0067] For Agrobacterium mediated transformation, soybean seeds are germinated overnight and the meristem explants excised (see U.S. Pat. No. 7,002,058). The meristems and the explants are placed in a wounding vessel. Soybean explants and induced Agrobacterium cells from a strain containing plasmid DNA with the expression cassettes of the present invention and a plant selectable marker cassette are mixed within about 14 hours from the time of initiation of seed germination and wounded using sonication. Following wounding, explants are placed in co-culture for 2-5 days at which point they are transferred to selection media for 6-8 weeks to allow selection and growth of transgenic shoots. Trait positive shoots are harvested after approximately 6-8 weeks and placed into selective rooting media for 2-3 weeks. Shoots producing roots are transferred to the greenhouse and potted in soil. Shoots that remain healthy on selection but that do not produce roots are transferred to non-selective rooting media for an additional two weeks. Roots from any shoots that produce roots off selection are tested for expression of the plant selectable marker before they are transferred to the greenhouse and potted in soil. Additionally, a DNA construct can be transferred into the genome of a soybean cell by particle bombardment and the cell regenerated into a fertile soybean plant as described in U.S. Pat. No. 5,015,580.
[0068] Transgenic soybean plant cells are transformed with recombinant DNA of this invention. Progeny transgenic plants and seed of the transformed plant cells are selected that provide pest resistance, especially nematode resistance.
Example 5
[0069] A soybean cyst nematode pot assay is used to evaluate the resistance of transgenic soybean plants comprising the methylketone synthase coding sequence to infection by and reproduction of the soybean cyst nematode (Heterodera glycines) on roots. Three or four inch diameter square pots are filled with clean sand and watered thoroughly. Transgenic and control soybean seeds, or alternatively any rooted plant parts, are planted one per pot in the center of the pot and watered well to remove air pockets. The pots are incubated in the greenhouse or growth chamber at 20° C. to 30° C. until the plants reached a suitable age for inoculation. Soybeans started from seed are typically inoculated 2-3 weeks after planting, while transplants are inoculated 1-3 days after planting. The test inoculum consists of eggs from ripe H. glycines cysts collected from the soil and roots of infested soybean plants. A 250 micron mesh sieve is used to collect the cysts, which are then crushed in a Tenbroeck glass tissue homogenizer to release the eggs. The eggs are further purified by sieving and centrifugation over 40 percent sucrose solution at 4000 RPM for 5 minutes. Inoculum for an experiment consisted of water containing 500 vermiform eggs per mL. Five mL of the egg suspension is applied over the surface of the sand containing the test plants and the eggs arc lightly watered in. The test plants are then returned to the greenhouse or growth chamber and incubated for 3-4 weeks to allow for root infection and cyst formation. The roots are then harvested by gently removing the pot and sand and rinsing in water. The severity of nematode infection is measured by counting the number of nematode cysts adhering to the root system. Alternatively, the sand and roots could be diluted in water and passed over a 250 micron sieve to collect and concentrate the cysts for storage or counting.
Example 6
[0070] This example describes the detection and measurement of the recombinant DNA construct in the transgenic plant cell. Detecting or measuring transcription of the recombinant DNA construct in the transgenic plant cell of the invention can be achieved by any suitable method, including protein detection methods (for example, western blots, ELISAs, and other immunochemical methods), measurements of enzymatic activity, or nucleic acid detection methods (for example, Southern blots, northern blots, PCR, RT-PCR, fluorescent in situ hybridization). Such methods are well known to those of ordinary skill in the art as evidenced by the numerous handbooks available; see, for example, Joseph Sambrook and David W. Russell, "Molecular Cloning: A Laboratory Manual" (third edition), Cold Spring Harbor Laboratory Press, NY, 2001; Frederick M. Ausubel et al. (editors) "Short Protocols in Molecular Biology" (fifth edition), John Wiley and Sons, 2002; John M. Walker (editor) "Protein Protocols Handbook" (second edition), Humana Press, 2002; and Leandro Pena (editor) "Transgenic Plants: Methods and Protocols", Humana Press, 2004.
[0071] DNA sequence information provided by the invention allows for the preparation of relatively short DNA (or RNA) sequences having the ability to specifically hybridize to DNA sequences of the selected polynucleotides disclosed herein. The polynucleotides disclosed in the present invention include SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 59, 60, and 61. In these aspects, nucleic acid probes of an appropriate length are prepared. The ability of the nucleic acid probes to specifically hybridize to one or more of these gene coding sequences lends them particular utility in a variety of embodiments. Most importantly, the probes may be used in a variety of assays for detecting the presence of complementary sequences in a given sample.
[0072] In certain embodiments, it is advantageous to use oligonucleotide primers. The sequence of such primers is designed using a portion of a polynucleotide sequence of the present invention to be homologous or complementary to the sequence for use in detecting, amplifying a defined polynucleotide segment using PCR® technology (A Guide to Methods and Applications, Academic Press: San Diego, 1990). PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5,© (1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.). Primers and probes based on the sequences disclosed herein can be used to confirm and, if necessary, to modify the disclosed sequences by conventional methods, for example, by re-cloning and re-sequencing. Exemplary PCR reaction conditions may include: Component Amount/Volume required sub-library aliquot 1 μl Gene-specific primer 1, 1 μl (100 pmol, GenomeWalker®) Adaptor primer 1 (AP1), 1 μl dNTP mix (10 mM of each dNTP), 1 μl DMSO 2.5 μl (or 2-5% final concentration) 10× PCR buffer, 5 μl (final concentration of 1×) Amplitaq Gold®, 0.5 μl distilled water for final reaction volume of 50 μl reaction conditions for primary PCR:
[0073] A. 9 minutes at 95° C.;
[0074] B. 94° C. for 2 seconds, 70° C. for 3 minutes; repeat 94° C/70° C. cycling for total of 7 times;
[0075] C. 94° C. for 2 seconds, 65° C. for 3 minutes; repeat 94° C/65° C. cycling for total of 36 times;
[0076] D. 65° C. for 4 minutes as a final extension;
[0077] E. 10° C. for an extended incubation
[0078] NESTED PCR (secondary PCR reaction) Component Amount/Volume Required 1:50 dilution of the primary PCR reaction; 1 μl Gene-specific primer 2; 1 μl (100 pmol, GenomeWalker® Adaptor primer 2; 1 μl or 3 (AP2 or AP3), dNTP mix (10 mM of each dNTP); 1 μl DMSO; 2.5 μl 10× PCR buffer containing MgC12; 5 μl (final concentration of 1×) Amplitaq Gold®; 0.5 μl distilled water to final reaction volume of 50 μl reaction. Conditions for Nested PCR:
[0079] A. 9 minutes at 95° C.;
[0080] B. 94° C. for 2 seconds, 70° C. for 3 minutes; repeat 94° C/70° C. cycling for total of 5 times;
[0081] C. 94° C. for 2 seconds, 65° C. for 3 minutes; repeat 94° C/65° C. cycling for total of 24 times;
[0082] D. 65° C. for 4 minutes as a final extension;
[0083] E. 10° C. for an extended incubation.
PCR conditions can be modified from the described conditions by those skilled in the method to produce an amplicon.
[0084] Detection of foreign gene expression in transgenic plant is monitored by an immunological method for example ELISA (enzyme-linked immunosorbent assays) for a quantitative determination of the level of corresponding protein obtained. Quantitative determination of the encoded protein in the leaves of transgenic plants is performed using ELISA, for example as disclosed in Clark et al.,: ELISA Techniques. In: Weissbach A, Weissbach H (eds) Methods in Enzymology 118:742-766, Academic Press, Florida (1986).
[0085] All publications and patents referenced herein are intended to be herein incorporated by reference in their entirety.
Sequence CWU
1
1
621798DNAArtificial sequenceSynthetic sequence 1atggaaaagt ctatgtcacc
cttcgtcaag aaacacttcg tgcttgttca tactgctttc 60cacggtgcct ggtgctggta
caagatcgtc gcccttatga gaagttcagg ccacaacgtg 120accgcactgg acctcggggc
ctccgggata aaccccaaac aagctttgca gattcctaac 180ttcagtgatt atctttctcc
acttatggag tttatggcct ctcttcccgc taacgaaaag 240atcatactcg ttggtcatgc
tcttggtggt cttgccattt ctaaagcaat ggaaaccttc 300cctgagaaaa tatcagtggc
tgttttcttg tccggtctta tgcccggacc taatattgac 360gcaacaaccg tttgtaccaa
ggctgggtcc gccgtgttgg gccaactcga caactgcgtg 420acctacgaaa atggtcctac
taatccacct actaccctta tcgcaggacc taagtttctc 480gctacaaacg tgtaccacct
ctctccaata gaggatctgg ctcttgccac tgctttggtc 540aggcctcttt atctgtacct
