Patent application title: POLYNUCLEOTIDES AND METHODS FOR IMPROVING PLANTS
Inventors:
Sathish Puthigae (Auckland, NZ)
Sathish Puthigae (Auckland, NZ)
Jonathan Robert Phillips (Bonn, DE)
Claudia Jeannette Smith-Espinoza (Bonn, DE)
Catherine Jane Bryant (Papatoetoe, NZ)
Kieran Michael Elborough (Franklin, NZ)
Margaret Biswas (Auckland, NZ)
Assignees:
Fonterra Co-operative Group Limited
IPC8 Class: AA01H500FI
USPC Class:
800290
Class name: Multicellular living organisms and unmodified parts thereof and related processes method of introducing a polynucleotide molecule into or rearrangement of genetic material within a plant or plant part the polynucleotide alters plant part growth (e.g., stem or tuber length, etc.)
Publication date: 2012-10-11
Patent application number: 20120260369
Abstract:
The invention provides methods and compositions for producing plant with
altered biomass, the methods comprising the step of altering the
expression and/or activity of the polypeptide comprising the sequence of
SEQ ID NO:1, or a variant thereof, in a plant cell or plant. The
invention also provides a polypeptide comprising the sequence of SEQ ID
NO:1, and fragments of variants thereof the sequence. The invention also
provides polynucleotides encoding such polypeptide sequences. The
invention also provides constructs, cells and plants comprising such
polynucleotides.Claims:
1. A method of producing a plant with increased biomass the method
comprising the step of reducing the expression, in a plant cell or plant,
of a polypeptide comprising the sequence of SEQ ID NO: 20, wherein the
reducing of expression leads to the increased biomass.
2. The method of claim 1, wherein the expression is reduced by transforming a plant cell, or plant, with a polynucleotide that is complementary to an endogenous gene encoding the polypeptide, such that expression of the polynucleotide results in reduced expression of the polypeptide and leads to the increased biomass.
3. The method of claim 2, wherein the plant cell or plant is transformed with at least one of: a) a polynucleotide including a sequence encoding of a polypeptide comprising the amino acid sequence of SEQ ID NO:20; b) a polynucleotide comprising a fragment, of at least 15 nucleotides in length, of the polynucleotide of a); and c) a polynucleotide comprising a complement, of at least 15 nucleotides in length, of the polynucleotide of a); wherein the expression of the polynucleotide in the plant cell or plant results in reduced expression of the polypeptide and leads to the increased biomass.
4. A plant produced by the method of claim 1.
5. A plant produced by the method of claim 1, wherein the expression of an endogenous polypeptide comprising the amino acid sequence of SEQ ID NO:20 is down-regulated in the plant relative to a non-transformed control plant, and wherein the plant has increased biomass relative to a non-transformed control plant.
6. The plant of claim 5 that has an increased number of tillers relative to a non-transformed control plant.
7. A method for selecting a plant with altered biomass, the method comprising testing of a plant for reduced expression of a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:20, wherein the reduced expression is indicative of increased biomass.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. application Ser. No. 12/324,664, filed Nov. 26, 2008, and U.S. Provisional Application No. 60/990,590, filed Nov. 27, 2007. The priority applications are incorporated herein by reference in their entirety.
REFERENCE TO SEQUENCE LISTING, TABLE, OR COMPUTER PROGRAM LISTING
[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled Sequence-Listing-DAIRY94003AUS.txt, created Nov. 26, 2008, which is 76.6 Kb in size. The information in the electronic format 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 compositions and methods for producing plants with increased biomass.
[0005] 2. Description of the Related Art
[0006] As the population of the world increases, a major goal of agricultural research is to improve the biomass yield of crop and forage plant species.
[0007] Such improvements have until recently depended on selective breeding of plants for desirable characteristics. However for many plants the heterogeneous genetic complements produced in off-spring do not result in the same desirable traits as those of their parents, thus limiting the effectiveness of selective breeding approaches.
[0008] Advances in molecular biology now make it possible to genetically manipulate the germplasm of both plants and animals. Genetic engineering of plants involves the isolation and manipulation of genetic material and the subsequent introduction of such material into a plant. This technology has led to the development of plants that are capable of expressing pharmaceuticals and other chemicals, plants with increased pest resistance, increased stress tolerance, and plants that express other beneficial traits.
[0009] Whilst it is known in the art that certain growth factors may be applied to increase plant size, the application of such growth factors is both costly and time consuming. Thus, there exists a need for plants with increased biomass relative to their cultivated counterparts.
[0010] It is an object of the invention to provide improved compositions and/or methods for developing plant varieties with altered biomass or at least to provide the public with a useful choice.
SUMMARY OF THE INVENTION
[0011] In a first aspect the invention provides a method for producing a plant with altered biomass, the method comprising transformation of a plant with a: [0012] a) a polynucleotide including a sequence encoding of a polypeptide with the amino acid sequence of SEQ ID NO:1 or a variant of the polypeptide; or [0013] b) a polynucleotide comprising a fragment, of at least 15 nucleotides in length, of the polynucleotide of a); or [0014] c) a polynucleotide comprising a compliment, of at least 15 nucleotides in length, of the polynucleotide of a); or [0015] d) a polynucleotide comprising a sequence, of at least 15 nucleotides in length, capable of hybridising to the polynucleotide of a) under stringent conditions.
[0016] Preferably the polynucleotide is included as part of a genetic construct.
[0017] In one embodiment the variant has at least 70% sequence identity to a polypeptide with the amino acid sequence of SEQ ID NO: 1.
[0018] In a further embodiment the variant comprises the amino acid sequence of SEQ ID NO: 20.
[0019] In a further embodiment the variant is derived from a plant species and comprises the amino acid sequence of SEQ ID NO: 20.
[0020] In a further embodiment the variant is derived from a dicotyledonous plant species and comprises the amino acid sequence of SEQ ID NO: 21.
[0021] Preferably the variant is capable of modulating biomass in a plant
[0022] In a further embodiment the polynucleotide of a) encodes a polypeptide with the amino acid sequence of SEQ ID NO: 1.
[0023] Preferably expression of the polynucleotide in the plant results in down-regulation of an endogenous polynucleotide/polypeptide capable of modulating biomass production in the plant.
[0024] Preferably the reduced expression is effected by antisense suppression, sense suppression or RNA interference.
[0025] Preferably the plant produced has increased biomass relative to a suitable control plant.
[0026] In a further aspect the invention provides a method for producing a plant with increased biomass, the method comprising transformation of a plant with a polynucleotide with sufficient sequence similarity to an endogenous nucleic acid encoding a polypeptide with the sequence of SEQ ID NO:1 or a variant thereof, such that expression of the polynucleotide results in inhibition of expression of the endogenous nucleic acid.
[0027] Preferably the polynucleotide is included as part of a genetic construct.
[0028] In one embodiment the variant has at least 70% sequence identity to a polypeptide with the amino acid sequence of SEQ ID NO: 1.
[0029] In a further embodiment the variant comprises the amino acid sequence of SEQ ID NO: 20.
[0030] In a further embodiment the variant is derived from a plant species and comprises the amino acid sequence of SEQ ID NO: 20.
[0031] In a further embodiment the variant is derived from a dicotyledonous plant species and comprises the amino acid sequence of SEQ ID NO: 21.
[0032] Preferably the variant is capable of modulating biomass in a plant.
[0033] In a further embodiment the polypeptide has the sequence of SEQ ID NO:1
[0034] In a further aspect the invention provides a method of producing a plant with altered biomass, the method comprising transformation of a plant cell or plant with a: [0035] a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:10, or a variant thereof; or [0036] b) a polynucleotide comprising a fragment, of at least 15 nucleotides in length, of the polynucleotide of a); or [0037] c) a polynucleotide comprising a complement, of at least 15 nucleotides in length, of the polynucleotide of a); or [0038] d) a polynucleotide comprising a sequence, of at least 15 nucleotides in length, capable of hybridising to the polynucleotide of a) under stringent conditions.
[0039] Preferably the polynucleotide is included as part of a genetic construct.
[0040] Preferably the variant encodes a polypeptide capable of modulating biomass in a plant
[0041] In one embodiment the polynucleotide of a) comprises the sequence of SEQ ID NO:10.
[0042] Preferably expression of the polynucleotide in the plant results in down-regulation of an endogenous polynucleotide/polypeptide capable of modulating biomass production in the plant.
[0043] Preferably the down-regulation is effected by antisense suppression, sense suppression or RNA interference.
[0044] Preferably the plant produced by the method of the invention has increased biomass relative to a suitable control plant.
[0045] In a further aspect the invention provides a method for producing a plant with increased biomass the method comprising transformation of a plant with a polynucleotide with sufficient sequence similarity to an endogenous nucleic acid with the sequence of SEQ ID NO:10 or a variant thereof, such that in expression of the polynucleotide results in inhibition of expression of the endogenous nucleic acid.
[0046] Preferably the polynucleotide is included as part of a genetic construct.
[0047] In one embodiment the variant has at least 70% sequence identity with the full-length coding sequence of SEQ ID NO: 10.
[0048] In a further embodiment the variant encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 20.
[0049] In a further embodiment the variant is derived from a plant species and encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 20.
[0050] In a further embodiment the variant is derived from a dicotyledonous plant species and encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 21.
[0051] Preferably the variant encodes a polypeptide capable of modulating biomass in a plant In a further embodiment the endogenous nucleic acid comprises the full-length coding sequence of SEQ ID NO:10.
[0052] In a further aspect the invention provides a method for producing a plant cell or plant with altered biomass, the method comprising reducing the expression or activity of a polypeptide including the amino acid sequence of SEQ ID NO: 1 or variant thereof.
[0053] In one embodiment the variant has at least 70% sequence identity to a polypeptide with the amino acid sequence of SEQ ID NO: 1.
[0054] In a further embodiment the variant comprises the amino acid sequence of SEQ ID NO: 20.
[0055] In a further embodiment the variant is derived from a plant species and comprises the amino acid sequence of SEQ ID NO: 20.
[0056] In a further embodiment the variant is derived from a dicotyledonous plant species and comprises the amino acid sequence of SEQ ID NO: 21.
[0057] In a further embodiment the polypeptide has the sequence of SEQ ID NO:1
[0058] In a further aspect the invention provides a method of producing a plant with altered biomass the method comprising the step of reducing the expression or activity in a plant cell or plant of a polypeptide comprising the sequence of SEQ ID NO: 20.
[0059] In one embodiment the polypeptide comprises the sequence of SEQ ID NO: 21.
[0060] In a further embodiment the polypeptide comprises the sequence of with at least 70% identity to the sequence of SEQ ID NO: 1.
[0061] In a further embodiment the polypeptide comprises the sequence of SEQ ID NO: 1.
[0062] In a further embodiment the a polynucleotide capable of hybridising under stringent conditions to an endogenous nucleic acid encoding the polypeptide is introduced into the plant cell or plant to effect reduced expression of the polypeptide.
[0063] In a further embodiment the endogenous nucleic acid comprises a sequence with at least 70% identity to the full-length coding sequence of SEQ ID NO: 10.
[0064] In a further embodiment the endogenous nucleic acid comprises the full-length coding sequence of SEQ ID NO: 10.
[0065] In a further embodiment the polynucleotide comprises at least 15 contiguous nucleotides of a sequence with at least 70% identity to the sequence of SEQ ID NO: 10.
[0066] In a further embodiment the polynucleotide comprises at least 15 contiguous nucleotides of SEQ ID NO: 10.
[0067] In a further aspect the invention provides a plant cell or plant produced by a method of the invention.
[0068] Preferably the plant produced by the method of the invention has increased biomass production relative to a suitable control plant.
[0069] Preferably the plant produced by the method of the invention has an increased number of tillers relative to a suitable control plant.
[0070] In a further aspect the invention provides an isolated polynucleotide having at least 71% sequence identity to a nucleotide sequence that encodes a polyp eptide comprising the amino acid sequence of SEQ ID NO: 1.
[0071] Preferably the polynucleotide encodes a polypeptide capable of modulating biomass in a plant
[0072] In one embodiment the polypeptide comprises the amino acid sequence of SEQ ID NO:1
[0073] In a further embodiment the nucleotide sequence comprises the sequence of SEQ ID NO:10.
[0074] In a further embodiment said nucleotide sequence comprises the full-length coding sequence of SEQ ID NO:10.
[0075] In a further aspect the invention provides an isolated polynucleotide that encodes a polypeptide comprising an amino acid sequence SEQ ID NO: 1.
[0076] In one embodiment the polynucleotide comprises the sequence of SEQ ID NO:10.
[0077] In a further embodiment the polynucleotide comprises the full-length coding sequence of SEQ ID NO:10.
[0078] In a further aspect the invention provides an isolated polynucleotide comprising the full-length coding sequence of SEQ ID NO: 10 or a variant thereof, wherein the variant is derived from ryegrass or fescue, and encodes a polypeptide capable of modulating biomass in a plant.
[0079] In one embodiment the variant has at least 70% sequence identity to the full-length coding sequence of SEQ ID NO:10.
[0080] In one embodiment the isolated polynucleotide comprises the sequence of SEQ ID NO:10.
[0081] In a further aspect the invention provides an isolated polypeptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1, wherein the polypeptide is capable of modulating biomass in a plant.
[0082] In one embodiment the isolated polypeptide the amino acid sequence of SEQ ID NO: 1.
[0083] In a further aspect the invention provides an isolated polynucleotide encoding a polypeptide of the invention.
[0084] In a further aspect the invention provides an isolated polynucleotide comprising: [0085] a) a polynucleotide comprising a fragment, of at least 15 nucleotides in length, of a polynucleotide of the invention; or [0086] b) a polynucleotide comprising a complement, of at least 15 nucleotides in length, of the polynucleotide of the invention; or [0087] c) a polynucleotide comprising a sequence, of at least 15 nucleotides in length, capable of hybridising to the polynucleotide of the invention.
[0088] In a further aspect the invention provides a genetic construct which comprises a polynucleotide of the invention.
[0089] In one embodiment the genetic construct is an expression construct.
[0090] In a further aspect the invention provides a vector comprising an expression construct or genetic construct of the invention.
[0091] In a further aspect the invention provides a host cell genetically modified to express a polynucleotide of the invention, or a polypeptide of the invention.
[0092] In a further aspect the invention provides a host cell comprising an expression construct or genetic construct of the invention.
[0093] In a further aspect the invention provides a plant cell genetically modified to express a polynucleotide of the invention, or a polypeptide of the invention.
[0094] In a further aspect the invention provides a plant cell which comprises an expression construct of the invention or the genetic construct of the invention.
[0095] Preferably the expression construct is capable of expressing the polynucleotide, resulting in inhibition of expression of an endogenous polynucleotide/polypeptide which is capable of modulating biomass production in the plant.
[0096] In a further aspect the invention provides a plant which comprises a plant cell of the invention.
[0097] In a further aspect the invention provides a method for selecting a plant with altered biomass, the method comprising testing of a plant for altered expression of a polynucleotide of the invention.
[0098] In a further aspect the invention provides a method for selecting a plant with altered biomass, the method comprising testing of a plant for altered expression of a polypeptide of the invention.
[0099] In a further aspect the invention provides a plant cell or plant produced by the method of the invention.
[0100] In a further aspect the invention provides a plant selected by the method of the invention.
[0101] In a further aspect the invention provides an antibody raised against a polypeptide of the invention.
[0102] The polynucleotides and polynucleotide variants, of the invention may be derived from any species and/or may be produced recombinantly or synthetically.
[0103] In one embodiment the polynucleotide or variant, is derived from a plant species.
[0104] In a further embodiment the polynucleotide or variant, is derived from a gymnosperm plant species.
[0105] In a further embodiment the polynucleotide or variant, is derived from an angiosperm plant species.
[0106] In a further embodiment the polynucleotide or variant, is derived from a from dicotyledonous plant species.
[0107] In a further embodiment the polynucleotide or variant, is derived from a monocotyledonous plant species.
[0108] The polypeptide and polypeptide variants, of the invention may be derived from any species and/or may be produced recombinantly or synthetically.
[0109] In one embodiment the polypeptide or variant, is derived from a plant species.
[0110] In a further embodiment the polypeptide or variant, is derived from a gymnosperm plant species.
[0111] In a further embodiment the polypeptide or variant, is derived from an angiosperm plant species.
[0112] In a further embodiment the polypeptide or variant, is derived from a from dicotyledonous plant species.
[0113] In a further embodiment the polypeptide or variant, is derived from a monocotyledonous plant species.
[0114] The plant cell or plant may be derived from any plant species.
[0115] In a further embodiment the plant cell or plant, is derived from a gymnosperm plant species.
[0116] In a further embodiment the plant cell or plant, is derived from an angiosperm plant species.
[0117] In a further embodiment the plant cell or plant, is derived from a from dicotyledonous plant species.
[0118] In a further embodiment the plant cell or plant, is derived from a monocotyledonous plant species.
[0119] Preferred dicotyledonous genera include: Amygdalus, Anacardium, Arachis, Brassica, Cajanus, Cannabis, Carthamus, Carya, Ceiba, Cicer, Cocos, Coriandrum, Coronilla, Cossypium, Crotalaria, Dolichos, Elaeis, lycine, Gossypium, Helianthus, Lathyrus, Lens, Lespedeza, Linum, Lotus, Lupinus, Macadamia, Medicago, Melilotus, Mucuna, Oka, Onobrychis, Ornithopus, Papaver, Phaseolus, Phoenix, Pistacia, Pisum, Prunus, Pueraria, Ribes, Ricinus, Sesamum, Theobroma, Trifolium, Trigonella, Vicia and Vigna.