cgctgaggac atatctaaag aagttgtgct ttcttctaag 600cgttatggtt ccgtgaagcg
tgtcttcata gtggcaacag agaatgatgc tcttaagaaa 660gagtttctca aactgatgat
cgagaagaat ccacctgatg aagtgaaaga aattgagggt 720tctgaccatg ttaccatgat
gtcaaagcct caacagttgt ttaccacatt gcttagtata 780gctaataagt ataaataa
7982798DNAartificial
sequenceSynthetic sequence 2atggaaaagt ctatgtcacc cttcgttaag aaacactttg
tattggtcca cactgctttc 60cacggggctt ggtgctggta caagattgtc gcattgatga
gatcttccgg tcacaacgtc 120actgctttgg acctgggagc atctggaatt aaccctaagc
aagcattgca aatcccaaac 180ttcagtgatt acctctctcc tcttatggaa ttcatggcca
gccttccagc taacgaaaag 240atcatcttgg tgggtcacgc ccttggtgga ttggcaatat
caaaggccat ggaaacattt 300ccagaaaaga ttagcgtggc tgtctttctg agtggcctta
tgcccggccc taacatcgac 360gcaactacag tctgcacaaa agctggttcc gctgtattgg
gacaacttga caattgcgtg 420acctacgaga atggtcccac taacccaccc accacattga
tcgcaggacc taagttcctt 480gctaccaacg tctaccattt gtctccaatt gaggacttgg
ctctggccac cgcacttgtt 540agacctttgt acctctatct tgcagaagat atttccaagg
aagtggtttt gtcttctaaa 600agatatggtt ctgtgaagag ggtgttcatc gttgccacag
agaatgatgc tctgaagaaa 660gagtttctca aattgatgat tgaaaagaat cctccagacg
aagtgaagga gatagaagga 720agtgaccatg ttactatgat gagtaagcct caacaacttt
tcactacact gctgtctatt 780gcaaacaaat acaagtaa
7983265PRTLycopersicon hirsutum 3Met Glu Lys Ser
Met Ser Pro Phe Val Lys Lys His Phe Val Leu Val 1 5
10 15 His Thr Ala Phe His Gly Ala Trp Cys
Trp Tyr Lys Ile Val Ala Leu 20 25
30 Met Arg Ser Ser Gly His Asn Val Thr Ala Leu Asp Leu Gly
Ala Ser 35 40 45
Gly Ile Asn Pro Lys Gln Ala Leu Gln Ile Pro Asn Phe Ser Asp Tyr 50
55 60 Leu Ser Pro Leu Met
Glu Phe Met Ala Ser Leu Pro Ala Asn Glu Lys 65 70
75 80 Ile Ile Leu Val Gly His Ala Leu Gly Gly
Leu Ala Ile Ser Lys Ala 85 90
95 Met Glu Thr Phe Pro Glu Lys Ile Ser Val Ala Val Phe Leu Ser
Gly 100 105 110 Leu
Met Pro Gly Pro Asn Ile Asp Ala Thr Thr Val Cys Thr Lys Ala 115
120 125 Gly Ser Ala Val Leu Gly
Gln Leu Asp Asn Cys Val Thr Tyr Glu Asn 130 135
140 Gly Pro Thr Asn Pro Pro Thr Thr Leu Ile Ala
Gly Pro Lys Phe Leu 145 150 155
160 Ala Thr Asn Val Tyr His Leu Ser Pro Ile Glu Asp Leu Ala Leu Ala
165 170 175 Thr Ala
Leu Val Arg Pro Leu Tyr Leu Tyr Leu Ala Glu Asp Ile Ser 180
185 190 Lys Glu Val Val Leu Ser Ser
Lys Arg Tyr Gly Ser Val Lys Arg Val 195 200
205 Phe Ile Val Ala Thr Glu Asn Asp Ala Leu Lys Lys
Glu Phe Leu Lys 210 215 220
Leu Met Ile Glu Lys Asn Pro Pro Asp Glu Val Lys Glu Ile Glu Gly 225
230 235 240 Ser Asp His
Val Thr Met Met Ser Lys Pro Gln Gln Leu Phe Thr Thr 245
250 255 Leu Leu Ser Ile Ala Asn Lys Tyr
Lys 260 265 4801DNAArtificial
sequenceSynthetic sequence 4atggctgaga agtctatgag tcctttcgtt aagaagcact
tcgttctagt tcacactgct 60ttccacggtg cttggtgctg gtacaagatt gttgctttga
tgcgtagttc tggtcataac 120gtgactgctc ttgatctcgg tgctagtggt attaacccaa
agcaagcctt gcaaatcccc 180aactttagcg attacttgtc accactcatg gagtttatgg
cctcccttcc tgccaacgag 240aagatcatac ttgttggaca tgctttagga gggctggcca
tctcaaaagc tatggaaacc 300ttccctgaga agataagcgt ggcagtattt ctctcgggcc
tcatgcctgg accaaacatt 360gacgcaacta ccgtctgtac caaggctgga tctgcagtct
tggggcagtt ggataactgc 420gtgacttatg agaatgggcc gacaaatcct ccaacaaccc
ttattgccgg acctaagttc 480ttggcaacga atgtctatca tctttctccc atcgaggatc
tggccctcgc tactgctctt 540gtccgcccac tttacctcta tctcgctgag gacatctcaa
aagaagttgt actttcatcc 600aagagatacg gctccgtgaa gagggttttc attgttgcga
cagagaatga tgcacttaag 660aaagaatttc tgaagctgat gatcgagaag aatccacctg
acgaagtgaa agaaatagaa 720ggctctgacc atgtgactat gatgtccaaa ccacaacagt
tgtttacaac gcttttgagt 780atcgcaaaca agtacaagta a
8015266PRTartificial sequenceSynthetic sequence
5Met Ala Glu Lys Ser Met Ser Pro Phe Val Lys Lys His Phe Val Leu 1
5 10 15 Val His Thr Ala
Phe His Gly Ala Trp Cys Trp Tyr Lys Ile Val Ala 20
25 30 Leu Met Arg Ser Ser Gly His Asn Val
Thr Ala Leu Asp Leu Gly Ala 35 40
45 Ser Gly Ile Asn Pro Lys Gln Ala Leu Gln Ile Pro Asn Phe
Ser Asp 50 55 60
Tyr Leu Ser Pro Leu Met Glu Phe Met Ala Ser Leu Pro Ala Asn Glu 65
70 75 80 Lys Ile Ile Leu Val
Gly His Ala Leu Gly Gly Leu Ala Ile Ser Lys 85
90 95 Ala Met Glu Thr Phe Pro Glu Lys Ile Ser
Val Ala Val Phe Leu Ser 100 105
110 Gly Leu Met Pro Gly Pro Asn Ile Asp Ala Thr Thr Val Cys Thr
Lys 115 120 125 Ala
Gly Ser Ala Val Leu Gly Gln Leu Asp Asn Cys Val Thr Tyr Glu 130
135 140 Asn Gly Pro Thr Asn Pro
Pro Thr Thr Leu Ile Ala Gly Pro Lys Phe 145 150
155 160 Leu Ala Thr Asn Val Tyr His Leu Ser Pro Ile
Glu Asp Leu Ala Leu 165 170
175 Ala Thr Ala Leu Val Arg Pro Leu Tyr Leu Tyr Leu Ala Glu Asp Ile
180 185 190 Ser Lys
Glu Val Val Leu Ser Ser Lys Arg Tyr Gly Ser Val Lys Arg 195
200 205 Val Phe Ile Val Ala Thr Glu
Asn Asp Ala Leu Lys Lys Glu Phe Leu 210 215
220 Lys Leu Met Ile Glu Lys Asn Pro Pro Asp Glu Val
Lys Glu Ile Glu 225 230 235
240 Gly Ser Asp His Val Thr Met Met Ser Lys Pro Gln Gln Leu Phe Thr
245 250 255 Thr Leu Leu
Ser Ile Ala Asn Lys Tyr Lys 260 265
6801DNALycopersicon esculentum 6atggctaaga agtctacttc acctttcgtg
aagaagcact tcgttctagt tcacactggt 60ttccacggtg cttggtgctg gtacaagatt
gttgctttga tgcgtagttc tggtcacaac 120gtgactgctc ttgatctagg tgctagtggt
attaacccta agcaagccct tgaaatccca 180catttcagcg actacttgtc cccactcatg
gagttcatgg ctagtttgcc ggccaatgag 240aagataattc ttgtcggtca tgcactcgga
ggcttagcca ttagcaaggc tatggagact 300ttcccagaga aaatttcagt tgccgtattc
ctctcaggac ttatgcccgg acctaacatc 360gacgcaacca cagtttacac taaggcagcg
tctgctgtga ttgggcaatt ggacaattac 420gttacctacg agaacggccc cactaaccca
cctactacct taatcgccgg gcctaagttt 480ctcgctacca atgtttatca tctttccccc
atcgaggatc ttgccctggc aacggctctg 540gtgagacctg tctatctcta tcttgctgag
gacatatcca aggaaatcgt actgtcctct 600aaacgttatg gatctgttaa aagggtcttt
attgtggcaa cccagaacga tgctttcaag 660aaagagtttc tcaaattgat gattgagaaa
aatcctccag acgaagtcaa ggagatcgag 720ggctcagatc atgtgacaat gatgtcaaaa
ccacaacagt tgtttacaac attgctgtcg 780atagcaaata agtacaagtg a
8017266PRTLycopersicon esculentum 7Met
Ala Lys Lys Ser Thr Ser Pro Phe Val Lys Lys His Phe Val Leu 1
5 10 15 Val His Thr Gly Phe His
Gly Ala Trp Cys Trp Tyr Lys Ile Val Ala 20
25 30 Leu Met Arg Ser Ser Gly His Asn Val Thr
Ala Leu Asp Leu Gly Ala 35 40
45 Ser Gly Ile Asn Pro Lys Gln Ala Leu Glu Ile Pro His Phe
Ser Asp 50 55 60
Tyr Leu Ser Pro Leu Met Glu Phe Met Ala Ser Leu Pro Ala Asn Glu 65
70 75 80 Lys Ile Ile Leu Val
Gly His Ala Leu Gly Gly Leu Ala Ile Ser Lys 85
90 95 Ala Met Glu Thr Phe Pro Glu Lys Ile Ser
Val Ala Val Phe Leu Ser 100 105
110 Gly Leu Met Pro Gly Pro Asn Ile Asp Ala Thr Thr Val Tyr Thr
Lys 115 120 125 Ala
Ala Ser Ala Val Ile Gly Gln Leu Asp Asn Tyr Val Thr Tyr Glu 130
135 140 Asn Gly Pro Thr Asn Pro
Pro Thr Thr Leu Ile Ala Gly Pro Lys Phe 145 150
155 160 Leu Ala Thr Asn Val Tyr His Leu Ser Pro Ile
Glu Asp Leu Ala Leu 165 170
175 Ala Thr Ala Leu Val Arg Pro Val Tyr Leu Tyr Leu Ala Glu Asp Ile
180 185 190 Ser Lys
Glu Ile Val Leu Ser Ser Lys Arg Tyr Gly Ser Val Lys Arg 195
200 205 Val Phe Ile Val Ala Thr Gln
Asn Asp Ala Phe Lys Lys Glu Phe Leu 210 215
220 Lys Leu Met Ile Glu Lys Asn Pro Pro Asp Glu Val
Lys Glu Ile Glu 225 230 235
240 Gly Ser Asp His Val Thr Met Met Ser Lys Pro Gln Gln Leu Phe Thr
245 250 255 Thr Leu Leu
Ser Ile Ala Asn Lys Tyr Lys 260 265
8228DNAArabidopsis thaliana 8atggcgcaag ttagcagaat ctgcaatggt gtgcagaacc
catctcttat ctccaatctc 60tcgaaatcca gtcaacgcaa atctccctta tcggtttctc
tgaagacgca gcagcatcca 120cgagcttatc cgatttcgtc gtcgtgggga ttgaagaaga
gtgggatgac gttaattggc 180tctgagcttc gtcctcttaa ggtcatgtct tctgtttcca
cggcgtgc 228976PRTArabidopsis thaliana 9Met Ala Gln Val
Ser Arg Ile Cys Asn Gly Val Gln Asn Pro Ser Leu 1 5
10 15 Ile Ser Asn Leu Ser Lys Ser Ser Gln
Arg Lys Ser Pro Leu Ser Val 20 25
30 Ser Leu Lys Thr Gln Gln His Pro Arg Ala Tyr Pro Ile Ser
Ser Ser 35 40 45
Trp Gly Leu Lys Lys Ser Gly Met Thr Leu Ile Gly Ser Glu Leu Arg 50
55 60 Pro Leu Lys Val Met
Ser Ser Val Ser Thr Ala Cys 65 70 75
10216DNAPetunia hybrida 10atggcccaga tcaacaacat ggcccagggc atccagaccc
tgaaccctaa ctctaacttc 60cacaagccgc aagtgcccaa gtctagctcc ttcctcgtgt
tcggctccaa gaagctcaag 120aatagcgcca attccatgct ggtcctgaag aaagactcga
tcttcatgca gaagttctgc 180tcctttcgca tcagtgcttc ggttgcgact gcctgc
2161172PRTPetunia hybrida 11Met Ala Gln Ile Asn
Asn Met Ala Gln Gly Ile Gln Thr Leu Asn Pro 1 5
10 15 Asn Ser Asn Phe His Lys Pro Gln Val Pro
Lys Ser Ser Ser Phe Leu 20 25
30 Val Phe Gly Ser Lys Lys Leu Lys Asn Ser Ala Asn Ser Met Leu
Val 35 40 45 Leu
Lys Lys Asp Ser Ile Phe Met Gln Lys Phe Cys Ser Phe Arg Ile 50
55 60 Ser Ala Ser Val Ala Thr
Ala Cys 65 70 121026DNAartificial
sequenceSynthetic sequence 12atggcgcaag ttagcagaat ctgcaatggt gtgcagaacc
catctcttat ctccaatctc 60tcgaaatcca gtcaacgcaa atctccctta tcggtttctc
tgaagacgca gcagcatcca 120cgagcttatc cgatttcgtc gtcgtgggga ttgaagaaga
gtgggatgac gttaattggc 180tctgagcttc gtcctcttaa ggtcatgtct tctgtttcca
cggcgtgcat ggaaaagtct 240atgtcaccct tcgtcaagaa acacttcgtg cttgttcata
ctgctttcca cggtgcctgg 300tgctggtaca agatcgtcgc ccttatgaga agttcaggcc
acaacgtgac cgcactggac 360ctcggggcct ccgggataaa ccccaaacaa gctttgcaga
ttcctaactt cagtgattat 420ctttctccac ttatggagtt tatggcctct cttcccgcta
acgaaaagat catactcgtt 480ggtcatgctc ttggtggtct tgccatttct aaagcaatgg
aaaccttccc tgagaaaata 540tcagtggctg ttttcttgtc cggtcttatg cccggaccta
atattgacgc aacaaccgtt 600tgtaccaagg ctgggtccgc cgtgttgggc caactcgaca
actgcgtgac ctacgaaaat 660ggtcctacta atccacctac tacccttatc gcaggaccta
agtttctcgc tacaaacgtg 720taccacctct ctccaataga ggatctggct cttgccactg
ctttggtcag gcctctttat 780ctgtacctcg ctgaggacat atctaaagaa gttgtgcttt
cttctaagcg ttatggttcc 840gtgaagcgtg tcttcatagt ggcaacagag aatgatgctc
ttaagaaaga gtttctcaaa 900ctgatgatcg agaagaatcc acctgatgaa gtgaaagaaa
ttgagggttc tgaccatgtt 960accatgatgt caaagcctca acagttgttt accacattgc
ttagtatagc taataagtat 1020aaataa
102613341PRTartificial sequenceSynthetic sequence
13Met Ala Gln Val Ser Arg Ile Cys Asn Gly Val Gln Asn Pro Ser Leu 1
5 10 15 Ile Ser Asn Leu
Ser Lys Ser Ser Gln Arg Lys Ser Pro Leu Ser Val 20
25 30 Ser Leu Lys Thr Gln Gln His Pro Arg
Ala Tyr Pro Ile Ser Ser Ser 35 40
45 Trp Gly Leu Lys Lys Ser Gly Met Thr Leu Ile Gly Ser Glu
Leu Arg 50 55 60
Pro Leu Lys Val Met Ser Ser Val Ser Thr Ala Cys Met Glu Lys Ser 65
70 75 80 Met Ser Pro Phe Val
Lys Lys His Phe Val Leu Val His Thr Ala Phe 85
90 95 His Gly Ala Trp Cys Trp Tyr Lys Ile Val
Ala Leu Met Arg Ser Ser 100 105
110 Gly His Asn Val Thr Ala Leu Asp Leu Gly Ala Ser Gly Ile Asn
Pro 115 120 125 Lys
Gln Ala Leu Gln Ile Pro Asn Phe Ser Asp Tyr Leu Ser Pro Leu 130
135 140 Met Glu Phe Met Ala Ser
Leu Pro Ala Asn Glu Lys Ile Ile Leu Val 145 150
155 160 Gly His Ala Leu Gly Gly Leu Ala Ile Ser Lys
Ala Met Glu Thr Phe 165 170
175 Pro Glu Lys Ile Ser Val Ala Val Phe Leu Ser Gly Leu Met Pro Gly
180 185 190 Pro Asn
Ile Asp Ala Thr Thr Val Cys Thr Lys Ala Gly Ser Ala Val 195
200 205 Leu Gly Gln Leu Asp Asn Cys
Val Thr Tyr Glu Asn Gly Pro Thr Asn 210 215
220 Pro Pro Thr Thr Leu Ile Ala Gly Pro Lys Phe Leu
Ala Thr Asn Val 225 230 235
240 Tyr His Leu Ser Pro Ile Glu Asp Leu Ala Leu Ala Thr Ala Leu Val
245 250 255 Arg Pro Leu
Tyr Leu Tyr Leu Ala Glu Asp Ile Ser Lys Glu Val Val 260
265 270 Leu Ser Ser Lys Arg Tyr Gly Ser
Val Lys Arg Val Phe Ile Val Ala 275 280
285 Thr Glu Asn Asp Ala Leu Lys Lys Glu Phe Leu Lys Leu
Met Ile Glu 290 295 300
Lys Asn Pro Pro Asp Glu Val Lys Glu Ile Glu Gly Ser Asp His Val 305
310 315 320 Thr Met Met Ser
Lys Pro Gln Gln Leu Phe Thr Thr Leu Leu Ser Ile 325
330 335 Ala Asn Lys Tyr Lys 340
141014DNAartificial sequenceSynthetic sequence 14atggcccaga
tcaacaacat ggcccagggc atccagaccc tgaaccctaa ctctaacttc 60cacaagccgc
aagtgcccaa gtctagctcc ttcctcgtgt tcggctccaa gaagctcaag 120aatagcgcca
attccatgct ggtcctgaag aaagactcga tcttcatgca gaagttctgc 180tcctttcgca
tcagtgcttc ggttgcgact gcctgcatgg aaaagtctat gtcacccttc 240gtcaagaaac
acttcgtgct tgttcatact gctttccacg gtgcctggtg ctggtacaag 300atcgtcgccc
ttatgagaag ttcaggccac aacgtgaccg cactggacct cggggcctcc 360gggataaacc
ccaaacaagc tttgcagatt cctaacttca gtgattatct ttctccactt 420atggagttta
tggcctctct tcccgctaac gaaaagatca tactcgttgg tcatgctctt 480ggtggtcttg
ccatttctaa agcaatggaa accttccctg agaaaatatc agtggctgtt 540ttcttgtccg
gtcttatgcc cggacctaat attgacgcaa caaccgtttg taccaaggct 600gggtccgccg
tgttgggcca actcgacaac tgcgtgacct acgaaaatgg tcctactaat 660ccacctacta
cccttatcgc aggacctaag tttctcgcta caaacgtgta ccacctctct 720ccaatagagg
atctggctct tgccactgct ttggtcaggc ctctttatct gtacctcgct 780gaggacatat
ctaaagaagt tgtgctttct tctaagcgtt atggttccgt gaagcgtgtc 840ttcatagtgg
caacagagaa tgatgctctt aagaaagagt ttctcaaact gatgatcgag 900aagaatccac
ctgatgaagt gaaagaaatt gagggttctg accatgttac catgatgtca 960aagcctcaac
agttgtttac cacattgctt agtatagcta ataagtataa ataa
101415337PRTartificial sequenceSynthetic sequence 15Met Ala Gln Ile Asn
Asn Met Ala Gln Gly Ile Gln Thr Leu Asn Pro 1 5
10 15 Asn Ser Asn Phe His Lys Pro Gln Val Pro
Lys Ser Ser Ser Phe Leu 20 25
30 Val Phe Gly Ser Lys Lys Leu Lys Asn Ser Ala Asn Ser Met Leu
Val 35 40 45 Leu
Lys Lys Asp Ser Ile Phe Met Gln Lys Phe Cys Ser Phe Arg Ile 50
55 60 Ser Ala Ser Val Ala Thr
Ala Cys Met Glu Lys Ser Met Ser Pro Phe 65 70
75 80 Val Lys Lys His Phe Val Leu Val His Thr Ala
Phe His Gly Ala Trp 85 90
95 Cys Trp Tyr Lys Ile Val Ala Leu Met Arg Ser Ser Gly His Asn Val
100 105 110 Thr Ala
Leu Asp Leu Gly Ala Ser Gly Ile Asn Pro Lys Gln Ala Leu 115
120 125 Gln Ile Pro Asn Phe Ser Asp
Tyr Leu Ser Pro Leu Met Glu Phe Met 130 135
140 Ala Ser Leu Pro Ala Asn Glu Lys Ile Ile Leu Val
Gly His Ala Leu 145 150 155
160 Gly Gly Leu Ala Ile Ser Lys Ala Met Glu Thr Phe Pro Glu Lys Ile
165 170 175 Ser Val Ala
Val Phe Leu Ser Gly Leu Met Pro Gly Pro Asn Ile Asp 180
185 190 Ala Thr Thr Val Cys Thr Lys Ala
Gly Ser Ala Val Leu Gly Gln Leu 195 200
205 Asp Asn Cys Val Thr Tyr Glu Asn Gly Pro Thr Asn Pro
Pro Thr Thr 210 215 220
Leu Ile Ala Gly Pro Lys Phe Leu Ala Thr Asn Val Tyr His Leu Ser 225
230 235 240 Pro Ile Glu Asp
Leu Ala Leu Ala Thr Ala Leu Val Arg Pro Leu Tyr 245
250 255 Leu Tyr Leu Ala Glu Asp Ile Ser Lys
Glu Val Val Leu Ser Ser Lys 260 265
270 Arg Tyr Gly Ser Val Lys Arg Val Phe Ile Val Ala Thr Glu
Asn Asp 275 280 285
Ala Leu Lys Lys Glu Phe Leu Lys Leu Met Ile Glu Lys Asn Pro Pro 290
295 300 Asp Glu Val Lys Glu
Ile Glu Gly Ser Asp His Val Thr Met Met Ser 305 310
315 320 Lys Pro Gln Gln Leu Phe Thr Thr Leu Leu
Ser Ile Ala Asn Lys Tyr 325 330
335 Lys 161026DNAartificial sequenceSynthetic sequence
16atggcgcaag ttagcagaat ctgcaatggt gtgcagaacc catctcttat ctccaatctc
60tcgaaatcca gtcaacgcaa atctccctta tcggtttctc tgaagacgca gcagcatcca
120cgagcttatc cgatttcgtc gtcgtgggga ttgaagaaga gtgggatgac gttaattggc
180tctgagcttc gtcctcttaa ggtcatgtct tctgtttcca cggcgtgcat ggaaaagtct
240atgtcaccct tcgttaagaa acactttgta ttggtccaca ctgctttcca cggggcttgg
300tgctggtaca agattgtcgc attgatgaga tcttccggtc acaacgtcac tgctttggac
360ctgggagcat ctggaattaa ccctaagcaa gcattgcaaa tcccaaactt cagtgattac
420ctctctcctc ttatggaatt catggccagc cttccagcta acgaaaagat catcttggtg
480ggtcacgccc ttggtggatt ggcaatatca aaggccatgg aaacatttcc agaaaagatt
540agcgtggctg tctttctgag tggccttatg cccggcccta acatcgacgc aactacagtc
600tgcacaaaag ctggttccgc tgtattggga caacttgaca attgcgtgac ctacgagaat
660ggtcccacta acccacccac cacattgatc gcaggaccta agttccttgc taccaacgtc
720taccatttgt ctccaattga ggacttggct ctggccaccg cacttgttag acctttgtac
780ctctatcttg cagaagatat ttccaaggaa gtggttttgt cttctaaaag atatggttct
840gtgaagaggg tgttcatcgt tgccacagag aatgatgctc tgaagaaaga gtttctcaaa
900ttgatgattg aaaagaatcc tccagacgaa gtgaaggaga tagaaggaag tgaccatgtt
960actatgatga gtaagcctca acaacttttc actacactgc tgtctattgc aaacaaatac
1020aagtaa
102617341PRTartificial sequenceSynthetic sequence 17Met Ala Gln Val Ser
Arg Ile Cys Asn Gly Val Gln Asn Pro Ser Leu 1 5
10 15 Ile Ser Asn Leu Ser Lys Ser Ser Gln Arg
Lys Ser Pro Leu Ser Val 20 25
30 Ser Leu Lys Thr Gln Gln His Pro Arg Ala Tyr Pro Ile Ser Ser
Ser 35 40 45 Trp
Gly Leu Lys Lys Ser Gly Met Thr Leu Ile Gly Ser Glu Leu Arg 50
55 60 Pro Leu Lys Val Met Ser
Ser Val Ser Thr Ala Cys Met Glu Lys Ser 65 70
75 80 Met Ser Pro Phe Val Lys Lys His Phe Val Leu
Val His Thr Ala Phe 85 90
95 His Gly Ala Trp Cys Trp Tyr Lys Ile Val Ala Leu Met Arg Ser Ser
100 105 110 Gly His
Asn Val Thr Ala Leu Asp Leu Gly Ala Ser Gly Ile Asn Pro 115
120 125 Lys Gln Ala Leu Gln Ile Pro
Asn Phe Ser Asp Tyr Leu Ser Pro Leu 130 135
140 Met Glu Phe Met Ala Ser Leu Pro Ala Asn Glu Lys
Ile Ile Leu Val 145 150 155
160 Gly His Ala Leu Gly Gly Leu Ala Ile Ser Lys Ala Met Glu Thr Phe
165 170 175 Pro Glu Lys
Ile Ser Val Ala Val Phe Leu Ser Gly Leu Met Pro Gly 180
185 190 Pro Asn Ile Asp Ala Thr Thr Val
Cys Thr Lys Ala Gly Ser Ala Val 195 200
205 Leu Gly Gln Leu Asp Asn Cys Val Thr Tyr Glu Asn Gly
Pro Thr Asn 210 215 220
Pro Pro Thr Thr Leu Ile Ala Gly Pro Lys Phe Leu Ala Thr Asn Val 225
230 235 240 Tyr His Leu Ser
Pro Ile Glu Asp Leu Ala Leu Ala Thr Ala Leu Val 245
250 255 Arg Pro Leu Tyr Leu Tyr Leu Ala Glu
Asp Ile Ser Lys Glu Val Val 260 265
270 Leu Ser Ser Lys Arg Tyr Gly Ser Val Lys Arg Val Phe Ile
Val Ala 275 280 285
Thr Glu Asn Asp Ala Leu Lys Lys Glu Phe Leu Lys Leu Met Ile Glu 290
295 300 Lys Asn Pro Pro Asp
Glu Val Lys Glu Ile Glu Gly Ser Asp His Val 305 310
315 320 Thr Met Met Ser Lys Pro Gln Gln Leu Phe
Thr Thr Leu Leu Ser Ile 325 330
335 Ala Asn Lys Tyr Lys 340 181014DNAartificial
sequenceSynthetic sequence 18atggcccaga tcaacaacat ggcccagggc atccagaccc
tgaaccctaa ctctaacttc 60cacaagccgc aagtgcccaa gtctagctcc ttcctcgtgt
tcggctccaa gaagctcaag 120aatagcgcca attccatgct ggtcctgaag aaagactcga
tcttcatgca gaagttctgc 180tcctttcgca tcagtgcttc ggttgcgact gcctgcatgg
aaaagtctat gtcacccttc 240gttaagaaac actttgtatt ggtccacact gctttccacg
gggcttggtg ctggtacaag 300attgtcgcat tgatgagatc ttccggtcac aacgtcactg
ctttggacct gggagcatct 360ggaattaacc ctaagcaagc attgcaaatc ccaaacttca
gtgattacct ctctcctctt 420atggaattca tggccagcct tccagctaac gaaaagatca
tcttggtggg tcacgccctt 480ggtggattgg caatatcaaa ggccatggaa acatttccag
aaaagattag cgtggctgtc 540tttctgagtg gccttatgcc cggccctaac atcgacgcaa
ctacagtctg cacaaaagct 600ggttccgctg tattgggaca acttgacaat tgcgtgacct
acgagaatgg tcccactaac 660ccacccacca cattgatcgc aggacctaag ttccttgcta
ccaacgtcta ccatttgtct 720ccaattgagg acttggctct ggccaccgca cttgttagac
ctttgtacct ctatcttgca 780gaagatattt ccaaggaagt ggttttgtct tctaaaagat
atggttctgt gaagagggtg 840ttcatcgttg ccacagagaa tgatgctctg aagaaagagt
ttctcaaatt gatgattgaa 900aagaatcctc cagacgaagt gaaggagata gaaggaagtg
accatgttac tatgatgagt 960aagcctcaac aacttttcac tacactgctg tctattgcaa
acaaatacaa gtaa 101419337PRTartificial sequenceSynthetic sequence
19Met Ala Gln Ile Asn Asn Met Ala Gln Gly Ile Gln Thr Leu Asn Pro 1