[0120] Preferred dicotyledonous species include: Amygdalus communis, Anacardium occidentale, Arachis hypogaea, Arachis hypogea, Brassica napus Rape, Brassica nigra, Brassica campestris, Cajanus cajan, Cajanus indicus, Cannabis sativa, Carthamus tinctorius, Carya illinoinensis, Ceiba pentandra, Cicer arietinum, Cocos nucifera, Coriandrum sativum, Coronilla varia, Cossypium hirsutum, Crotalaria juncea, Dolichos lablab, Elaeis guineensis, Gossypium arboreum, Gossypium nanking, Gossypium barbadense, Gossypium herbaceum, Gossypium hirsutum, Glycine max, Glycine ussuriensis, Glycine gracilis, Helianthus annus, Lupinus angustifolius, Lupinus luteus, Lupinus mutabilis, Lespedeza sericea, Lespedeza striata, Lotus uliginosus, Lathyrus sativus, Lens culinaris, Lespedeza stipulacea, Linum usitatissimum, Lotus corniculatus, Lupinus albus, Medicago arborea, Medicago falcate, Medicago hispida, Medicago officinalis, Medicago sativa Alfalfa, Medicago tribuloides, Macadamia integrifolia, Medicago arabica, Melilotus albus, Mucuna pruriens, Olea europaea, Onobrychis viciifolia, Ornithopus sativus, Phaseolus aureus, Prunus cerasifera, Prunus cerasus, Phaseolus coccineus, Prunus domestica, Phaseolus lunatus, Prunus maheleb, Phaseolus mungo, Prunus persica, Prunus pseudocerasus, Phaseolus vulgaris, Papaver somniferum, Phaseolus acutifolius, Phoenix dactylifera, Pistacia vera, Pisum sativum, Prunus amygdalus, Prunus armeniaca, Pueraria thunbergiana, Ribes nigrum, Ribes rubrum, Ribes grossularia, Ricinus communis, Sesamum indicum, Trifolium augustifolium, Trifolium diffusum, Trifolium hybridum, Trifolium incarnatum, Trifolium ingrescens, Trifolium pratense, Trifolium repens, Trifolium resupinatum, Trifolium subterraneum, Theobroma cacao, Trifolium alexandrinum, Trigonella foenumgraecum, Vicia angustifolia, Vicia atropurpurea, Vicia calcarata, Vicia dasycarpa, Vicia ervilia, Vaccinium oxycoccos, Vicia pannonica, Vigna sesquipedalis, Vigna sinensis, Vicia villosa, Vicia faba, Vicia sative and Vigna angularis.
[0121] Preferred monocotyledonous genera include: Agropyron, Allium, Alopecurus, Andropogon, Arrhenatherum, Asparagus, Avena, Bambusa, Bothrichloa, Bouteloua, Bromus, Calamovilfa, Cenchrus, Chloris, Cymbopogon, Cynodon, Dactylis, Dichanthium, Digitaria, Eleusine, Eragrostis, Fagopyrum, Festuca, Helianthus, Hordeum, Lolium, Miscanthis, Miscanthus×giganteus, Oryza, Panicum, Paspalum, Pennisetum, Phalaris, Phleum, Poa, Saccharum, Secale, Setaria, Sorgahastum, Sorghum, Triticum, Vanilla, X Triticosecale Triticale and Zea.
[0122] Preferred monocotyledonous species include: Agropyron cristatum, Agropyron desertorum, Agropyron elongatum, Agropyron intermedium, Agropyron smithii, Agropyron spicatum, Agropyron trachycaulum, Agropyron trichophorum, Allium ascalonicum, Allium cepa, Allium chinense, Allium porrum, Allium schoenoprasum, Allium fistulosum, Allium sativum, Alopecurus pratensis, Andropogon gerardi, Andropogon Gerardii, Andropogon scoparious, Arrhenatherum elatius, Asparagus officinalis, Avena nuda, Avena sativa, Bambusa vulgaris, Bothrichloa barbinodis, Bothrichloa ischaemum, Bothrichloa saccharoides, Bouteloua curipendula, Bouteloua eriopoda, Bouteloua gracilis, Bromus erectus, Bromus inermis, Bromus riparius, Calamovilfa longifilia, Cenchrus ciliaris, Chloris gayana, Cymbopogon nardus, Cynodon dactylon, Dactylis glomerata, Dichanthium annulatum, Dichanthium aristatum, Dichanthium sericeum, Digitaria decumbens, Digitaria smutsii, Eleusine coracan, Elymus angustus, Elymus junceus, Eragrostis curvula, Eragrostis tef, Fagopyrum esculentum, Fagopyrum tataricum, Festuca arundinacea, Festuca ovina, Festuca pratensis, Festuca rubra, Helianthus annuus sunflower, Hordeum distichum, Hordeum vulgare, Lolium multiflorum, Lolium perenn, Miscanthis sinensis, Miscanthus×giganteus, Oryza sativa, Panicum italicium, Panicum maximum, Panicum miliaceum, Panicum purpurascens, Panicum virgatum, Panicum virgatum, Paspalum dilatatum, Paspalum notatum, Pennisetum clandestinum, Pennisetum glaucum, Pennisetum purpureum, Pennisetum spicatum, Phalaris arundinacea, Phleum bertolinii, Phleum pratense, Poa fendleriana, Poa pratensis, Poa nemoralis, Saccharum officinarum, Saccharum robustum, Saccharum sinense, Saccharum spontaneum, Secale cereale, Setaria sphacelata, Sorgahastum nutans, Sorghastrum nutans, Sorghum dochna, Sorghum halepense, Sorghum sudanense, Sorghum vulgare, Sorghum vulgare, Triticum aestivum, Triticum dicoccum, Triticum durum, Triticum monococcum, Vanilla fragrans, X Triticosecale and Zea mays.
[0123] Preferred plants are forage plant species from a group comprising but not limited to the following genera: Lolium, Festuca, Dactylis, Bromus, Trifolium, Medicago, Phleum, Phalaris, Holcus, Lotus, Plantago and Cichorium.
[0124] Particularly preferred plants are from the genera Lolium and Trifolium. Particularly preferred species are Lolium perenne and Trifolium repens.
[0125] Particularly preferred monocotyledonous plant species are: Lolium perenne and Oryza sativa.
[0126] The term "plant" is intended to include a whole plant, any part of a plant, propagules and progeny of a plant.
[0127] The term `propagule` means any part of a plant that may be used in reproduction or propagation, either sexual or asexual, including seeds and cuttings.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
Polynucleotides and Fragments
[0128] The term "polynucleotide(s)," as used herein, means a single or double-stranded deoxyribonucleotide or ribonucleotide polymer of any length but preferably at least 15 nucleotides, and include as non-limiting examples, coding and non-coding sequences of a gene, sense and antisense sequences complements, exons, introns, genomic DNA, cDNA, pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polypeptides, isolated and purified naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid probes, primers and fragments.
[0129] A "fragment" of a polynucleotide sequence provided herein is a subsequence of contiguous nucleotides that is capable of specific hybridization to a target of interest, e.g., a sequence that is at least 15 nucleotides in length. The fragments of the invention comprise 15 nucleotides, preferably at least 20 nucleotides, more preferably at least 30 nucleotides, more preferably at least 50 nucleotides, more preferably at least 50 nucleotides and most preferably at least 60 nucleotides of contiguous nucleotides of a polynucleotide of the invention. A fragment of a polynucleotide sequence can be used in antisense, gene silencing, triple helix or ribozyme technology, or as a primer, a probe, included in a microarray, or used in polynucleotide-based selection methods of the invention.
[0130] The term "primer" refers to a short polynucleotide, usually having a free 3'OH group, that is hybridized to a template and used for priming polymerization of a polynucleotide complementary to the target.
[0131] The term "probe" refers to a short polynucleotide that is used to detect a polynucleotide sequence, that is complementary to the probe, in a hybridization-based assay. The probe may consist of a "fragment" of a polynucleotide as defined herein.
Polypeptides and Fragments
[0132] The term "polypeptide", as used herein, encompasses amino acid chains of any length but preferably at least 5 amino acids, including full-length proteins, in which amino acid residues are linked by covalent peptide bonds. Polypeptides of the present invention may be purified natural products, or may be produced partially or wholly using recombinant or synthetic techniques. The term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or derivative thereof.
[0133] A "fragment" of a polypeptide is a subsequence of the polypeptide that performs a function that is required for the biological activity and/or provides three dimensional structure of the polypeptide. The term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or derivative thereof capable of performing the above enzymatic activity.
[0134] The term "isolated" as applied to the polynucleotide or polypeptide sequences disclosed herein is used to refer to sequences that are removed from their natural cellular environment. An isolated molecule may be obtained by any method or combination of methods including biochemical, recombinant, and synthetic techniques.
[0135] The term "recombinant" refers to a polynucleotide sequence that is removed from sequences that surround it in its natural context and/or is recombined with sequences that are not present in its natural context.
[0136] A "recombinant" polypeptide sequence is produced by translation from a "recombinant" polynucleotide sequence.
[0137] The term "derived from" with respect to polynucleotides and polypeptides of the invention being "derived from" a particular genera or species, means that the polynucleotide or polypeptide has the same sequence as a polynucleotide or polypeptide found naturally in that genera or species. The polynucleotide or polypeptide which is derived from a genera or species may therefore be produced synthetically or recombinantly.
Variants
[0138] As used herein, the term "variant" refers to polynucleotide or polypeptide sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or added. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variants may be from the same or from other species and may encompass homologues, paralogues and orthologues. In certain embodiments, variants of the inventive polypeptides and polynucleotides possess biological activities that are the same or similar to those of the inventive polypeptides or polynucleotides. The term "variant" with reference to polypeptides and polynucleotides encompasses all forms of polypeptides and polynucleotides as defined herein.
Polynucleotide Variants
[0139] Variant polynucleotide sequences preferably exhibit at least 50%, more preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably at least 62%, more preferably at least 63%, more preferably at least 64%, more preferably at least 65%, more preferably at least 66%, more preferably at least 67%, more preferably at least 68%, more preferably at least 69%, more preferably at least 70%, more preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% identity to a specified polynucleotide sequence. Identity is found over a comparison window of at least 20 nucleotide positions, preferably at least 50 nucleotide positions, more preferably at least 100 nucleotide positions, and most preferably over the entire length of the specified polynucleotide sequence.