5 10 15 Asn Ser Asn Phe
His Lys Pro Gln Val Pro Lys Ser Ser Ser Phe Leu 20
25 30 Val Phe Gly Ser Lys Lys Leu Lys Asn
Ser Ala Asn Ser Met Leu Val 35 40
45 Leu Lys Lys Asp Ser Ile Phe Met Gln Lys Phe Cys Ser Phe
Arg Ile 50 55 60
Ser Ala Ser Val Ala Thr Ala Cys Met Glu Lys Ser Met Ser Pro Phe 65
70 75 80 Val Lys Lys His Phe
Val Leu Val His Thr Ala Phe His Gly Ala Trp 85
90 95 Cys Trp Tyr Lys Ile Val Ala Leu Met Arg
Ser Ser Gly His Asn Val 100 105
110 Thr Ala Leu Asp Leu Gly Ala Ser Gly Ile Asn Pro Lys Gln Ala
Leu 115 120 125 Gln
Ile Pro Asn Phe Ser Asp Tyr Leu Ser Pro Leu Met Glu Phe Met 130
135 140 Ala Ser Leu Pro Ala Asn
Glu Lys Ile Ile Leu Val Gly His Ala Leu 145 150
155 160 Gly Gly Leu Ala Ile Ser Lys Ala Met Glu Thr
Phe Pro Glu Lys Ile 165 170
175 Ser Val Ala Val Phe Leu Ser Gly Leu Met Pro Gly Pro Asn Ile Asp
180 185 190 Ala Thr
Thr Val Cys Thr Lys Ala Gly Ser Ala Val Leu Gly Gln Leu 195
200 205 Asp Asn Cys Val Thr Tyr Glu
Asn Gly Pro Thr Asn Pro Pro Thr Thr 210 215
220 Leu Ile Ala Gly Pro Lys Phe Leu Ala Thr Asn Val
Tyr His Leu Ser 225 230 235
240 Pro Ile Glu Asp Leu Ala Leu Ala Thr Ala Leu Val Arg Pro Leu Tyr
245 250 255 Leu Tyr Leu
Ala Glu Asp Ile Ser Lys Glu Val Val Leu Ser Ser Lys 260
265 270 Arg Tyr Gly Ser Val Lys Arg Val
Phe Ile Val Ala Thr Glu Asn Asp 275 280
285 Ala Leu Lys Lys Glu Phe Leu Lys Leu Met Ile Glu Lys
Asn Pro Pro 290 295 300
Asp Glu Val Lys Glu Ile Glu Gly Ser Asp His Val Thr Met Met Ser 305
310 315 320 Lys Pro Gln Gln
Leu Phe Thr Thr Leu Leu Ser Ile Ala Asn Lys Tyr 325
330 335 Lys 201029DNAartificial
sequenceSynthetic sequence 20atggcgcaag ttagcagaat ctgcaatggt gtgcagaacc
catctcttat ctccaatctc 60tcgaaatcca gtcaacgcaa atctccctta tcggtttctc
tgaagacgca gcagcatcca 120cgagcttatc cgatttcgtc gtcgtgggga ttgaagaaga
gtgggatgac gttaattggc 180tctgagcttc gtcctcttaa ggtcatgtct tctgtttcca
cggcgtgcat ggctgagaag 240tctatgagtc ctttcgttaa gaagcacttc gttctagttc
acactgcttt ccacggtgct 300tggtgctggt acaagattgt tgctttgatg cgtagttctg
gtcataacgt gactgctctt 360gatctcggtg ctagtggtat taacccaaag caagccttgc
aaatccccaa ctttagcgat 420tacttgtcac cactcatgga gtttatggcc tcccttcctg
ccaacgagaa gatcatactt 480gttggacatg ctttaggagg gctggccatc tcaaaagcta
tggaaacctt ccctgagaag 540ataagcgtgg cagtatttct ctcgggcctc atgcctggac
caaacattga cgcaactacc 600gtctgtacca aggctggatc tgcagtcttg gggcagttgg
ataactgcgt gacttatgag 660aatgggccga caaatcctcc aacaaccctt attgccggac
ctaagttctt ggcaacgaat 720gtctatcatc tttctcccat cgaggatctg gccctcgcta
ctgctcttgt ccgcccactt 780tacctctatc tcgctgagga catctcaaaa gaagttgtac
tttcatccaa gagatacggc 840tccgtgaaga gggttttcat tgttgcgaca gagaatgatg
cacttaagaa agaatttctg 900aagctgatga tcgagaagaa tccacctgac gaagtgaaag
aaatagaagg ctctgaccat 960gtgactatga tgtccaaacc acaacagttg tttacaacgc
ttttgagtat cgcaaacaag 1020tacaagtaa
102921342PRTartificial sequenceSynthetic sequence
21Met Ala Gln Val Ser Arg Ile Cys Asn Gly Val Gln Asn Pro Ser Leu 1
5 10 15 Ile Ser Asn Leu
Ser Lys Ser Ser Gln Arg Lys Ser Pro Leu Ser Val 20
25 30 Ser Leu Lys Thr Gln Gln His Pro Arg
Ala Tyr Pro Ile Ser Ser Ser 35 40
45 Trp Gly Leu Lys Lys Ser Gly Met Thr Leu Ile Gly Ser Glu
Leu Arg 50 55 60
Pro Leu Lys Val Met Ser Ser Val Ser Thr Ala Cys Met Ala Glu Lys 65
70 75 80 Ser Met Ser Pro Phe
Val Lys Lys His Phe Val Leu Val His Thr Ala 85
90 95 Phe His Gly Ala Trp Cys Trp Tyr Lys Ile
Val Ala Leu Met Arg Ser 100 105
110 Ser Gly His Asn Val Thr Ala Leu Asp Leu Gly Ala Ser Gly Ile
Asn 115 120 125 Pro
Lys Gln Ala Leu Gln Ile Pro Asn Phe Ser Asp Tyr Leu Ser Pro 130
135 140 Leu Met Glu Phe Met Ala
Ser Leu Pro Ala Asn Glu Lys Ile Ile Leu 145 150
155 160 Val Gly His Ala Leu Gly Gly Leu Ala Ile Ser
Lys Ala Met Glu Thr 165 170
175 Phe Pro Glu Lys Ile Ser Val Ala Val Phe Leu Ser Gly Leu Met Pro
180 185 190 Gly Pro
Asn Ile Asp Ala Thr Thr Val Cys Thr Lys Ala Gly Ser Ala 195
200 205 Val Leu Gly Gln Leu Asp Asn
Cys Val Thr Tyr Glu Asn Gly Pro Thr 210 215
220 Asn Pro Pro Thr Thr Leu Ile Ala Gly Pro Lys Phe
Leu Ala Thr Asn 225 230 235
240 Val Tyr His Leu Ser Pro Ile Glu Asp Leu Ala Leu Ala Thr Ala Leu
245 250 255 Val Arg Pro
Leu Tyr Leu Tyr Leu Ala Glu Asp Ile Ser Lys Glu Val 260
265 270 Val Leu Ser Ser Lys Arg Tyr Gly
Ser Val Lys Arg Val Phe Ile Val 275 280
285 Ala Thr Glu Asn Asp Ala Leu Lys Lys Glu Phe Leu Lys
Leu Met Ile 290 295 300
Glu Lys Asn Pro Pro Asp Glu Val Lys Glu Ile Glu Gly Ser Asp His 305
310 315 320 Val Thr Met Met
Ser Lys Pro Gln Gln Leu Phe Thr Thr Leu Leu Ser 325
330 335 Ile Ala Asn Lys Tyr Lys
340 221017DNAartificial sequenceSynthetic sequence 22atggcccaga
tcaacaacat ggcccagggc atccagaccc tgaaccctaa ctctaacttc 60cacaagccgc
aagtgcccaa gtctagctcc ttcctcgtgt tcggctccaa gaagctcaag 120aatagcgcca
attccatgct ggtcctgaag aaagactcga tcttcatgca gaagttctgc 180tcctttcgca
tcagtgcttc ggttgcgact gcctgcatgg ctgagaagtc tatgagtcct 240ttcgttaaga
agcacttcgt tctagttcac actgctttcc acggtgcttg gtgctggtac 300aagattgttg
ctttgatgcg tagttctggt cataacgtga ctgctcttga tctcggtgct 360agtggtatta
acccaaagca agccttgcaa atccccaact ttagcgatta cttgtcacca 420ctcatggagt
ttatggcctc ccttcctgcc aacgagaaga tcatacttgt tggacatgct 480ttaggagggc
tggccatctc aaaagctatg gaaaccttcc ctgagaagat aagcgtggca 540gtatttctct
cgggcctcat gcctggacca aacattgacg caactaccgt ctgtaccaag 600gctggatctg
cagtcttggg gcagttggat aactgcgtga cttatgagaa tgggccgaca 660aatcctccaa
caacccttat tgccggacct aagttcttgg caacgaatgt ctatcatctt 720tctcccatcg
aggatctggc cctcgctact gctcttgtcc gcccacttta cctctatctc 780gctgaggaca
tctcaaaaga agttgtactt tcatccaaga gatacggctc cgtgaagagg 840gttttcattg
ttgcgacaga gaatgatgca cttaagaaag aatttctgaa gctgatgatc 900gagaagaatc
cacctgacga agtgaaagaa atagaaggct ctgaccatgt gactatgatg 960tccaaaccac
aacagttgtt tacaacgctt ttgagtatcg caaacaagta caagtaa
101723338PRTartificial sequenceSynthetic sequence 23Met Ala Gln Ile Asn
Asn Met Ala Gln Gly Ile Gln Thr Leu Asn Pro 1 5
10 15 Asn Ser Asn Phe His Lys Pro Gln Val Pro
Lys Ser Ser Ser Phe Leu 20 25
30 Val Phe Gly Ser Lys Lys Leu Lys Asn Ser Ala Asn Ser Met Leu
Val 35 40 45 Leu
Lys Lys Asp Ser Ile Phe Met Gln Lys Phe Cys Ser Phe Arg Ile 50
55 60 Ser Ala Ser Val Ala Thr
Ala Cys Met Ala Glu Lys Ser Met Ser Pro 65 70
75 80 Phe Val Lys Lys His Phe Val Leu Val His Thr
Ala Phe His Gly Ala 85 90
95 Trp Cys Trp Tyr Lys Ile Val Ala Leu Met Arg Ser Ser Gly His Asn
100 105 110 Val Thr
Ala Leu Asp Leu Gly Ala Ser Gly Ile Asn Pro Lys Gln Ala 115
120 125 Leu Gln Ile Pro Asn Phe Ser
Asp Tyr Leu Ser Pro Leu Met Glu Phe 130 135
140 Met Ala Ser Leu Pro Ala Asn Glu Lys Ile Ile Leu
Val Gly His Ala 145 150 155
160 Leu Gly Gly Leu Ala Ile Ser Lys Ala Met Glu Thr Phe Pro Glu Lys
165 170 175 Ile Ser Val
Ala Val Phe Leu Ser Gly Leu Met Pro Gly Pro Asn Ile 180
185 190 Asp Ala Thr Thr Val Cys Thr Lys
Ala Gly Ser Ala Val Leu Gly Gln 195 200
205 Leu Asp Asn Cys Val Thr Tyr Glu Asn Gly Pro Thr Asn
Pro Pro Thr 210 215 220
Thr Leu Ile Ala Gly Pro Lys Phe Leu Ala Thr Asn Val Tyr His Leu 225
230 235 240 Ser Pro Ile Glu
Asp Leu Ala Leu Ala Thr Ala Leu Val Arg Pro Leu 245
250 255 Tyr Leu Tyr Leu Ala Glu Asp Ile Ser
Lys Glu Val Val Leu Ser Ser 260 265
270 Lys Arg Tyr Gly Ser Val Lys Arg Val Phe Ile Val Ala Thr
Glu Asn 275 280 285
Asp Ala Leu Lys Lys Glu Phe Leu Lys Leu Met Ile Glu Lys Asn Pro 290
295 300 Pro Asp Glu Val Lys
Glu Ile Glu Gly Ser Asp His Val Thr Met Met 305 310
315 320 Ser Lys Pro Gln Gln Leu Phe Thr Thr Leu
Leu Ser Ile Ala Asn Lys 325 330
335 Tyr Lys 241029DNAartificial sequenceSynthetic sequence
24atggcgcaag ttagcagaat ctgcaatggt gtgcagaacc catctcttat ctccaatctc
60tcgaaatcca gtcaacgcaa atctccctta tcggtttctc tgaagacgca gcagcatcca
120cgagcttatc cgatttcgtc gtcgtgggga ttgaagaaga gtgggatgac gttaattggc
180tctgagcttc gtcctcttaa ggtcatgtct tctgtttcca cggcgtgcat ggctaagaag
240tctacttcac ctttcgtgaa gaagcacttc gttctagttc acactggttt ccacggtgct
300tggtgctggt acaagattgt tgctttgatg cgtagttctg gtcacaacgt gactgctctt
360gatctaggtg ctagtggtat taaccctaag caagcccttg aaatcccaca tttcagcgac
420tacttgtccc cactcatgga gttcatggct agtttgccgg ccaatgagaa gataattctt
480gtcggtcatg cactcggagg cttagccatt agcaaggcta tggagacttt cccagagaaa
540atttcagttg ccgtattcct ctcaggactt atgcccggac ctaacatcga cgcaaccaca
600gtttacacta aggcagcgtc tgctgtgatt gggcaattgg acaattacgt tacctacgag
660aacggcccca ctaacccacc tactacctta atcgccgggc ctaagtttct cgctaccaat
720gtttatcatc tttcccccat cgaggatctt gccctggcaa cggctctggt gagacctgtc
780tatctctatc ttgctgagga catatccaag gaaatcgtac tgtcctctaa acgttatgga
840tctgttaaaa gggtctttat tgtggcaacc cagaacgatg ctttcaagaa agagtttctc
900aaattgatga ttgagaaaaa tcctccagac gaagtcaagg agatcgaggg ctcagatcat
960gtgacaatga tgtcaaaacc acaacagttg tttacaacat tgctgtcgat agcaaataag
1020tacaagtga
102925341PRTartificial sequenceSynthetic sequence 25Met Ala Gln Val Ser
Arg Ile Cys Asn Gly Val Gln Asn Pro Ser Leu 1 5
10 15 Ile Ser Asn Leu Ser Lys Ser Ser Gln Arg
Lys Ser Pro Leu Ser Val 20 25
30 Ser Leu Lys Thr Gln Gln His Pro Arg Ala Tyr Pro Ile Ser Ser
Ser 35 40 45 Trp
Gly Leu Lys Lys Ser Gly Met Thr Leu Ile Gly Ser Glu Leu Arg 50
55 60 Pro Leu Lys Val Met Ser
Ser Val Ser Thr Ala Cys Met Ala Lys Lys 65 70
75 80 Ser Thr Ser Pro Phe Val Lys Lys His Phe Val
Leu Val His Thr Gly 85 90
95 Phe His Gly Ala Trp Cys Trp Tyr Lys Ile Val Ala Leu Met Arg Ser
100 105 110 Ser Gly
His Asn Val Thr Ala Leu Asp Leu Gly Ala Ser Gly Ile Asn 115
120 125 Pro Lys Gln Ala Leu Glu Ile
Pro His Phe Ser Asp Tyr Leu Ser Pro 130 135
140 Leu Met Glu Phe Met Ala Ser Leu Pro Ala Asn Glu
Lys Ile Ile Leu 145 150 155
160 Val Gly His Ala Leu Gly Gly Leu Ala Ile Ser Lys Ala Met Glu Thr
165 170 175 Phe Pro Glu
Lys Ile Ser Val Ala Val Phe Leu Ser Gly Leu Met Pro 180
185 190 Gly Pro Asn Ile Asp Ala Thr Thr
Val Tyr Thr Lys Ala Ala Ser Ala 195 200
205 Val Ile Gly Gln Leu Asp Asn Tyr Val Thr Tyr Glu Asn
Gly Pro Thr 210 215 220
Asn Pro Pro Thr Thr Leu Ile Ala Gly Pro Lys Phe Leu Ala Thr Asn 225
230 235 240 Val Tyr His Leu
Ser Pro Ile Glu Asp Leu Ala Leu Ala Thr Ala Leu 245
250 255 Val Arg Pro Val Tyr Leu Tyr Leu Ala
Glu Asp Ile Ser Lys Glu Ile 260 265
270 Val Leu Ser Ser Lys Arg Tyr Gly Ser Val Lys Arg Val Phe
Ile Val 275 280 285
Ala Thr Gln Asn Asp Ala Phe Lys Lys Glu Phe Leu Lys Leu Met Ile 290
295 300 Glu Lys Asn Pro Pro
Asp Glu Val Lys Glu Ile Glu Gly Ser Asp His 305 310
315 320 Val Thr Met Met Ser Lys Pro Gln Gln Leu
Phe Thr Thr Leu Leu Ser 325 330
335 Ile Ala Asn Lys Tyr 340 261017DNAartificial
sequenceSynthetic sequence 26atggcccaga tcaacaacat ggcccagggc atccagaccc
tgaaccctaa ctctaacttc 60cacaagccgc aagtgcccaa gtctagctcc ttcctcgtgt
tcggctccaa gaagctcaag 120aatagcgcca attccatgct ggtcctgaag aaagactcga
tcttcatgca gaagttctgc 180tcctttcgca tcagtgcttc ggttgcgact gcctgcatgg
ctaagaagtc tacttcacct 240ttcgtgaaga agcacttcgt tctagttcac actggtttcc
acggtgcttg gtgctggtac 300aagattgttg ctttgatgcg tagttctggt cacaacgtga
ctgctcttga tctaggtgct 360agtggtatta accctaagca agcccttgaa atcccacatt
tcagcgacta cttgtcccca 420ctcatggagt tcatggctag tttgccggcc aatgagaaga
taattcttgt cggtcatgca 480ctcggaggct tagccattag caaggctatg gagactttcc
cagagaaaat ttcagttgcc 540gtattcctct caggacttat gcccggacct aacatcgacg
caaccacagt ttacactaag 600gcagcgtctg ctgtgattgg gcaattggac aattacgtta
cctacgagaa cggccccact 660aacccaccta ctaccttaat cgccgggcct aagtttctcg
ctaccaatgt ttatcatctt 720tcccccatcg aggatcttgc cctggcaacg gctctggtga
gacctgtcta tctctatctt 780gctgaggaca tatccaagga aatcgtactg tcctctaaac
gttatggatc tgttaaaagg 840gtctttattg tggcaaccca gaacgatgct ttcaagaaag
agtttctcaa attgatgatt 900gagaaaaatc ctccagacga agtcaaggag atcgagggct
cagatcatgt gacaatgatg 960tcaaaaccac aacagttgtt tacaacattg ctgtcgatag
caaataagta caagtga 101727338PRTartificial sequenceSynthetic sequence
27Met Ala Gln Ile Asn Asn Met Ala Gln Gly Ile Gln Thr Leu Asn Pro 1
5 10 15 Asn Ser Asn Phe
His Lys Pro Gln Val Pro Lys Ser Ser Ser Phe Leu 20
25 30 Val Phe Gly Ser Lys Lys Leu Lys Asn
Ser Ala Asn Ser Met Leu Val 35 40
45 Leu Lys Lys Asp Ser Ile Phe Met Gln Lys Phe Cys Ser Phe
Arg Ile 50 55 60
Ser Ala Ser Val Ala Thr Ala Cys Met Ala Lys Lys Ser Thr Ser Pro 65
70 75 80 Phe Val Lys Lys His
Phe Val Leu Val His Thr Gly Phe His Gly Ala 85
90 95 Trp Cys Trp Tyr Lys Ile Val Ala Leu Met
Arg Ser Ser Gly His Asn 100 105
110 Val Thr Ala Leu Asp Leu Gly Ala Ser Gly Ile Asn Pro Lys Gln
Ala 115 120 125 Leu
Glu Ile Pro His Phe Ser Asp Tyr Leu Ser Pro Leu Met Glu Phe 130
135 140 Met Ala Ser Leu Pro Ala
Asn Glu Lys Ile Ile Leu Val Gly His Ala 145 150
155 160 Leu Gly Gly Leu Ala Ile Ser Lys Ala Met Glu
Thr Phe Pro Glu Lys 165 170
175 Ile Ser Val Ala Val Phe Leu Ser Gly Leu Met Pro Gly Pro Asn Ile
180 185 190 Asp Ala
Thr Thr Val Tyr Thr Lys Ala Ala Ser Ala Val Ile Gly Gln 195
200 205 Leu Asp Asn Tyr Val Thr Tyr
Glu Asn Gly Pro Thr Asn Pro Pro Thr 210 215
220 Thr Leu Ile Ala Gly Pro Lys Phe Leu Ala Thr Asn
Val Tyr His Leu 225 230 235
240 Ser Pro Ile Glu Asp Leu Ala Leu Ala Thr Ala Leu Val Arg Pro Val
245 250 255 Tyr Leu Tyr
Leu Ala Glu Asp Ile Ser Lys Glu Ile Val Leu Ser Ser 260
265 270 Lys Arg Tyr Gly Ser Val Lys Arg
Val Phe Ile Val Ala Thr Gln Asn 275 280
285 Asp Ala Phe Lys Lys Glu Phe Leu Lys Leu Met Ile Glu
Lys Asn Pro 290 295 300
Pro Asp Glu Val Lys Glu Ile Glu Gly Ser Asp His Val Thr Met Met 305
310 315 320 Ser Lys Pro Gln
Gln Leu Phe Thr Thr Leu Leu Ser Ile Ala Asn Lys 325
330 335 Tyr Lys 281026DNAartificial
sequenceSynthetic sequence 28atggcgcaag ttagcagaat ctgcaatggt gtgcagaacc
catctcttat ctccaatctc 60tcgaaatcca gtcaacgcaa atctccctta tcggtttctc
tgaagacgca gcagcatcca 120cgagcttatc cgatttcgtc gtcgtgggga ttgaagaaga
gtgggatgac gttaattggc 180tctgagcttc gtcctcttaa ggtcatgtct tctgtttcca
cggcgtgcat ggaaaagtct 240atgtcaccct tcgtcaagaa acacttcgtg cttgttcata
ctgctttcca cggtgcctgg 300tgctggtaca agatcgtcgc ccttatgaga agttcaggcc
acaacgtgac cgcactggac 360ctcggggcct ccgggataaa ccccaaacaa gctttgcaga
ttcctaactt cagtgattat 420ctttctccac ttatggagtt tatggcctct cttcccgcta
acgaaaagat catactcgtt 480ggtcattctc ttggtggtct tgccatttct aaagcaatgg
aaaccttccc tgagaaaata 540tcagtggctg ttttcttgtc cggtcttatg cccggaccta
atattgacgc aacaaccgtt 600tgtaccaagg ctgggtccgc cgtgttgggc caactcgaca
actgcgtgac ctacgaaaat 660ggtcctacta atccacctac tacccttatc gcaggaccta
agtttctcgc tacaaacgtg 720taccacctct ctccaataga ggatctggct cttgccactg
ctttggtcag gcctctttat 780ctgtacctcg ctgaggacat atctaaagaa gttgtgcttt
cttctaagcg ttatggttcc 840gtgaagcgtg tcttcatagt ggcaacagag aatgatgctc
ttaagaaaga gtttctcaaa 900ctgatgatcg agaagaatcc acctgatgaa gtgaaagaaa
ttgagggttc tgaccatgtt 960accatgatgt caaagcctca acagttgttt accacattgc
ttagtatagc taataagtat 1020aaataa
102629341PRTartificial sequenceSynthetic sequence
29Met Ala Gln Val Ser Arg Ile Cys Asn Gly Val Gln Asn Pro Ser Leu 1
5 10 15 Ile Ser Asn Leu
Ser Lys Ser Ser Gln Arg Lys Ser Pro Leu Ser Val 20
25 30 Ser Leu Lys Thr Gln Gln His Pro Arg
Ala Tyr Pro Ile Ser Ser Ser 35 40
45 Trp Gly Leu Lys Lys Ser Gly Met Thr Leu Ile Gly Ser Glu
Leu Arg 50 55 60
Pro Leu Lys Val Met Ser Ser Val Ser Thr Ala Cys Met Glu Lys Ser 65
70 75 80 Met Ser Pro Phe Val
Lys Lys His Phe Val Leu Val His Thr Ala Phe 85
90 95 His Gly Ala Trp Cys Trp Tyr Lys Ile Val
Ala Leu Met Arg Ser Ser 100 105
110 Gly His Asn Val Thr Ala Leu Asp Leu Gly Ala Ser Gly Ile Asn
Pro 115 120 125 Lys
Gln Ala Leu Gln Ile Pro Asn Phe Ser Asp Tyr Leu Ser Pro Leu 130
135 140 Met Glu Phe Met Ala Ser
Leu Pro Ala Asn Glu Lys Ile Ile Leu Val 145 150
155 160 Gly His Ser Leu Gly Gly Leu Ala Ile Ser Lys
Ala Met Glu Thr Phe 165 170
175 Pro Glu Lys Ile Ser Val Ala Val Phe Leu Ser Gly Leu Met Pro Gly
180 185 190 Pro Asn
Ile Asp Ala Thr Thr Val Cys Thr Lys Ala Gly Ser Ala Val 195
200 205 Leu Gly Gln Leu Asp Asn Cys
Val Thr Tyr Glu Asn Gly Pro Thr Asn 210 215
220 Pro Pro Thr Thr Leu Ile Ala Gly Pro Lys Phe Leu
Ala Thr Asn Val 225 230 235
240 Tyr His Leu Ser Pro Ile Glu Asp Leu Ala Leu Ala Thr Ala Leu Val
245 250 255 Arg Pro Leu
Tyr Leu Tyr Leu Ala Glu Asp Ile Ser Lys Glu Val Val 260
265 270 Leu Ser Ser Lys Arg Tyr Gly Ser
Val Lys Arg Val Phe Ile Val Ala 275 280
285 Thr Glu Asn Asp Ala Leu Lys Lys Glu Phe Leu Lys Leu
Met Ile Glu 290 295 300
Lys Asn Pro Pro Asp Glu Val Lys Glu Ile Glu Gly Ser Asp His Val 305
310 315 320 Thr Met Met Ser
Lys Pro Gln Gln Leu Phe Thr Thr Leu Leu Ser Ile 325
330 335 Ala Asn Lys Tyr Lys 340
301026DNAartificial sequenceSynthetic sequence 30atggcgcaag
ttagcagaat ctgcaatggt gtgcagaacc catctcttat ctccaatctc 60tcgaaatcca
gtcaacgcaa atctccctta tcggtttctc tgaagacgca gcagcatcca 120cgagcttatc
cgatttcgtc gtcgtgggga ttgaagaaga gtgggatgac gttaattggc 180tctgagcttc
gtcctcttaa ggtcatgtct tctgtttcca cggcgtgcat ggaaaagtct 240atgtcaccct
tcgtcaagaa acacttcgtg cttgttcata ctgctttcca cggtgcctgg 300tgctggtaca
agatcgtcgc ccttatgaga agttcaggcc acaacgtgac cgcactggac 360ctcggggcct
ccgggataaa ccccaaacaa gctttgcaga ttcctaactt cagtgattat 420ctttctccac
ttatggagtt tatggcctct cttcccgcta acgaaaagat catactcgtt 480ggtcatgctc
ttggtggtct tgccatttct aaagcaatgg aaaccttccc tgagaaaata 540tcagtggctg
ttttcttgtc cggtcttatg cccggaccta atattgacgc aacaaccgtt 600tgtaccaagg
ctgggtccgc cgtgttgggc caactcgaca actgcgtgac ctacgaaaat 660ggtcctacta
atccacctac tacccttatc gcaggaccta agtttctcgc tacaaacgtg 720taccacctct
ctccaataga ggatctggct cttgccactg ctttggtcag gcctctttat 780ctgtacctcg
ctgaggacat atctaaagaa gttgtgcttt cttctaagcg ttatggttcc 840gtgaagcgtg
tcttcatagt ggcaacagag gatgatgctc ttaagaaaga gtttctcaaa 900ctgatgatcg
agaagaatcc acctgatgaa gtgaaagaaa ttgagggttc tgaccatgtt 960accatgatgt
caaagcctca acagttgttt accacattgc ttagtatagc taataagtat 1020aaataa
102631341PRTartificial sequenceSynthetic sequence 31Met Ala Gln Val Ser
Arg Ile Cys Asn Gly Val Gln Asn Pro Ser Leu 1 5
10 15 Ile Ser Asn Leu Ser Lys Ser Ser Gln Arg
Lys Ser Pro Leu Ser Val 20 25
30 Ser Leu Lys Thr Gln Gln His Pro Arg Ala Tyr Pro Ile Ser Ser
Ser 35 40 45 Trp
Gly Leu Lys Lys Ser Gly Met Thr Leu Ile Gly Ser Glu Leu Arg 50
55 60 Pro Leu Lys Val Met Ser
Ser Val Ser Thr Ala Cys Met Glu Lys Ser 65 70
75 80 Met Ser Pro Phe Val Lys Lys His Phe Val Leu
Val His Thr Ala Phe 85 90
95 His Gly Ala Trp Cys Trp Tyr Lys Ile Val Ala Leu Met Arg Ser Ser
100 105 110 Gly His
Asn Val Thr Ala Leu Asp Leu Gly Ala Ser Gly Ile Asn Pro 115
120 125 Lys Gln Ala Leu Gln Ile Pro
Asn Phe Ser Asp Tyr Leu Ser Pro Leu 130 135
140 Met Glu Phe Met Ala Ser Leu Pro Ala Asn Glu Lys
Ile Ile Leu Val 145 150 155
160 Gly His Ala Leu Gly Gly Leu Ala Ile Ser Lys Ala Met Glu Thr Phe
165 170 175 Pro Glu Lys
Ile Ser Val Ala Val Phe Leu Ser Gly Leu Met Pro Gly 180
185 190 Pro Asn Ile Asp Ala Thr Thr Val
Cys Thr Lys Ala Gly Ser Ala Val 195 200
205 Leu Gly Gln Leu Asp Asn Cys Val Thr Tyr Glu Asn Gly