[0140] Polynucleotide sequence identity can be determined in the following manner. The subject polynucleotide sequence is compared to a candidate polynucleotide sequence using BLASTN (from the BLAST suite of programs, version 2.2.5 [November 2002]) in bl2seq (Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast 2 sequences--a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250), which is publicly available from NCBI (ftp://ftp.ncbi.nih.gov/blast/). The default parameters of bl2seq are utilized except that filtering of low complexity parts should be turned off.
[0141] The identity of polynucleotide sequences may be examined using the following unix command line parameters:
bl2seq-i nucleotideseq1-j nucleotideseq2-F F-p blastn
[0142] The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. The bl2seq program reports sequence identity as both the number and percentage of identical nucleotides in a line "Identities=".
[0143] Polynucleotide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs (e.g. Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). A full implementation of the Needleman-Wunsch global alignment algorithm is found in the needle program in the EMBOSS package (Rice, P. Longden, I. and Bleasby, A. EMBOSS: The European Molecular Biology Open Software Suite, Trends in Genetics June 2000, vol 16, No 6. pp. 276-277) which can be obtained from http://www.hgmp.mrc.ac.uk/Software/EMBOSS/. The European Bioinformatics Institute server also provides the facility to perform EMBOSS-needle global alignments between two sequences on line at http:/www.ebi.ac.uk/emboss/align/.
[0144] Alternatively the GAP program may be used which computes an optimal global alignment of two sequences without penalizing terminal gaps. GAP is described in the following paper: Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.
[0145] Use of BLASTN as described above is preferred for use in the determination of sequence identity for polynucleotide variants according to the present invention.
[0146] Polynucleotide variants of the present invention also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance. Such sequence similarity with respect to polypeptides may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [November 2002]) from NCBI (ftp://ftp.ncbi.nih.gov/blast/).
[0147] The similarity of polynucleotide sequences may be examined using the following unix command line parameters: [0148] bl2seq-i nucleotideseq1-j nucleotideseq2-F F-p tblastx
[0149] The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an "E value" which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. The size of this database is set by default in the bl2seq program. For small E values, much less than one, the E value is approximately the probability of such a random match.
[0150] Variant polynucleotide sequences preferably exhibit an E value of less than 1×10-10 more preferably less than 1×10-20, more preferably less than 1×10-30, more preferably less than 1×10-40, more preferably less than 1×10-50, more preferably less than 1×10-60, more preferably less than 1×10-70, more preferably less than 1×10-80, more preferably less than 1×10-90 and most preferably less than 1×10-100 when compared with any one of the specifically identified sequences.
[0151] Alternatively, variant polynucleotides of the present invention hybridize to a specified polynucleotide sequence, or complements thereof under stringent conditions.
[0152] The term "hybridize under stringent conditions", and grammatical equivalents thereof, refers to the ability of a polynucleotide molecule to hybridize to a target polynucleotide molecule (such as a target polynucleotide molecule immobilized on a DNA or RNA blot, such as a Southern blot or Northern blot) under defined conditions of temperature and salt concentration. The ability to hybridize under stringent hybridization conditions can be determined by initially hybridizing under less stringent conditions then increasing the stringency to the desired stringency.
[0153] With respect to polynucleotide molecules greater than about 100 bases in length, typical stringent hybridization conditions are no more than 25 to 30° C. (for example, 10° C.) below the melting temperature (Tm) of the native duplex (see generally, Sambrook et al., Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Ausubel et al., 1987, Current Protocols in Molecular Biology, Greene Publishing,). Tm for polynucleotide molecules greater than about 100 bases can be calculated by the formula Tm=81.5+0.41% (G+C-log(Na+). (Sambrook et al., Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Bolton and McCarthy, 1962, PNAS 84:1390). Typical stringent conditions for polynucleotide of greater than 100 bases in length would be hybridization conditions such as prewashing in a solution of 6×SSC, 0.2% SDS; hybridizing at 65° C., 6×SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in 1×SSC, 0.1% SDS at 65° C. and two washes of 30 minutes each in 0.2×SSC, 0.1% SDS at 65° C.
[0154] With respect to polynucleotide molecules having a length less than 100 bases, exemplary stringent hybridization conditions are 5 to 10° C. below Tm. On average, the Tm of a polynucleotide molecule of length less than 100 bp is reduced by approximately (500/oligonucleotide length)° C.
[0155] With respect to the DNA mimics known as peptide nucleic acids (PNAs) (Nielsen et al., Science. 1991 Dec. 6; 254(5037):1497-500) Tm values are higher than those for DNA-DNA or DNA-RNA hybrids, and can be calculated using the formula described in Giesen et al., Nucleic Acids Res. 1998 Nov. 1; 26(21):5004-6. Exemplary stringent hybridization conditions for a DNA-PNA hybrid having a length less than 100 bases are 5 to 10° C. below the Tm.
[0156] Variant polynucleotides of the present invention also encompasses polynucleotides that differ from the sequences of the invention but that, as a consequence of the degeneracy of the genetic code, encode a polypeptide having similar activity to a polypeptide encoded by a polynucleotide of the present invention. A sequence alteration that does not change the amino acid sequence of the polypeptide is a "silent variation". Except for ATG (methionine) and TGG (tryptophan), other codons for the same amino acid may be changed by art recognized techniques, e.g., to optimize codon expression in a particular host organism.
[0157] Polynucleotide sequence alterations resulting in conservative substitutions of one or several amino acids in the encoded polypeptide sequence without significantly altering its biological activity are also included in the invention. A skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, e.g., Bowie et al., 1990, Science 247, 1306).
[0158] Variant polynucleotides due to silent variations and conservative substitutions in the encoded polypeptide sequence may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [November 2002]) from NCBI (ftp://ftp.ncbi.nih.gov/blast/) via the tblastx algorithm as previously described.
Polypeptide Variants
[0159] The term "variant" with reference to polypeptides encompasses naturally occurring, recombinantly and synthetically produced polypeptides. Variant polypeptide sequences preferably exhibit at least 50%, more preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably at least 62%, more preferably at least 63%, more preferably at least 64%, more preferably at least 65%, more preferably at least 66%, more preferably at least 67%, more preferably at least 68%, more preferably at least 69%, more preferably at least 70%, more preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more preferably at least %, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% identity to a sequences of the present invention. Identity is found over a comparison window of at least 20 amino acid positions, preferably at least 50 amino acid positions, more preferably at least 100 amino acid positions, and most preferably over the entire length of a polypeptide of the invention.
[0160] Polypeptide sequence identity can be determined in the following manner. The subject polypeptide sequence is compared to a candidate polypeptide sequence using BLASTP (from the BLAST suite of programs, version 2.2.5 [November 2002]) in bl2seq, which is publicly available from NCBI (ftp://ftp.ncbi.nih.gov/blast/). The default parameters of bl2seq are utilized except that filtering of low complexity regions should be turned off.
[0161] Polypeptide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs. EMBOSS-needle (available at http:/www.ebi.ac.uk/emboss/align/) and GAP (Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.) as discussed above are also suitable global sequence alignment programs for calculating polypeptide sequence identity.
[0162] Use of BLASTP as described above is preferred for use in the determination of polypeptide variants according to the present invention.
[0163] Polypeptide variants of the present invention also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance. Such sequence similarity with respect to polypeptides may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [November 2002]) from NCBI (ftp://ftp.ncbi.nih.gov/blast/). The similarity of polypeptide sequences may be examined using the following unix command line parameters: [0164] bl2seq-i peptideseq1-j peptideseq2-F F-p blastp
[0165] Variant polypeptide sequences preferably exhibit an E value of less than 1×10-10 more preferably less than 1×10-20, more preferably less than 1×10-30, more preferably less than 1×10-40, more preferably less than 1×10-50, more preferably less than 1×10-60, more preferably less than 1×10-70, more preferably less than 1×10-80, more preferably less than 1×10-90 and most preferably less than 1×10-100 when compared with any one of the specifically identified sequences.
[0166] The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an "E value" which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. For small E values, much less than one, this is approximately the probability of such a random match.
[0167] Conservative substitutions of one or several amino acids of a described polypeptide sequence without significantly altering its biological activity are also included in the invention. A skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, e.g., Bowie et al., 1990, Science 247, 1306).
Constructs, Vectors and Components Thereof
[0168] The term "genetic construct" refers to a polynucleotide molecule, usually double-stranded DNA, which may have inserted into it another polynucleotide molecule (the insert polynucleotide molecule) such as, but not limited to, a cDNA molecule. A genetic construct may contain the necessary elements that permit transcribing the insert polynucleotide molecule, and, optionally, translating the transcript into a polypeptide. The insert polynucleotide molecule may be derived from the host cell, or may be derived from a different cell or organism and/or may be a recombinant polynucleotide. Once inside the host cell the genetic construct may become integrated in the host chromosomal DNA. The genetic construct may be linked to a vector.
[0169] The term "vector" refers to a polynucleotide molecule, usually double stranded DNA, which is used to transport the genetic construct into a host cell. The vector may be capable of replication in at least one additional host system, such as E. coli.
[0170] The term "expression construct" refers to a genetic construct that includes the necessary elements that permit transcribing the insert polynucleotide molecule, and, optionally, translating the transcript into a polypeptide. An expression construct typically comprises in a 5' to 3' direction: [0171] a) a promoter functional in the host cell into which the construct will be transformed, [0172] b) the polynucleotide to be expressed, and [0173] c) a terminator functional in the host cell into which the construct will be transformed.
[0174] The term "coding region" or "open reading frame" (ORF) refers to the sense strand of a genomic DNA sequence or a cDNA sequence that is capable of producing a transcription product and/or a polypeptide under the control of appropriate regulatory sequences. The coding sequence is identified by the presence of a 5' translation start codon and a 3' translation stop codon. When inserted into a genetic construct, a "coding sequence" is capable of being expressed when it is operably linked to promoter and terminator sequences.
[0175] "Operably-linked" means that the sequenced to be expressed is placed under the control of regulatory elements that include promoters, tissue-specific regulatory elements, temporal regulatory elements, enhancers, repressors and terminators.
[0176] The term "noncoding region" refers to untranslated sequences that are upstream of the translational start site and downstream of the translational stop site. These sequences are also referred to respectively as the 5' UTR and the 3' UTR. These regions include elements required for transcription initiation and termination and for regulation of translation efficiency.
[0177] Terminators are sequences, which terminate transcription, and are found in the 3' untranslated ends of genes downstream of the translated sequence. Terminators are important determinants of mRNA stability and in some cases have been found to have spatial regulatory functions.
[0178] The term "promoter" refers to nontranscribed cis-regulatory elements upstream of the coding region that regulate gene transcription. Promoters comprise cis-initiator elements which specify the transcription initiation site and conserved boxes such as the TATA box, and motifs that are bound by transcription factors.
[0179] A "transgene" is a polynucleotide that is taken from one organism and introduced into a different organism by transformation. The transgene may be derived from the same species or from a different species as the species of the organism into which the transgene is introduced.
[0180] An "inverted repeat" is a sequence that is repeated, where the second half of the repeat is in the complementary strand, e.g.,
TABLE-US-00001 (5')GATCTA . . . TAGATC(3') (3')CTAGAT . . . ATCTAG(5')
[0181] Read-through transcription will produce a transcript that undergoes complementary base-pairing to form a hairpin structure provided that there is a 3-5 bp spacer between the repeated regions.
[0182] A "transgenic plant" refers to a plant which contains new genetic material as a result of genetic manipulation or transformation. The new genetic material may be derived from a plant of the same species as the resulting transgenic plant or from a different species.
[0183] The terms "to alter expression of" and "altered expression" of a polynucleotide or polypeptide of the invention, are intended to encompass the situation where genomic DNA corresponding to a polynucleotide of the invention is modified thus leading to altered expression of a polynucleotide or polypeptide of the invention. Modification of the genomic DNA may be through genetic transformation or other methods known in the art for inducing mutations. The "altered expression" can be related to an increase or decrease in the amount of messenger RNA and/or polypeptide produced and may also result in altered activity of a polypeptide due to alterations in the sequence of a polynucleotide and polypeptide produced.
[0184] The term "biomass" refers to the size and/or mass and/or number of vegetative organs of the plant at a particular age or developmental stage. Thus a plant with increased biomass has increased size and/or mass and/or number of vegetative organs than a suitable control plant of the same age or at an equivalent developmental stage. Conversely, a plant with decreased biomass has decreased size and/or mass and/or number of vegetative organs than a suitable control. Altered biomass may also involve an alteration in rate of growth and/or rate of formation of vegetative organs during some or all periods of the life cycle of a plant relative to a suitable control. Thus altered biomass may result in an advance or delay in the time taken for such a plant to reach a certain developmental stage.
[0185] Suitable control plants may include non-transformed plants of the same species and variety, or plants of the same species or variety transformed with a control construct.
[0186] The invention provides methods for producing and selecting plants with altered biomass relative to suitable control plants, including plants with both increased and decreased biomass and plants produced by such methods.
[0187] The invention provides a polynucleotide (SEQ ID NO:10) encoding a polypeptide (SEQ ID NO:1) which modulates biomass in plants. The invention provides polynucleotide variants of SEQ ID NO:10 (SEQ ID NOs: 11 to 18) which encode polypeptide variants of SEQ ID NO:1 (SEQ ID NO:2 to 9). The applicants have also identified a consensus polypeptide sequence motif present in SEQ ID NO:1 and all of the polypeptide variants of SEQ ID NO:1, as shown in SEQ ID NO:20, and a further consensus motif (SEQ ID NO: 21) present in SEQ ID NO:1 (ORF54) and all polypeptide variants thereof that are derived from dicotyledonous plants.
Methods for Isolating Polynucleotides
[0188] The polynucleotide molecules of the invention can be isolated by using a variety of techniques known to those of ordinary skill in the art. By way of example, such polypeptides can be isolated through use of the polymerase chain reaction (PCR) described in Mullis et al., Eds. 1994 The Polymerase Chain Reaction, Birkhauser, incorporated herein by reference. The polypeptides of the invention can be amplified using primers, as defined herein, derived from the polynucleotide sequences of the invention.
[0189] Further methods for isolating polynucleotides of the invention, or polynucleotides useful in methods of the invention, include use of all, or portions of, the polynucleotides set forth herein as hybridization probes. The technique of hybridizing labelled polynucleotide probes to polynucleotides immobilized on solid supports such as nitrocellulose filters or nylon membranes, can be used to screen the genomic or cDNA libraries. Exemplary hybridization and wash conditions are: hybridization for 20 hours at 65° C. in 5.0×SSC, 0.5% sodium dodecyl sulfate, 1×Denhardt's solution; washing (three washes of twenty minutes each at 55° C.) in 1.0×SSC, 1% (w/v) sodium dodecyl sulfate, and optionally one wash (for twenty minutes) in 0.5×SSC, 1% (w/v) sodium dodecyl sulfate, at 60° C. An optional further wash (for twenty minutes) can be conducted under conditions of 0.1×SSC, 1% (w/v) sodium dodecyl sulfate, at 60° C.
[0190] The polynucleotide fragments of the invention may be produced by techniques well-known in the art such as restriction endonuclease digestion and oligonucleotide synthesis.
[0191] A partial polynucleotide sequence may be used, in methods well-known in the art to identify the corresponding full-length polynucleotide sequence. Such methods include PCR-based methods, 5'RACE (Frohman M A, 1993, Methods Enzymol. 218: 340-56) and hybridization-based method, computer/database-based methods. Further, by way of example, inverse PCR permits acquisition of unknown sequences, flanking the polynucleotide sequences disclosed herein, starting with primers based on a known region (Triglia et al., 1998, Nucleic Acids Res 16, 8186, incorporated herein by reference). The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template. Divergent primers are designed from the known region. In order to physically assemble full-length clones, standard molecular biology approaches can be utilized (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987).
[0192] It may be beneficial, when producing a transgenic plant from a particular species, to transform such a plant with a sequence or sequences derived from that species. The benefit may be to alleviate public concerns regarding cross-species transformation in generating transgenic organisms. Additionally when down-regulation of a gene is the desired result, it may be necessary to utilise a sequence identical (or at least highly similar) to that in the plant, for which reduced expression is desired. For these reasons among others, it is desirable to be able to identify and isolate orthologues of a particular gene in several different plant species. Variants (including orthologues) may be identified by the methods described.
Methods for Identifying Variants
Physical Methods
[0193] Variant polynucleotides may be identified using PCR-based methods (Mullis et al., Eds. 1994 The Polymerase Chain Reaction, Birkhauser). Typically, the polynucleotide sequence of a primer, useful to amplify variant polynucleotide molecules by PCR, may be based on a sequence encoding a conserved region of the corresponding amino acid sequence.
[0194] Alternatively library screening methods will be known to those skilled in the art (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987) may be employed. When identifying variants of the probe sequence hybridisation and/or wash stringency conditions will typically be reduced relative to when exact sequence matches are sought.
[0195] Polypeptide variants of the invention may be identified by physical methods, for example by screening expression libraries using antibodies raised against polypeptides of the invention (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987) or by identifying polypeptides from natural sources with the aid of such antibodies.
Computer Based Methods
[0196] The variant sequences of the invention, including both polynucleotide and polypeptide variants, may also be identified by computer-based methods well-known to those skilled in the art, using public domain sequence alignment algorithms and sequence similarity search tools to search sequence databases (public domain databases include Genbank, EMBL, Swiss-Prot, PIR and others). See, e.g., Nucleic Acids Res. 29: 1-10 and 11-16, 2001 for examples of online resources. Similarity searches retrieve and align target sequences for comparison with a sequence to be analyzed (i.e., a query sequence). Sequence comparison algorithms use scoring matrices to assign an overall score to each of the alignments.
[0197] An exemplary family of programs useful for identifying variants in sequence databases is the BLAST suite of programs (version 2.2.5 [November 2002]) including BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX, which are publicly available from (ftp://ftp.ncbi.nih.gov/blast/) or from the National Center for Biotechnology Information (NCBI), National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894 USA. The NCBI server also provides the facility to use the programs to screen a number of publicly available sequence databases. BLASTN compares a nucleotide query sequence against a nucleotide sequence database. BLASTP compares an amino acid query sequence against a protein sequence database. BLASTX compares a nucleotide query sequence translated in all reading frames against a protein sequence database. tBLASTN compares a protein query sequence against a nucleotide sequence database dynamically translated in all reading frames. tBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database. The BLAST programs may be used with default parameters or the parameters may be altered as required to refine the screen.
[0198] The use of the BLAST family of algorithms, including BLASTN, BLASTP, and BLASTX, is described in the publication of Altschul et al., Nucleic Acids Res. 25: 3389-3402, 1997.