Pro Thr Asn 210 215 220
Pro Pro Thr Thr Leu Ile Ala Gly Pro Lys Phe Leu Ala Thr Asn Val 225
230 235 240 Tyr His Leu Ser
Pro Ile Glu Asp Leu Ala Leu Ala Thr Ala Leu Val 245
250 255 Arg Pro Leu Tyr Leu Tyr Leu Ala Glu
Asp Ile Ser Lys Glu Val Val 260 265
270 Leu Ser Ser Lys Arg Tyr Gly Ser Val Lys Arg Val Phe Ile
Val Ala 275 280 285
Thr Glu Asp Asp Ala Leu Lys Lys Glu Phe Leu Lys Leu Met Ile Glu 290
295 300 Lys Asn Pro Pro Asp
Glu Val Lys Glu Ile Glu Gly Ser Asp His Val 305 310
315 320 Thr Met Met Ser Lys Pro Gln Gln Leu Phe
Thr Thr Leu Leu Ser Ile 325 330
335 Ala Asn Lys Tyr Lys 340 321026DNAartificial
sequenceSynthetic sequence 32atggcgcaag ttagcagaat ctgcaatggt gtgcagaacc
catctcttat ctccaatctc 60tcgaaatcca gtcaacgcaa atctccctta tcggtttctc
tgaagacgca gcagcatcca 120cgagcttatc cgatttcgtc gtcgtgggga ttgaagaaga
gtgggatgac gttaattggc 180tctgagcttc gtcctcttaa ggtcatgtct tctgtttcca
cggcgtgcat ggaaaagtct 240atgtcaccct tcgtcaagaa acacttcgtg cttgttcata
ctgctttcca cggtgcctgg 300tgctggtaca agatcgtcgc ccttatgaga agttcaggcc
acaacgtgac cgcactggac 360ctcggggcct ccgggataaa ccccaaacaa gctttgcaga
ttcctaactt cagtgattat 420ctttctccac ttatggagtt tatggcctct cttcccgcta
acgaaaagat catactcgtt 480ggtcattctc ttggtggtct tgccatttct aaagcaatgg
aaaccttccc tgagaaaata 540tcagtggctg ttttcttgtc cggtcttatg cccggaccta
atattgacgc aacaaccgtt 600tgtaccaagg ctgggtccgc cgtgttgggc caactcgaca
actgcgtgac ctacgaaaat 660ggtcctacta atccacctac tacccttatc gcaggaccta
agtttctcgc tacaaacgtg 720taccacctct ctccaataga ggatctggct cttgccactg
ctttggtcag gcctctttat 780ctgtacctcg ctgaggacat atctaaagaa gttgtgcttt
cttctaagcg ttatggttcc 840gtgaagcgtg tcttcatagt ggcaacagag gatgatgctc
ttaagaaaga gtttctcaaa 900ctgatgatcg agaagaatcc acctgatgaa gtgaaagaaa
ttgagggttc tgaccatgtt 960accatgatgt caaagcctca acagttgttt accacattgc
ttagtatagc taataagtat 1020aaataa
102633341PRTartificial sequenceSynthetic sequence
33Met Ala Gln Val Ser Arg Ile Cys Asn Gly Val Gln Asn Pro Ser Leu 1
5 10 15 Ile Ser Asn Leu
Ser Lys Ser Ser Gln Arg Lys Ser Pro Leu Ser Val 20
25 30 Ser Leu Lys Thr Gln Gln His Pro Arg
Ala Tyr Pro Ile Ser Ser Ser 35 40
45 Trp Gly Leu Lys Lys Ser Gly Met Thr Leu Ile Gly Ser Glu
Leu Arg 50 55 60
Pro Leu Lys Val Met Ser Ser Val Ser Thr Ala Cys Met Glu Lys Ser 65
70 75 80 Met Ser Pro Phe Val
Lys Lys His Phe Val Leu Val His Thr Ala Phe 85
90 95 His Gly Ala Trp Cys Trp Tyr Lys Ile Val
Ala Leu Met Arg Ser Ser 100 105
110 Gly His Asn Val Thr Ala Leu Asp Leu Gly Ala Ser Gly Ile Asn
Pro 115 120 125 Lys
Gln Ala Leu Gln Ile Pro Asn Phe Ser Asp Tyr Leu Ser Pro Leu 130
135 140 Met Glu Phe Met Ala Ser
Leu Pro Ala Asn Glu Lys Ile Ile Leu Val 145 150
155 160 Gly His Ser Leu Gly Gly Leu Ala Ile Ser Lys
Ala Met Glu Thr Phe 165 170
175 Pro Glu Lys Ile Ser Val Ala Val Phe Leu Ser Gly Leu Met Pro Gly
180 185 190 Pro Asn
Ile Asp Ala Thr Thr Val Cys Thr Lys Ala Gly Ser Ala Val 195
200 205 Leu Gly Gln Leu Asp Asn Cys
Val Thr Tyr Glu Asn Gly Pro Thr Asn 210 215
220 Pro Pro Thr Thr Leu Ile Ala Gly Pro Lys Phe Leu
Ala Thr Asn Val 225 230 235
240 Tyr His Leu Ser Pro Ile Glu Asp Leu Ala Leu Ala Thr Ala Leu Val
245 250 255 Arg Pro Leu
Tyr Leu Tyr Leu Ala Glu Asp Ile Ser Lys Glu Val Val 260
265 270 Leu Ser Ser Lys Arg Tyr Gly Ser
Val Lys Arg Val Phe Ile Val Ala 275 280
285 Thr Glu Asp Asp Ala Leu Lys Lys Glu Phe Leu Lys Leu
Met Ile Glu 290 295 300
Lys Asn Pro Pro Asp Glu Val Lys Glu Ile Glu Gly Ser Asp His Val 305
310 315 320 Thr Met Met Ser
Lys Pro Gln Gln Leu Phe Thr Thr Leu Leu Ser Ile 325
330 335 Ala Asn Lys Tyr Lys 340
341029DNAartificial sequenceSynthetic sequence 34atggcgcaag
ttagcagaat ctgcaatggt gtgcagaacc catctcttat ctccaatctc 60tcgaaatcca
gtcaacgcaa atctccctta tcggtttctc tgaagacgca gcagcatcca 120cgagcttatc
cgatttcgtc gtcgtgggga ttgaagaaga gtgggatgac gttaattggc 180tctgagcttc
gtcctcttaa ggtcatgtct tctgtttcca cggcgtgcat ggctaagaag 240tctacttcac
ctttcgtgaa gaagcacttc gttctagttc acactggttt ccacggtgct 300tggtgctggt
acaagattgt tgctttgatg cgtagttctg gtcacaacgt gactgctctt 360gatctaggtg
ctagtggtat taaccctaag caagcccttg aaatcccaca tttcagcgac 420tacttgtccc
cactcatgga gttcatggct agtttgccgg ccaatgagaa gataattctt 480gtcggtcatt
cactcggagg cttagccatt agcaaggcta tggagacttt cccagagaaa 540atttcagttg
ccgtattcct ctcaggactt atgcccggac ctaacatcga cgcaaccaca 600gtttacacta
aggcagcgtc tgctgtgatt gggcaattgg acaattacgt tacctacgag 660aacggcccca
ctaacccacc tactacctta atcgccgggc ctaagtttct cgctaccaat 720gtttatcatc
tttcccccat cgaggatctt gccctggcaa cggctctggt gagacctgtc 780tatctctatc
ttgctgagga catatccaag gaaatcgtac tgtcctctaa acgttatgga 840tctgttaaaa
gggtctttat tgtggcaacc cagaacgatg ctttcaagaa agagtttctc 900aaattgatga
ttgagaaaaa tcctccagac gaagtcaagg agatcgaggg ctcagatcat 960gtgacaatga
tgtcaaaacc acaacagttg tttacaacat tgctgtcgat agcaaataag 1020tacaagtga
102935342PRTartificial sequenceSynthetic sequence 35Met Ala Gln Val Ser
Arg Ile Cys Asn Gly Val Gln Asn Pro Ser Leu 1 5
10 15 Ile Ser Asn Leu Ser Lys Ser Ser Gln Arg
Lys Ser Pro Leu Ser Val 20 25
30 Ser Leu Lys Thr Gln Gln His Pro Arg Ala Tyr Pro Ile Ser Ser
Ser 35 40 45 Trp
Gly Leu Lys Lys Ser Gly Met Thr Leu Ile Gly Ser Glu Leu Arg 50
55 60 Pro Leu Lys Val Met Ser
Ser Val Ser Thr Ala Cys Met Ala Lys Lys 65 70
75 80 Ser Thr Ser Pro Phe Val Lys Lys His Phe Val
Leu Val His Thr Gly 85 90
95 Phe His Gly Ala Trp Cys Trp Tyr Lys Ile Val Ala Leu Met Arg Ser
100 105 110 Ser Gly
His Asn Val Thr Ala Leu Asp Leu Gly Ala Ser Gly Ile Asn 115
120 125 Pro Lys Gln Ala Leu Glu Ile
Pro His Phe Ser Asp Tyr Leu Ser Pro 130 135
140 Leu Met Glu Phe Met Ala Ser Leu Pro Ala Asn Glu
Lys Ile Ile Leu 145 150 155
160 Val Gly His Ser Leu Gly Gly Leu Ala Ile Ser Lys Ala Met Glu Thr
165 170 175 Phe Pro Glu
Lys Ile Ser Val Ala Val Phe Leu Ser Gly Leu Met Pro 180
185 190 Gly Pro Asn Ile Asp Ala Thr Thr
Val Tyr Thr Lys Ala Ala Ser Ala 195 200
205 Val Ile Gly Gln Leu Asp Asn Tyr Val Thr Tyr Glu Asn
Gly Pro Thr 210 215 220
Asn Pro Pro Thr Thr Leu Ile Ala Gly Pro Lys Phe Leu Ala Thr Asn 225
230 235 240 Val Tyr His Leu
Ser Pro Ile Glu Asp Leu Ala Leu Ala Thr Ala Leu 245
250 255 Val Arg Pro Val Tyr Leu Tyr Leu Ala
Glu Asp Ile Ser Lys Glu Ile 260 265
270 Val Leu Ser Ser Lys Arg Tyr Gly Ser Val Lys Arg Val Phe
Ile Val 275 280 285
Ala Thr Gln Asn Asp Ala Phe Lys Lys Glu Phe Leu Lys Leu Met Ile 290
295 300 Glu Lys Asn Pro Pro
Asp Glu Val Lys Glu Ile Glu Gly Ser Asp His 305 310
315 320 Val Thr Met Met Ser Lys Pro Gln Gln Leu
Phe Thr Thr Leu Leu Ser 325 330
335 Ile Ala Asn Lys Tyr Lys 340
361029DNAartificial sequenceSynthetic sequence 36atggcgcaag ttagcagaat
ctgcaatggt gtgcagaacc catctcttat ctccaatctc 60tcgaaatcca gtcaacgcaa
atctccctta tcggtttctc tgaagacgca gcagcatcca 120cgagcttatc cgatttcgtc
gtcgtgggga ttgaagaaga gtgggatgac gttaattggc 180tctgagcttc gtcctcttaa
ggtcatgtct tctgtttcca cggcgtgcat ggctaagaag 240tctacttcac ctttcgtgaa
gaagcacttc gttctagttc acactggttt ccacggtgct 300tggtgctggt acaagattgt
tgctttgatg cgtagttctg gtcacaacgt gactgctctt 360gatctaggtg ctagtggtat
taaccctaag caagcccttg aaatcccaca tttcagcgac 420tacttgtccc cactcatgga
gttcatggct agtttgccgg ccaatgagaa gataattctt 480gtcggtcatg cactcggagg
cttagccatt agcaaggcta tggagacttt cccagagaaa 540atttcagttg ccgtattcct
ctcaggactt atgcccggac ctaacatcga cgcaaccaca 600gtttacacta aggcagcgtc
tgctgtgatt gggcaattgg acaattacgt tacctacgag 660aacggcccca ctaacccacc
tactacctta atcgccgggc ctaagtttct cgctaccaat 720gtttatcatc tttcccccat
cgaggatctt gccctggcaa cggctctggt gagacctgtc 780tatctctatc ttgctgagga
catatccaag gaaatcgtac tgtcctctaa acgttatgga 840tctgttaaaa gggtctttat
tgtggcaacc caggacgatg ctttcaagaa agagtttctc 900aaattgatga ttgagaaaaa
tcctccagac gaagtcaagg agatcgaggg ctcagatcat 960gtgacaatga tgtcaaaacc
acaacagttg tttacaacat tgctgtcgat agcaaataag 1020tacaagtga
102937342PRTartificial
sequenceSynthetic sequence 37Met Ala Gln Val Ser Arg Ile Cys Asn Gly Val
Gln Asn Pro Ser Leu 1 5 10
15 Ile Ser Asn Leu Ser Lys Ser Ser Gln Arg Lys Ser Pro Leu Ser Val
20 25 30 Ser Leu
Lys Thr Gln Gln His Pro Arg Ala Tyr Pro Ile Ser Ser Ser 35
40 45 Trp Gly Leu Lys Lys Ser Gly
Met Thr Leu Ile Gly Ser Glu Leu Arg 50 55
60 Pro Leu Lys Val Met Ser Ser Val Ser Thr Ala Cys
Met Ala Lys Lys 65 70 75
80 Ser Thr Ser Pro Phe Val Lys Lys His Phe Val Leu Val His Thr Gly
85 90 95 Phe His Gly
Ala Trp Cys Trp Tyr Lys Ile Val Ala Leu Met Arg Ser 100
105 110 Ser Gly His Asn Val Thr Ala Leu
Asp Leu Gly Ala Ser Gly Ile Asn 115 120
125 Pro Lys Gln Ala Leu Glu Ile Pro His Phe Ser Asp Tyr
Leu Ser Pro 130 135 140
Leu Met Glu Phe Met Ala Ser Leu Pro Ala Asn Glu Lys Ile Ile Leu 145
150 155 160 Val Gly His Ala
Leu Gly Gly Leu Ala Ile Ser Lys Ala Met Glu Thr 165
170 175 Phe Pro Glu Lys Ile Ser Val Ala Val
Phe Leu Ser Gly Leu Met Pro 180 185
190 Gly Pro Asn Ile Asp Ala Thr Thr Val Tyr Thr Lys Ala Ala
Ser Ala 195 200 205
Val Ile Gly Gln Leu Asp Asn Tyr Val Thr Tyr Glu Asn Gly Pro Thr 210
215 220 Asn Pro Pro Thr Thr
Leu Ile Ala Gly Pro Lys Phe Leu Ala Thr Asn 225 230
235 240 Val Tyr His Leu Ser Pro Ile Glu Asp Leu
Ala Leu Ala Thr Ala Leu 245 250
255 Val Arg Pro Val Tyr Leu Tyr Leu Ala Glu Asp Ile Ser Lys Glu
Ile 260 265 270 Val
Leu Ser Ser Lys Arg Tyr Gly Ser Val Lys Arg Val Phe Ile Val 275
280 285 Ala Thr Gln Asp Asp Ala
Phe Lys Lys Glu Phe Leu Lys Leu Met Ile 290 295
300 Glu Lys Asn Pro Pro Asp Glu Val Lys Glu Ile