[0199] The "hits" to one or more database sequences by a queried sequence produced by BLASTN, BLASTP, BLASTX, tBLASTN, tBLASTX, or a similar algorithm, align and identify similar portions of sequences. The hits are arranged in order of the degree of similarity and the length of sequence overlap. Hits to a database sequence generally represent an overlap over only a fraction of the sequence length of the queried sequence.
[0200] The BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX algorithms also produce "Expect" values for alignments. The Expect value (E) indicates the number of hits one can "expect" to see by chance when searching a database of the same size containing random contiguous sequences.
[0201] The Expect value is used as a significance threshold for determining whether the hit to a database indicates true similarity. For example, an E value of 0.1 assigned to a polynucleotide hit is interpreted as meaning that in a database of the size of the database screened, one might expect to see 0.1 matches over the aligned portion of the sequence with a similar score simply by chance. For sequences having an E value of 0.01 or less over aligned and matched portions, the probability of finding a match by chance in that database is 1% or less using the BLASTN, BLASTP, BLASTX, tBLASTN or tBLASTX algorithm.
[0202] Multiple sequence alignments of a group of related sequences can be carried out with CLUSTALW (Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994) CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22:4673-4680, http://www-igbmc.u-strasbg.fr/BioInfo/ClustalW/Top.html) or T-COFFEE (Cedric Notredame, Desmond G. Higgins, Jaap Hering a, T-Coffee: A novel method for fast and accurate multiple sequence alignment, J. Mol. Biol. (2000) 302: 205-217)) or PILEUP, which uses progressive, pairwise alignments. (Feng and Doolittle, 1987, J. Mol. Evol. 25, 351).
[0203] Pattern recognition software applications are available for finding motifs or signature sequences. For example, MEME (Multiple Em for Motif Elicitation) finds motifs and signature sequences in a set of sequences, and MAST (Motif Alignment and Search Tool) uses these motifs to identify similar or the same motifs in query sequences. The MAST results are provided as a series of alignments with appropriate statistical data and a visual overview of the motifs found. MEME and MAST were developed at the University of California, San Diego.
[0204] PROSITE (Bairoch and Bucher, 1994, Nucleic Acids Res. 22, 3583; Hofmann et al., 1999, Nucleic Acids Res. 27, 215) is a method of identifying the functions of uncharacterized proteins translated from genomic or cDNA sequences. The PROSITE database (www.expasy.org/prosite) contains biologically significant patterns and profiles and is designed so that it can be used with appropriate computational tools to assign a new sequence to a known family of proteins or to determine which known domain(s) are present in the sequence (Falquet et al., 2002, Nucleic Acids Res. 30, 235). Prosearch is a tool that can search SWISS-PROT and EMBL databases with a given sequence pattern or signature.
Methods for Isolating Polypeptides
[0205] The polypeptides of the invention, including variant polypeptides, may be prepared using peptide synthesis methods well known in the art such as direct peptide synthesis using solid phase techniques (e.g. Stewart et al., 1969, in Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco Calif., or automated synthesis, for example using an Applied Biosystems 431A Peptide Synthesizer (Foster City, Calif.). Mutated forms of the polypeptides may also be produced during such syntheses.
[0206] The polypeptides and variant polypeptides of the invention may also be purified from natural sources using a variety of techniques that are well known in the art (e.g. Deutscher, 1990, Ed, Methods in Enzymology, Vol. 182, Guide to Protein Purification,).
[0207] Alternatively the polypeptides and variant polypeptides of the invention may be expressed recombinantly in suitable host cells and separated from the cells as discussed below.
Methods for Producing Constructs and Vectors
[0208] The genetic constructs of the present invention comprise one or more polynucleotide sequences of the invention and/or polynucleotides encoding polypeptides of the invention, and may be useful for transforming, for example, bacterial, fungal, insect, mammalian or plant organisms. The genetic constructs of the invention are intended to include expression constructs as herein defined.
[0209] Methods for producing and using genetic constructs and vectors are well known in the art and are described generally in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, 1987).
Methods for Producing Host Cells Comprising Constructs and Vectors
[0210] The invention provides a host cell which comprises a genetic construct or vector of the invention. Host cells may be derived from, for example, bacterial, fungal, insect, mammalian or plant organisms.
[0211] Host cells comprising genetic constructs, such as expression constructs, of the invention are useful in methods well known in the art (e.g. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, 1987) for recombinant production of polypeptides of the invention. Such methods may involve the culture of host cells in an appropriate medium in conditions suitable for or conducive to expression of a polypeptide of the invention. The expressed recombinant polypeptide, which may optionally be secreted into the culture, may then be separated from the medium, host cells or culture medium by methods well known in the art (e.g. Deutscher, Ed, 1990, Methods in Enzymology, Vol 182, Guide to Protein Purification).
[0212] Host cells of the invention may also be useful in methods for production of an enzymatic product generated by an expressed polypeptide of the invention. Such methods may involve culturing the host cells of the invention in a medium suitable for expression of a recombinant polypeptide of the invention, optionally in the presence of additional enzymatic substrate for the expressed polypeptide of the invention. The enzymatic product produced may then be separated from the host cells or medium by a variety of art standard methods.
Methods for Producing Plant Cells and Plants Comprising Constructs and Vectors
[0213] The invention further provides plant cells which comprise a genetic construct of the invention, and plant cells modified to alter expression of a polynucleotide or polypeptide of the invention. Plants comprising such cells also form an aspect of the invention.
[0214] Production of plants altered in biomass may be achieved through methods of the invention. Such methods may involve the transformation of plant cells and plants, with a construct of the invention designed to alter expression of a polynucleotide or polypeptide capable of modulating biomass production in such plant cells and plants. Such methods also include the transformation of plant cells and plants with a combination of the construct of the invention and one or more other constructs designed to alter expression of one or more polypeptides or polypeptides capable of modulating biomass production in such plant cells and plants.
[0215] Methods for transforming plant cells, plants and portions thereof with polynucleotides are described in Draper et al., 1988, Plant Genetic Transformation and Gene Expression. A Laboratory Manual, Blackwell Sci. Pub. Oxford, p. 365; Potrykus and Spangenburg, 1995, Gene Transfer to Plants. Springer-Verlag, Berlin.; and Gelvin et al., 1993, Plant Molecular Biol. Manual. Kluwer Acad. Pub. Dordrecht. A review of transgenic plants, including transformation techniques, is provided in Galun and Breiman, 1997, Transgenic Plants. Imperial College Press, London.
Methods for Genetic Manipulation of Plants
[0216] A number of strategies for genetically manipulating plants are available (e.g. Birch, 1997, Ann Rev Plant Phys Plant Mol Biol, 48, 297). For example, strategies may be designed to increase expression of a polynucleotide/polypeptide in a plant cell, organ and/or at a particular developmental stage where/when it is normally expressed or to ectopically express a polynucleotide/polypeptide in a cell, tissue, organ and/or at a particular developmental stage which/when it is not normally expressed. The expressed polynucleotide/polypeptide may be derived from the plant species to be transformed or may be derived from a different plant species.
[0217] Transformation strategies may be designed to reduce expression of a polynucleotide/polypeptide in a plant cell, tissue, organ or at a particular developmental stage which/when it is normally expressed. Such strategies are known as gene silencing strategies.
[0218] Genetic constructs for expression of genes in transgenic plants typically include promoters for driving the expression of one or more cloned polynucleotide, terminators and selectable marker sequences to detest presence of the genetic construct in the transformed plant.
[0219] The promoters suitable for use in the constructs of this invention are functional in a cell, tissue or organ of a monocot or dicot plant and include cell-, tissue- and organ-specific promoters, cell cycle specific promoters, temporal promoters, inducible promoters, constitutive promoters that are active in most plant tissues, and recombinant promoters. Choice of promoter will depend upon the temporal and spatial expression of the cloned polynucleotide, so desired. The promoters may be those normally associated with a transgene of interest, or promoters which are derived from genes of other plants, viruses, and plant pathogenic bacteria and fungi. Those skilled in the art will, without undue experimentation, be able to select promoters that are suitable for use in modifying and modulating plant traits using genetic constructs comprising the polynucleotide sequences of the invention. Examples of constitutive plant promoters include the CaMV 35S promoter, the nopaline synthase promoter and the octopine synthase promoter, and the Ubi 1 promoter from maize. Plant promoters which are active in specific tissues, respond to internal developmental signals or external abiotic or biotic stresses are described in the scientific literature. Exemplary promoters are described, e.g., in WO 02/00894, which is herein incorporated by reference.
[0220] Exemplary terminators that are commonly used in plant transformation genetic construct include, e.g., the cauliflower mosaic virus (CaMV) 35S terminator, the Agrobacterium tumefaciens nopaline synthase or octopine synthase terminators, the Zea mays zein gene terminator, the Oryza sativa ADP-glucose pyrophosphorylase terminator and the Solanum tuberosum PI-II terminator.
[0221] Selectable markers commonly used in plant transformation include the neomycin phophotransferase II gene (NPT II) which confers kanamycin resistance, the aadA gene, which confers spectinomycin and streptomycin resistance, the phosphinothricin acetyl transferase (bar gene) for Ignite (AgrEvo) and Basta (Hoechst) resistance, and the hygromycin phosphotransferase gene (hpt) for hygromycin resistance.
[0222] Use of genetic constructs comprising reporter genes (coding sequences which express an activity that is foreign to the host, usually an enzymatic activity and/or a visible signal (e.g., luciferase, GUS, GFP) which may be used for promoter expression analysis in plants and plant tissues are also contemplated. The reporter gene literature is reviewed in Herrera-Estrella et al., 1993, Nature 303, 209, and Schrott, 1995, In: Gene Transfer to Plants (Potrykus, T., Spangenberg. Eds) Springer Verlag. Berline, pp. 325-336.
[0223] Gene silencing strategies may be focused on the gene itself or regulatory elements which effect expression of the encoded polypeptide. "Regulatory elements" is used here in the widest possible sense and includes other genes which interact with the gene of interest.
[0224] Genetic constructs designed to decrease or silence the expression of a polynucleotide/polypeptide of the invention may include an antisense copy of a polynucleotide of the invention. In such constructs the polynucleotide is placed in an antisense orientation with respect to the promoter and terminator.
[0225] An "antisense" polynucleotide is obtained by inverting a polynucleotide or a segment of the polynucleotide so that the transcript produced will be complementary to the mRNA transcript of the gene, e.g.,
TABLE-US-00002 5'GATCTA 3' (coding 3'CTAGAT 5' (antisense strand) strand) 3'CUAGAU 5' mRNA 5'GAUCUCG 3' antisense RNA
[0226] Genetic constructs designed for gene silencing may also include an inverted repeat. An `inverted repeat` is a sequence that is repeated where the second half of the repeat is in the complementary strand, e.g.,
TABLE-US-00003 5'-GATCTA . . . TAGATC-3' 3'-CTAGAT . . . ATCTAG-5'
[0227] The transcript formed may undergo complementary base pairing to form a hairpin structure. Usually a spacer of at least 3-5 bp between the repeated region is required to allow hairpin formation.
[0228] Another silencing approach involves the use of a small antisense RNA targeted to the transcript equivalent to an miRNA (Llave et al., 2002, Science 297, 2053). Use of such small antisense RNA corresponding to polynucleotide of the invention is expressly contemplated.
[0229] The term genetic construct as used herein also includes small antisense RNAs and other such polynucleotides useful for effecting gene silencing.
[0230] Transformation with an expression construct, as herein defined, may also result in gene silencing through a process known as sense suppression (e.g. Napoli et al., 1990, Plant Cell 2, 279; de Carvalho Niebel et al., 1995, Plant Cell, 7, 347). In some cases sense suppression may involve over-expression of the whole or a partial coding sequence but may also involve expression of non-coding region of the gene, such as an intron or a 5' or 3' untranslated region (UTR). Chimeric partial sense constructs can be used to coordinately silence multiple genes (Abbott et al., 2002, Plant Physiol. 128(3): 844-53; Jones et al., 1998, Planta 204: 499-505). The use of such sense suppression strategies to silence the expression of a polynucleotide of the invention is also contemplated.
[0231] The polynucleotide inserts in genetic constructs designed for gene silencing may correspond to coding sequence and/or non-coding sequence, such as promoter and/or intron and/or 5' or 3' UTR sequence, or the corresponding gene.
[0232] Other gene silencing strategies include dominant negative approaches and the use of ribozyme constructs (McIntyre, 1996, Transgenic Res, 5, 257).
[0233] Pre-transcriptional silencing may be brought about through mutation of the gene itself or its regulatory elements. Such mutations may include point mutations, frameshifts, insertions, deletions and substitutions.
[0234] The following are representative publications disclosing genetic transformation protocols that can be used to genetically transform the following plant species: Rice (Alam et al., 1999, Plant Cell Rep. 18, 572); maize (U.S. Pat. Nos. 5,177,010 and 5,981,840); wheat (Ortiz et al., 1996, Plant Cell Rep. 15, 1996, 877); tomato (U.S. Pat. No. 5,159,135); potato (Kumar et al., 1996 Plant J. 9,: 821); cassava (Li et al., 1996 Nat. Biotechnology 14, 736); lettuce (Michelmore et al., 1987, Plant Cell Rep. 6, 439); tobacco (Horsch et al., 1985, Science 227, 1229); cotton (U.S. Pat. Nos. 5,846,797 and 5,004,863); grasses (U.S. Pat. Nos. 5,187,073, 6,020,539); peppermint (Niu et al., 1998, Plant Cell Rep. 17, 165); citrus plants (Pena et al., 1995, Plant Sci. 104, 183); caraway (Krens et al., 1997, Plant Cell Rep, 17, 39); banana (U.S. Pat. No. 5,792,935); soybean (U.S. Pat. Nos. 5,416,011; 5,569,834; 5,824,877; 5,563,04455 and 5,968,830); pineapple (U.S. Pat. No. 5,952,543); poplar (U.S. Pat. No. 4,795,855); monocots in general (U.S. Pat. Nos. 5,591,616 and 6,037,522); brassica (U.S. Pat. Nos. 5,188,958; 5,463,174 and 5,750,871); alfalfa (Weeks et al., (2008) Transgenic Research 17: 587-597; Samac et al., (2006) Methods Mol Biol, Vol 343, Agrobacterium Protocols. 2nd edition. Totowa, N.J.: Humana Press. p 301-311.); and cereals (U.S. Pat. No. 6,074,877). Other species are contemplated and suitable methods and protocols are available in the scientific literature for use by those skilled in the art.
[0235] Several further methods known in the art may be employed to alter expression of a nucleotide and/or alter expression or activity of a polypeptide of the invention, or used in a method of the invention. Such methods include but are not limited to Tilling (Till et al., 2003, Methods Mol Biol, 2%, 205), so called "Deletagene" technology (Li et al., 2001, Plant Journal 27(3), 235) and the use of artificial transcription factors such as synthetic zinc finger transcription factors. (e.g. Jouvenot et al., 2003, Gene Therapy 10, 513). Additionally antibodies or fragments thereof, targeted to a particular polypeptide may also be expressed in plants to modulate the activity of that polypeptide (Jobling et al., 2003, Nat. Biotechnol., 21(1), 35). Transposon tagging approaches may also be applied. Additionally peptides interacting with a polypeptide of the invention may be identified through technologies such as phage-display (Dyax Corporation). Such interacting peptides may be expressed in or applied to a plant to affect activity of a polypeptide of the invention. Plantibodies (Stoger et al., Current Opinion in Biotechnology Volume 13, Issue 2, 1 Apr. 2002, Pages 161-166; Sudarshana et al., Methods Mol Biol. 2007; 354:183-95.) may also be used to modulate expression or activity of a polypeptide in a plant. Use of each of the above approaches, including the silencing methods discussed, in alteration of expression of a nucleotide and/or expression or activity of a polypeptide of the invention is specifically contemplated.
Methods for Selecting Plants
[0236] Methods are also provided for selecting plants with altered biomass. Such methods involve testing of plants for altered for the expression of a polynucleotide or polypeptide of the invention. Such methods may be applied at a young age or early developmental stage when the altered biomass may not necessarily be visible, to accelerate breeding programs directed toward improving biomass.
[0237] The expression of a polynucleotide, such as a messenger RNA, is often used as an indicator of expression of a corresponding polypeptide. Exemplary methods for measuring the expression of a polynucleotide include but are not limited to Northern analysis, RT-PCR and dot-blot analysis (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987). Polynucleotides or portions of the polynucleotides of the invention are thus useful as probes or primers, as herein defined, in methods for the identification of plants with altered biomass. The polypeptides of the invention may be used as probes in hybridization experiments, or as primers in PCR based experiments, designed to identify such plants.
[0238] Alternatively antibodies may be raised against polypeptides of the invention. Methods for raising and using antibodies are standard in the art (see for example: Antibodies, A Laboratory Manual, Harlow A Lane, Eds, Cold Spring Harbour Laboratory, 1998). Such antibodies may be used in methods to detect altered expression of polypeptides which modulate biomass in plants. Such methods may include ELISA (Kemeny, 1991, A Practical Guide to ELISA, NY Pergamon Press) and Western analysis (Towbin & Gordon, 1994, J Immunol Methods, 72, 313).
[0239] These approaches for analysis of polynucleotide or polypeptide expression and the selection of plants with altered expression are useful in conventional breeding programs designed to produce varieties with altered biomass.
Plants
[0240] The plants of the invention may be grown and either self-ed or crossed with a different plant strain and the resulting hybrids, with the desired phenotypic characteristics, may be identified. Two or more generations may be grown to ensure that the subject phenotypic characteristics are stably maintained and inherited. Plants resulting from such standard breeding approaches also form an aspect of the present invention.
[0241] It may be desirable to either increase or decrease biomass in a particular plant species. Increased biomass would be advantageous for example in human food, forage and forestry crops as well as in ornamental plants. Decreased biomass may also be desirable in certain of the above cases, for example in the miniaturization of ornamental plants.
[0242] Biomass in a plant may also be altered through methods of the invention. Such methods may involve the transformation of plant cells and plants, with a construct of the invention designed to alter expression of a polynucleotide or polypeptide which modulates biomass in such plant cells and plants. Such methods also include the transformation of plant cells and plants with a combination of the construct of the invention and one or more other constructs designed to alter expression of one or more polynucleotides or polypeptides which modulates biomass in plants.