Glu Gly Ser Asp His 305 310 315
320 Val Thr Met Met Ser Lys Pro Gln Gln Leu Phe Thr Thr Leu Leu Ser
325 330 335 Ile Ala
Asn Lys Tyr Lys 340 381029DNAartificial
sequenceSynthetic sequence 38atggcgcaag ttagcagaat ctgcaatggt gtgcagaacc
catctcttat ctccaatctc 60tcgaaatcca gtcaacgcaa atctccctta tcggtttctc
tgaagacgca gcagcatcca 120cgagcttatc cgatttcgtc gtcgtgggga ttgaagaaga
gtgggatgac gttaattggc 180tctgagcttc gtcctcttaa ggtcatgtct tctgtttcca
cggcgtgcat ggctaagaag 240tctacttcac ctttcgtgaa gaagcacttc gttctagttc
acactggttt ccacggtgct 300tggtgctggt acaagattgt tgctttgatg cgtagttctg
gtcacaacgt gactgctctt 360gatctaggtg ctagtggtat taaccctaag caagcccttg
aaatcccaca tttcagcgac 420tacttgtccc cactcatgga gttcatggct agtttgccgg
ccaatgagaa gataattctt 480gtcggtcatt cactcggagg cttagccatt agcaaggcta
tggagacttt cccagagaaa 540atttcagttg ccgtattcct ctcaggactt atgcccggac
ctaacatcga cgcaaccaca 600gtttacacta aggcagcgtc tgctgtgatt gggcaattgg
acaattacgt tacctacgag 660aacggcccca ctaacccacc tactacctta atcgccgggc
ctaagtttct cgctaccaat 720gtttatcatc tttcccccat cgaggatctt gccctggcaa
cggctctggt gagacctgtc 780tatctctatc ttgctgagga catatccaag gaaatcgtac
tgtcctctaa acgttatgga 840tctgttaaaa gggtctttat tgtggcaacc caggacgatg
ctttcaagaa agagtttctc 900aaattgatga ttgagaaaaa tcctccagac gaagtcaagg
agatcgaggg ctcagatcat 960gtgacaatga tgtcaaaacc acaacagttg tttacaacat
tgctgtcgat agcaaataag 1020tacaagtga
102939342PRTartificial sequenceSynthetic sequence
39Met Ala Gln Val Ser Arg Ile Cys Asn Gly Val Gln Asn Pro Ser Leu 1
5 10 15 Ile Ser Asn Leu
Ser Lys Ser Ser Gln Arg Lys Ser Pro Leu Ser Val 20
25 30 Ser Leu Lys Thr Gln Gln His Pro Arg
Ala Tyr Pro Ile Ser Ser Ser 35 40
45 Trp Gly Leu Lys Lys Ser Gly Met Thr Leu Ile Gly Ser Glu
Leu Arg 50 55 60
Pro Leu Lys Val Met Ser Ser Val Ser Thr Ala Cys Met Ala Lys Lys 65
70 75 80 Ser Thr Ser Pro Phe
Val Lys Lys His Phe Val Leu Val His Thr Gly 85
90 95 Phe His Gly Ala Trp Cys Trp Tyr Lys Ile
Val Ala Leu Met Arg Ser 100 105
110 Ser Gly His Asn Val Thr Ala Leu Asp Leu Gly Ala Ser Gly Ile
Asn 115 120 125 Pro
Lys Gln Ala Leu Glu Ile Pro His Phe Ser Asp Tyr Leu Ser Pro 130
135 140 Leu Met Glu Phe Met Ala
Ser Leu Pro Ala Asn Glu Lys Ile Ile Leu 145 150
155 160 Val Gly His Ser Leu Gly Gly Leu Ala Ile Ser
Lys Ala Met Glu Thr 165 170
175 Phe Pro Glu Lys Ile Ser Val Ala Val Phe Leu Ser Gly Leu Met Pro
180 185 190 Gly Pro
Asn Ile Asp Ala Thr Thr Val Tyr Thr Lys Ala Ala Ser Ala 195
200 205 Val Ile Gly Gln Leu Asp Asn
Tyr Val Thr Tyr Glu Asn Gly Pro Thr 210 215
220 Asn Pro Pro Thr Thr Leu Ile Ala Gly Pro Lys Phe
Leu Ala Thr Asn 225 230 235
240 Val Tyr His Leu Ser Pro Ile Glu Asp Leu Ala Leu Ala Thr Ala Leu
245 250 255 Val Arg Pro
Val Tyr Leu Tyr Leu Ala Glu Asp Ile Ser Lys Glu Ile 260
265 270 Val Leu Ser Ser Lys Arg Tyr Gly
Ser Val Lys Arg Val Phe Ile Val 275 280
285 Ala Thr Gln Asp Asp Ala Phe Lys Lys Glu Phe Leu Lys
Leu Met Ile 290 295 300
Glu Lys Asn Pro Pro Asp Glu Val Lys Glu Ile Glu Gly Ser Asp His 305
310 315 320 Val Thr Met Met
Ser Lys Pro Gln Gln Leu Phe Thr Thr Leu Leu Ser 325
330 335 Ile Ala Asn Lys Tyr Lys
340 40411DNALycopersicon hirsutum 40atggcttctg ttactggatc
ttccttcgcc atctcttccc tttcctcctc cttcaacccc 60aacaaggcat gtttgaagac
atcctccctt tctatcaagg gaataagctt cccttccctc 120agagtgaagc cagctactcg
tcgcttcagc attagctgtg cggctaaacc agagacagtt 180gacaaagtgt gtgaaattgt
gaagaaacaa ctggctcttc ccaatgattc tgcagtcaat 240ggagagtcca aatttatagc
acttggtgct gattctcttg acacggttga gattgtcatg 300ggacttgagg aagcatttgg
aatcactgtt gaagaagaga atgcacagac tattgcaaca 360gttcaagatg ctgctgactt
gattgaggat ctcgttgcca agaaatgtta a 41141136PRTLycopersicon
hirsutum 41Met Ala Ser Val Thr Gly Ser Ser Phe Ala Ile Ser Ser Leu Ser
Ser 1 5 10 15 Ser
Phe Asn Pro Asn Lys Ala Cys Leu Lys Thr Ser Ser Leu Ser Ile
20 25 30 Lys Gly Ile Ser Phe
Pro Ser Leu Arg Val Lys Pro Ala Thr Arg Arg 35
40 45 Phe Ser Ile Ser Cys Ala Ala Lys Pro
Glu Thr Val Asp Lys Val Cys 50 55
60 Glu Ile Val Lys Lys Gln Leu Ala Leu Pro Asn Asp Ser
Ala Val Asn 65 70 75
80 Gly Glu Ser Lys Phe Ile Ala Leu Gly Ala Asp Ser Leu Asp Thr Val
85 90 95 Glu Ile Val Met
Gly Leu Glu Glu Ala Phe Gly Ile Thr Val Glu Glu 100
105 110 Glu Asn Ala Gln Thr Ile Ala Thr Val
Gln Asp Ala Ala Asp Leu Ile 115 120
125 Glu Asp Leu Val Ala Lys Lys Cys 130
135 42411DNALycopersicon hirsutum 42atggcttctg ttacaggctc atcagctacc
gccatgtcct gctccttcaa ggcacctaac 60cgtgtatcga atttgaagac atcttctctt
tcattcaagg ggattagcta ctcttccatc 120agagtgaatc cagccactcg tcgcctcagt
attacttgtg cggctaagcc agagactgtc 180aacaaagtgt gtgacattgt gaagaaacaa
ctagctcttt ctgctgatac tgatgtctgt 240ggagattcaa agtttgctgc gcttggtgct
gattctcttg atacggtgga gattgtcatg 300ggacttgagg aagagtttgg catctcggtg
gaagaagaca gtgctcagag tattgcaaca 360gttcaagatg ctgcagacct gattgaggat
ctcatttcaa agaaagcttg a 41143136PRTLycopersicon hirsutum
43Met Ala Ser Val Thr Gly Ser Ser Ala Thr Ala Met Ser Cys Ser Phe 1
5 10 15 Lys Ala Pro Asn
Arg Val Ser Asn Leu Lys Thr Ser Ser Leu Ser Phe 20
25 30 Lys Gly Ile Ser Tyr Ser Ser Ile Arg
Val Asn Pro Ala Thr Arg Arg 35 40
45 Leu Ser Ile Thr Cys Ala Ala Lys Pro Glu Thr Val Asn Lys
Val Cys 50 55 60
Asp Ile Val Lys Lys Gln Leu Ala Leu Ser Ala Asp Thr Asp Val Cys 65
70 75 80 Gly Asp Ser Lys Phe
Ala Ala Leu Gly Ala Asp Ser Leu Asp Thr Val 85
90 95 Glu Ile Val Met Gly Leu Glu Glu Glu Phe
Gly Ile Ser Val Glu Glu 100 105
110 Asp Ser Ala Gln Ser Ile Ala Thr Val Gln Asp Ala Ala Asp Leu
Ile 115 120 125 Glu
Asp Leu Ile Ser Lys Lys Ala 130 135
44411DNALycopersicon hirsutum 44atggcttctg ttactggatc ttccttcgcc
atctcctccc tttcctcctc cttcaacccc 60aacaaggcat gtttgaagac atcctccctt
tctatcaagg gaataagctt cccttccctc 120agagtgaagc cagctactcg tcacttcagc
attagctgtg cggctaaacc agagacagtt 180gacaaagtgt gtgaaattgt gaagaaacaa
ctggctcttc ccaatgattc tgcagtcaat 240ggagagtcca aatttatagc acttggtgct
gattctcttg acacggttga gattgtcatg 300ggacttgagg aagcatttgg aatcactgtt
gaagaagaga acgcacagac tattgcaaca 360gttcaagatg ctgctgactt gattgaggat
cttgtcgcca agaaatgtta a 41145136PRTLycopersicon hirsutum
45Met Ala Ser Val Thr Gly Ser Ser Phe Ala Ile Ser Ser Leu Ser Ser 1
5 10 15 Ser Phe Asn Pro
Asn Lys Ala Cys Leu Lys Thr Ser Ser Leu Ser Ile 20
25 30 Lys Gly Ile Ser Phe Pro Ser Leu Arg
Val Lys Pro Ala Thr Arg His 35 40
45 Phe Ser Ile Ser Cys Ala Ala Lys Pro Glu Thr Val Asp Lys
Val Cys 50 55 60
Glu Ile Val Lys Lys Gln Leu Ala Leu Pro Asn Asp Ser Ala Val Asn 65
70 75 80 Gly Glu Ser Lys Phe
Ile Ala Leu Gly Ala Asp Ser Leu Asp Thr Val 85
90 95 Glu Ile Val Met Gly Leu Glu Glu Ala Phe
Gly Ile Thr Val Glu Glu 100 105
110 Glu Asn Ala Gln Thr Ile Ala Thr Val Gln Asp Ala Ala Asp Leu
Ile 115 120 125 Glu
Asp Leu Val Ala Lys Lys Cys 130 135
46411DNALycopersicon esculentum 46atggcttcca ttacaggctc atcagctacc
gccatgtcct gctccttcaa ggcacctaac 60cgtgtatcga atttgaaaac atcttctctt
tcattcaagg ggattagcta ctcttccatc 120agtgtgaatc cagccactcg tcgcctcagt
attacttgtg cggctaagcc agagactgtc 180aacaaagtgt gtgacattgt gaagaaacaa
ctggctcttt ctgctgatac tgatgtctgt 240ggagattcaa agtttgctgc gcttggtgct
gattctcttg atacggtgga gattgtcatg 300ggacttgagg aagaatttgg catctcggtg
gaagaagaca gtgctcagag tattgcaacc 360gttcaagatg ctgcagacct gattgaggat
ctcatttcaa agaaagcttg a 41147136PRTLycopersicon esculentum
47Met Ala Ser Ile Thr Gly Ser Ser Ala Thr Ala Met Ser Cys Ser Phe 1
5 10 15 Lys Ala Pro Asn
Arg Val Ser Asn Leu Lys Thr Ser Ser Leu Ser Phe 20
25 30 Lys Gly Ile Ser Tyr Ser Ser Ile Ser
Val Asn Pro Ala Thr Arg Arg 35 40
45 Leu Ser Ile Thr Cys Ala Ala Lys Pro Glu Thr Val Asn Lys
Val Cys 50 55 60
Asp Ile Val Lys Lys Gln Leu Ala Leu Ser Ala Asp Thr Asp Val Cys 65
70 75 80 Gly Asp Ser Lys Phe
Ala Ala Leu Gly Ala Asp Ser Leu Asp Thr Val 85
90 95 Glu Ile Val Met Gly Leu Glu Glu Glu Phe
Gly Ile Ser Val Glu Glu 100 105
110 Asp Ser Ala Gln Ser Ile Ala Thr Val Gln Asp Ala Ala Asp Leu
Ile 115 120 125 Glu
Asp Leu Ile Ser Lys Lys Ala 130 135
48411DNASolanum tuberosum 48atggcttcta ttacaggctc atcagctacc gccatgtcct
gctccttcaa ggaacttgac 60cgtgtctcga atttgaagac atcttctctt tcattcaagg
ggattagcta ctcttccatc 120agagtgaatc cagccactcg tcgcctcagt attacttgtg
cggctaagcc agagactgtc 180aacaaagtgt gtgacattgt gaagaaacaa ctggctctct
ctgctggtac tgaagtctgt 240ggagaatcaa agtttgctgc gcttggtgct gattctcttg
atacggtgga gattgtcatg 300ggacttgagg aagagtttgg catctccgtg gaagaagaca
gtgctaacag tattgcaaca 360gttcaagatg ctgcagacct gattgaggat ctcatttcaa
agaaagcttg a 41149136PRTSolanum tuberosum 49Met Ala Ser Ile
Thr Gly Ser Ser Ala Thr Ala Met Ser Cys Ser Phe 1 5
10 15 Lys Glu Leu Asp Arg Val Ser Asn Leu
Lys Thr Ser Ser Leu Ser Phe 20 25
30 Lys Gly Ile Ser Tyr Ser Ser Ile Arg Val Asn Pro Ala Thr
Arg Arg 35 40 45
Leu Ser Ile Thr Cys Ala Ala Lys Pro Glu Thr Val Asn Lys Val Cys 50
55 60 Asp Ile Val Lys Lys
Gln Leu Ala Leu Ser Ala Gly Thr Glu Val Cys 65 70
75 80 Gly Glu Ser Lys Phe Ala Ala Leu Gly Ala
Asp Ser Leu Asp Thr Val 85 90
95 Glu Ile Val Met Gly Leu Glu Glu Glu Phe Gly Ile Ser Val Glu
Glu 100 105 110 Asp
Ser Ala Asn Ser Ile Ala Thr Val Gln Asp Ala Ala Asp Leu Ile 115
120 125 Glu Asp Leu Ile Ser Lys
Lys Ala 130 135 50411DNASolanum chacoense
50atggcttcta ttacaggctc atcagctacc gccatgtcct gctccttcaa ggaacttaac
60cgtgtctcga atttgaagac atcttctctt tcattcaagg ggattagcta ctcttccatc
120agagtgaatc cagccactcg tcgcctcagt attacttgtg cggctaagcc agagactgtc
180aacaaagtgt gtgacattgt gaagaaacaa ctggctcttt ctgctgatac tgaagtctgt
240ggagattcaa agtttgctgc gcttggtgct gattctcttg atacggtgga gattgtcatg
300ggacttgagg aagagtttgg catctccgtg gaagaagata gtgctcagag tattgcaaca
360gttcaagatg ctgcagacct gattgaggat ctcatttcaa agaaagcttg a
41151136PRTSolanum chacoense 51Met Ala Ser Ile Thr Gly Ser Ser Ala Thr
Ala Met Ser Cys Ser Phe 1 5 10
15 Lys Glu Leu Asn Arg Val Ser Asn Leu Lys Thr Ser Ser Leu Ser
Phe 20 25 30 Lys
Gly Ile Ser Tyr Ser Ser Ile Arg Val Asn Pro Ala Thr Arg Arg 35
40 45 Leu Ser Ile Thr Cys Ala
Ala Lys Pro Glu Thr Val Asn Lys Val Cys 50 55
60 Asp Ile Val Lys Lys Gln Leu Ala Leu Ser Ala
Asp Thr Glu Val Cys 65 70 75
80 Gly Asp Ser Lys Phe Ala Ala Leu Gly Ala Asp Ser Leu Asp Thr Val
85 90 95 Glu Ile
Val Met Gly Leu Glu Glu Glu Phe Gly Ile Ser Val Glu Glu 100
105 110 Asp Ser Ala Gln Ser Ile Ala
Thr Val Gln Asp Ala Ala Asp Leu Ile 115 120
125 Glu Asp Leu Ile Ser Lys Lys Ala 130
135 52417DNANicotiana tabacum 52atggcttcta ttacaggttc
atctgttacc atgtcatgct ccttgaagca aaatcaggca 60cttaaccgag tctctaattt
gaagacgtct tctctttcat tcaaggggat aagcttccct 120tccattcgat tgaatcgagc
cactggccac ctcactatca cttgcgcggc taagccagaa 180acggtcaaca aggtgtgtga
aattgtgaag aaacaactgg ctctttctgg tgaaactgaa 240gtcagtggag agtcaaagtt
tgctgcgctt ggtgctgatt ctcttgatac ggtggagata 300gtcatggggc ttgaggaaga
gtttggtatt tccgtagaag aagacagtgc tcagagtatt 360gcaacagttc aagatgctgc
agacctgatt gaggatctca tttcaaagaa agcttga 41753138PRTNicotiana
tabacum 53Met Ala Ser Ile Thr Gly Ser Ser Val Thr Met Ser Cys Ser Leu Lys
1 5 10 15 Gln Asn
Gln Ala Leu Asn Arg Val Ser Asn Leu Lys Thr Ser Ser Leu 20
25 30 Ser Phe Lys Gly Ile Ser Phe
Pro Ser Ile Arg Leu Asn Arg Ala Thr 35 40
45 Gly His Leu Thr Ile Thr Cys Ala Ala Lys Pro Glu
Thr Val Asn Lys 50 55 60
Val Cys Glu Ile Val Lys Lys Gln Leu Ala Leu Ser Gly Glu Thr Glu 65
70 75 80 Val Ser Gly
Glu Ser Lys Phe Ala Ala Leu Gly Ala Asp Ser Leu Asp 85
90 95 Thr Val Glu Ile Val Met Gly Leu
Glu Glu Glu Phe Gly Ile Ser Val 100 105
110 Glu Glu Asp Ser Ala Gln Ser Ile Ala Thr Val Gln Asp
Ala Ala Asp 115 120 125
Leu Ile Glu Asp Leu Ile Ser Lys Lys Ala 130 135
54408DNAPetunia hybrida 54atggcttcta ttactgcctc atccctttgc
tcctttaagc caaatcatgc acttaaccga 60gtctctaatt tgaagacgtc gtctcttact
ttcaagggga taagtttctc ttccatcaga 120ttgattccaa ctactcgtcg cctctgtatt
acttgcgcgg ctaagccaga gacagtaaat 180aaagtgtgtg aaattgtgaa gaaacagctg
gctctttctg ctgattctga agtcagtgga 240gagtcaaagt ttgctgcgct tggtgctgat
tctcttgata cggtggaaat tgtcatggga 300cttgaggaag agttcggtat ttccgtggaa
gaagatagtg ctcagagtat tgcaacagtt 360caagatgctg cagacctgat tgaggatctc
atttcaaaga aagcttga 40855135PRTPetunia hybrida 55Met Ala
Ser Ile Thr Ala Ser Ser Leu Cys Ser Phe Lys Pro Asn His 1 5
10 15 Ala Leu Asn Arg Val Ser Asn
Leu Lys Thr Ser Ser Leu Thr Phe Lys 20 25
30 Gly Ile Ser Phe Ser Ser Ile Arg Leu Ile Pro Thr
Thr Arg Arg Leu 35 40 45
Cys Ile Thr Cys Ala Ala Lys Pro Glu Thr Val Asn Lys Val Cys Glu
50 55 60 Ile Val Lys
Lys Gln Leu Ala Leu Ser Ala Asp Ser Glu Val Ser Gly 65
70 75 80 Glu Ser Lys Phe Ala Ala Leu
Gly Ala Asp Ser Leu Asp Thr Val Glu 85
90 95 Ile Val Met Gly Leu Glu Glu Glu Phe Gly Ile
Ser Val Glu Glu Asp 100 105
110 Ser Ala Gln Ser Ile Ala Thr Val Gln Asp Ala Ala Asp Leu Ile
Glu 115 120 125 Asp
Leu Ile Ser Lys Lys Ala 130 135 56411DNACapsicum
annum 56atggcttcta tcacaggctc atccatctcc tcctccttca aacttaatca ggcacttaac
60cgtgtctgtt gtttgaagac accttctatt tctttcaagg ggataagctt gtcttccagc
120agattgaatc aagccactcg tcgcctcagt attacttgtg cggctaaacc agagactgtc
180aacaaagtgt gtgacattgt gaagaaacaa ctggctcttt ccgctgatac tgcagtttgt
240ggagagtcca agtttgctgc gcttggtgct gattctcttg atacggtgga gattgtcatg
300ggacttgagg aagagtttgg catctccgtg gaagaagata gtgctcagaa tattgcaaca
360gttcaagatg ctgcagacct gattgaggat ctcatttcaa agaatgcttg a
41157136PRTCapsicum annum 57Met Ala Ser Ile Thr Gly Ser Ser Ile Ser Ser
Ser Phe Lys Leu Asn 1 5 10
15 Gln Ala Leu Asn Arg Val Cys Cys Leu Lys Thr Pro Ser Ile Ser Phe
20 25 30 Lys Gly
Ile Ser Leu Ser Ser Ser Arg Leu Asn Gln Ala Thr Arg Arg 35
40 45 Leu Ser Ile Thr Cys Ala Ala
Lys Pro Glu Thr Val Asn Lys Val Cys 50 55
60 Asp Ile Val Lys Lys Gln Leu Ala Leu Ser Ala Asp
Thr Ala Val Cys 65 70 75
80 Gly Glu Ser Lys Phe Ala Ala Leu Gly Ala Asp Ser Leu Asp Thr Val
85 90 95 Glu Ile Val
Met Gly Leu Glu Glu Glu Phe Gly Ile Ser Val Glu Glu 100
105 110 Asp Ser Ala Gln Asn Ile Ala Thr
Val Gln Asp Ala Ala Asp Leu Ile 115 120
125 Glu Asp Leu Ile Ser Lys Asn Ala 130
135 58638DNALycopersicon esculentum 58gggattactt gagtccgcta
atggagttca tgacttcact tcctgttgat gaaaaaatag 60ttcttgttgg ccatagcgtt
ggtggactcg ccatttctaa agccatggaa accttccctg 120aaaagatttc tgttgctgta
tttcttagtg gtgtaatgcc tggtccaaat attagtgcat 180caatcgtcta tactgaggca
atcaatgcaa taatacgtga acttgataat cgggttacat 240accacaacgg atctgagaat
cctccaacga ccttcaacct aggtcccaag ttcttggaaa 300ctaatgctta ccatctgagc
ccaattgagg atttggcgct ggctactaca ctagtaaggc 360cattttattt atacagtgcg
gaagatgttt ctaaagagat agtactttca agcaaaaaat 420atggatcagt taagagagtg
ttcatctttg ctgctaaaaa tgaagttgtg aagaaggaat 480ttttccaaac gatgattgaa
aagaatccac caaatgaaat agaagtaatc gaggggtctg 540accatgcgac catgacgtct
aagccccaac agctttatac tactcttctc aacattgcca 600acaagtatac ctgagccctt
tcaatttgat atttcatc 638591281DNASolanum
tuberosum 59caattgtcta acaagtcttg ttgttctaat acttgtcctg ccatatgtaa
atgcaacctt 60gtcaaggcca aaagctgcaa agcactttgt gttggttcat aaggcatgtc
atggagcctg 120gtcttggtac aaaattatag cattgatgaa atcttcaggg cataatgtca
cagctcttga 180cttaggtgct tctggaatca actcgaaaca ggcccccgaa atcacacatt
tttctgattt 240catgagtcct ttaatcgagt tcatgacttc tcttcctgca cataaaaatg
ttgttcttgt 300tggccatagc attggtggtc tagccatttc gaaagccatg gaactttttc
cagaaaagat 360ttcaggtgct gtatttgtag ctggtctaat gcctggtcca aatatcaatg
caagcactgt 420ctacattgag ttatgcaatg cagtagtatc gaaacttgat aatcgtgtta
tataccataa 480tgggacttcg aatcctccaa ccttcatttt aggtccgaag tacttagcaa
gtaatgttta 540ccaacagagc ccaattgagg atttggcgtt ggccactaca ctagtaaggg
aaatattttt 600ttacagtgtg gaggatgttt ctaaggagat aattctttcg agaaaaagat
atggatcaat 660taggcgagcg tttgttgtta ctccggaaga taaacttcta aaaaaggaat
tcaacaattg 720atgattgaca gaaatccacc ttgatgaagt gaaagagatc caagggcgct
gaccataatg 780gccatgatgt ctaaggcccc atgaactttt ttaacatttc ttctgagatt
tgctgacaag 840ggacttactc acgtgtctgt tgcatctggt ttgacgaacc taaagttctt
tccatggaaa 900gtccttcccg agctaagttg gaattggaat gtcccaattc attttccttc
aagttggtcg 960caggttccct tcaatgggga ctaattaggg ctggggaatg cacgtcctta
aacgattttt 1020tttcccgata aaaaagaaga gaggaacgaa cgaaaatcgc ggggggggcg
cggggaccca 1080aattccgact aaaatggagg aagttttaaa cggcgcgcaa gaggcgggtt
aaaaccgtcg 1140gctcggaaga caccggggtt cccacaataa ggcttgggag agacaccttt
tcgcaggggt 1200taagaaaaga ccccggtcct ctcaagaagg acgtcagaga ggaaggccgc
ttgacaaaca 1260ccgggtcgtt ggcaccacgc a
128160729DNANicotiana tabacumunsure(1)..(729)unsure at all n
locations 60aaaaaaagat atggagaaaa gcaagtttct aacaagtcta gttctaatac
tttgttttca 60tatgtaaatg caacaacatc agtgactata tggcctaagg cgagcaagca
tttcgtgcta 120gttcacgggg cttgccatgg agcttggtct tggtacaaga ttatggcgtc
gttaaaaact 180tcaggacata atgtcacagc tctggactta ggtgcttcgg gagtcaaccc
aaagcagggc 240cttgaaatcc cacatttatc ggactacgtt agtccgctaa tgaagttcat
ggcttctctt 300cctgcagatg aaaaagtgat tcttgtaggt catagccttg gtggatttgc
catttctaaa 360gcgatggaaa cttttccaga aaagatttca gttgctgtat ttgtcactgc
tctaatgcct 420ggtccaactc tcaatgcaac caccatattt attgagtcat ccaaggcagc
aatatcagta 480cttgataatc gtgttacatt cgatagtgga cctatgaatc ctccaacaac
cttcagcttt 540ggtccgaagt acttggcgag ttatctttat ccactgagcc caattcagga
ctgggcactg 600gctactacag tagtaaggcc attatattta tacagttcgg atgacatatc
aaaggaaata 660gttctttcaa gcaaaaagta tgcatcagtt taacgagtgt tccttgtggt
ggtgaanata 720aagtctaaa
72961719DNACapsicum annum 61ggcacgagga agatatggaa aaaagcaagt
ttctaacaag tctagttcta atacttctgt 60tgtcatatgt aaatgcaaca acatcaggcc
ctaaatggcc taaagctcgt aagcactttg 120tgctagttca tggggcttgc catggagcct
gggcttggta caagataata gcatcgataa 180aaacttcagg gcataacgta acagctctgg
acttgggtgc ttcaggggtc aacccaaaac 240aggtccttga aatcccacat ttatcggatt
actttagtcc gctaatggag ttcatgactt 300ctcttccaac agatgaaaaa gtgattcttg
ttggtaacag tcttggtgga tttgccattg 360ctaaagctat ggaaatcttt cctgagaaga
tttcagttgc tgtatttgtc gctgctctca 420tgcctggtct aattctcgat gcggccacca
tctataatga gacatccagc ggaacattta 480tacttgataa tcttattaca ttcgataatg
gacctaccaa tcctccaaca accgtcagct 540ttggtccgaa gttcttggcg agttatattt
atccactgag cccaattcaa gactgggcac 600tggctacgac actagtaagg ccattgtatc
tttacagttt ggatgacata tcaaaggaga 660tggttcttac aagcaagaag tatggatcag
ttaggcgagc atatatggtg gcggctgaa 71962588DNAArabidopsis thaliana
62ggtaccatct tctgtatcat cttcttcttc ttcaaggtga gtctctagat ccgttcgctt
60gattttgctg ctcgttagtc gttattgttg attctctatg ccgatttcgc tagatccgtt
120tagcatgcgt tgtggtttta tgagaaaatc tttgttttgg gggttgcttg ttatgtgatt
180cgatccgtgc ttgttggatc gatctgagct aattcttaag gtttatgtgt tagatcaatg
240gagtttgagg attcttctcg cttctgtcga tctctcgctg ttatttttgt ttttttcagt
300gaagtgaagt tgtttagttc gaaatgactt cgtgtatgct cgattgatct ggttttaatc
360ttcgatctgt taggtgttga tgtttacaag tgaattgatg tgttttctcg ttgtgatctg
420tgaagtttga acctagtttt ctcaataatc aacatatgaa gcgatgtttg agtttcaata
480aacgctgcta atcttcgaaa ctaagttgtg atctgattcg tgtttacttc atgagcttat
540ccaattcatt tcggtttcat tttacttttt ttttagtgaa ggcgcgcc
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