[0243] Exemplary methods for assessing growth rate and biomass in plants of the invention are provided in Boyes D C et al., 2001, Plant Cell. 13(7):1499-510; Lancashire P. D et al., 1991, Ann. Appl. Biol. 119: 560-601, and in Example 1 below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0244] The present invention will be better understood with reference to the accompanying drawings in which:
[0245] FIG. 1 shows the summary output of a BLAST-P search of the NR-PLANT database (release date 1 Jan. 2005) in which the ORF54 polypeptide (SEQ ID NO:1) was used as a seed sequence.
[0246] FIG. 2 shows PrettyPlot alignment of polypeptides (SEQ ID NO: 1 to 9), including ORF54 and variants thereof.
[0247] FIG. 3 shows a "T-COFFEE" alignment of the seven repeat elements, termed RCC repeats, found in the polypeptide sequences (SEQ ID NO: 1 to 9) as used by the applicants to identify a consensus region (SEQ ID NO: 10) present in each repeat of all the sequences.
[0248] FIG. 4 shows a map of a vector, for plant transformation, comprising ORF54 cloned in anti-sense orientation relative to the CaMV 35S promoter. The sequence of the vector is represented in SEQ ID NO:20.
[0249] FIG. 5 shows a DNA gel-blot analysis on genomic DNA from ORF54 TO transgenic plants digested with HindIII and probed with a fragment of ORF54 coding sequence to determine gene copy number and to identify independent transformation events.
[0250] FIG. 5a-5e show growth parameters observed for these ORF54 T1 plant lines compared to the best performing wild type control (Nipponbare) in two separate experiments. Where FIG. 5a Plant height measurements from Experiment 1, FIG. 5b Plant tiller number from Experiment 1, FIG. 5c Tiller number in ORF54 lines that exceeded the tillering capacity in the best performing wild-type control (Nipponbare), FIG. 5d Plant height measurements from Experiment 2 and FIG. 5e Plant tiller number from Experiment 2.
[0251] FIG. 6a shows shoot biomass analysis between ORF54 plants and wild-type control (Nipponbare). Student's t-test: *1 Mean differences between sample and Nipponbare is highly significant (p-0.01). *2 Mean differences between sample and Nipponbare is very highly significant (p=0.001).
[0252] FIG. 6b shows dry matter yield in ORF54 lines that exceeded the dry matter yielding capacity in the best performing wild-type control (Nipponbare).
[0253] FIG. 7 shows an alignment of the ORF54 polypeptide and variants thereof from monocotyledonous species. Highlighting shows the position a consensus sequence motif (SEQ ID NO: 20) completely conserved in all of the sequences.
[0254] FIG. 8 shows an alignment of the ORF54 polypeptide and variants thereof from dicotyledonous species. Highlighting shows the position a consensus sequence motif (SEQ ID NO: 21) completely conserved in all of the sequences.
[0255] FIG. 9 shows altered biomass (dry weight in grams) in the transgenic ryegrass lines (1V20, 1V5, 1V1, 1V8, 1V11, 1V3, 2V9, 2V8, 2V5, 2V7, 2V2, 3V5, 3V12, 3V11, 3V10 and 3V7) over the non-transgenic control lines (T40, T41 and T101) bars over the columns represent standard deviation.
EXAMPLES
[0256] The invention will now be illustrated with reference to the following non-limiting examples.
Example 1
Increased Biomass by Down Regulation of a Polynucleotide of the Invention in Transgenic Plants
ORF54
[0257] A polynucleotide sequence designated ORF54 (SEQ ID NO: 10), encoding the polypeptide of SEQ ID NO:1, was identified in a ViaLactia Biosciences Ltd proprietary ryegrass (Lolium perenne) GeneThresher (Orion Genomics) genomic library.
ORF54 Variants
[0258] The polypeptide sequence of ORF54 (SEQ ID NO:1) was used as a seed sequence to perform a BLASTP search against the NR-Plant database (release date 2005-01-01) to identify variants of ORF54. A cut-off value of less than or equal to 1e-140 was identified as distinguishing ORF54 variants based upon the applicant's assessment of the associated score value and annotations in the public data base. The BLASTP output summary is shown in FIG. 1.
[0259] The selected variant sequences were aligned using the EMBOSS tool EMMA (Thompson et al 1994), which is an interface to the popular multiple alignment program ClustalW. Aligned sequences were visualised using another EMBOSS tool called prettyplot as shown in FIG. 2.
[0260] The polypeptide sequences of ORF54 variants are listed as SEQ ID NO:2 to 9 in the sequence listing. The corresponding polynucleotide sequences are listed as SEQ ID NO:11 to 18 respectively.
[0261] Analysis of the polypeptide sequences revealed the presence of seven repeats per polypeptide sequence and the repeat sequences are termed RCC repeats. The repeat sequences from all polypeptides (SEQ ID NO:1-9) above were aligned using the T-COFFEE alignment programme Version--1.37 (Notredame et. al 2000, Higgins) and the outcome is presented in FIG. 3.
[0262] A polypeptide motif based on, and present in, all of the RCC repeats from ORF54 and each of the ORF54 variants was identified and is represented in FIG. 3.
A Vector Comprising ORF54
[0263] The ORF54 polynucleotide sequence (SEQ ID NO:10) was cloned in the anti-sense orientation upstream of the double 35S promoter in order to down-regulate expression of the ORF54 homologue in rice. The vector comprising ORF54 was produced by standard molecular biology techniques. A map of the binary vector is shown in FIG. 4. The sequence of the vector is represented in SEQ ID NO:19.
Plant Transformation--Rice
[0264] The purified binary vector (SEQ ID NO:19) was introduced into Agrobacterium strain EHA105 by electroporation (den Dulk-Ras A and Hooykaas P J.) and the suspension was incubated at 26° C. for 30 minutes. A small aliquot was plated on AB minimal medium (Schmidt-Eisenlohr et. al 1999) containing Kanamycin at 100 mg/L. Plates were incubated at 26° C. for 3 days and single colonies were tested for presence of the plasmid using construct specific primers and transformation confirmed.
[0265] Agrobacterium cultures were grown in AG minimal medium containing 100 mg/L kanamycin at 26° C. with shaking (200 rpm). The Agrobacterium suspensions were pelleted at 5,000 rpm for 5 minutes, washed once in basal MS medium containing 1% glucose and 3% sucrose, pH 5.2, and re-suspended in same medium containing 200 μM acetosyringone to OD600 0.6-0.8.
[0266] A. tumefaciens containing the binary vector ORF54 were used to co-cultivate at least 1,000 immature rice (Oryza sativa) cv. Nipponbare embryos. Immature seeds from rice were washed in sterile water and then surface sterilized with sodium hypochlorite containing 1.25% active chlorine with 10 μL Tween® 20 for 20 minutes. After sterilization, the seeds were washed several times with sterile water and blotted dry on sterile filter paper (3M). The seeds were de-husked manually using sterile pair of forceps and the embryo dissected out with sterile knife. The isolated embryos were immersed in Agrobacterium suspension for 30 minutes with continuous shaking at 100 rpm in a 10 mL culture tube. The excess liquid was drained off and the embryos blotted on to sterile filter paper before placing them on to co-cultivation medium containing MS medium (Murashige and Skoog, 1964) supplemented with 3% sucrose, 1% glucose, 2 mg/L 2,4-D, 0.1 mg/L BA, 400 μM acetosyringone, pH 5.2 for 4 days in dark. After co-cultivation, the calli forming embryos were sub-cultured once every two weeks on selection medium consisting of MS medium supplemented with 3% sucrose, 1% glucose, 2 mg/L 2,4-D (2,4-dichlorophenoxy acetic acid), 0.1 mg/L BA (benzyl adenine) and containing 50 mg/L hygromycin and 300 mg/L Timentin® (ticarcillin+clavulanic acid) till at-least 30 healthy calli showing green spots indicative of healthy shoot emergence was achieved. Calli containing the green spots were transferred to selection medium lacking 2,4-D to regenerate a minimum of 10 transformed plants. Regenerated plants were rooted and then transplanted to six inch pots containing soil and plants grown in greenhouse. DNA gel-blot analysis was carried out (FIG. 5) by digesting genomic DNA from transgenic plants with HindIII and probing with a fragment of ORF54 coding sequence to determine gene copy number and to identify five independent transformation events. T1 seeds were harvested from the transformed plants (T0).
T1 Plant Phenotyping
[0267] Thirty seeds from Southern positive T0 plants were sown in individual cups containing cocopeat and twenty healthy plants out of them were transplanted in the green house. These plants were arranged using a CRD using the random numbers from a random table.
[0268] T1 plant phenotyping was carried out in two separate experiments. The first experiment involved progeny lines from T0 events 1129503 and 123602 and Nipponbare (a wild-type control), and the second experiment involved progeny lines from T0 events 1164906, 1164914 and 1164922 and Nipponbare (a wild-type control.)
Phenotypic Analysis of T1 Lines
[0269] Plants height and tiller numbers were measured once every week post-transplanting until seed set was achieved. FIGS. 5a, b, c, d and e depict the growth parameters observed for these plants in two separate experiments. Transgenic ORF54 plants (T1) do not appear to be too different from the wildtype control (Nipponbare) in terms of plant height (FIG. 5a and d). However tillering capacity of ORF54 plants (T1) appear to be higher than in the wild-type control (Nipponbare) (FIG. 5b and e). A closer analysis revealed that 12.5% of the ORF54 T1 plants had out-performed the best tillering wild-type control plant (FIG. 5c) with tiller numbers more than doubling. As a result, the biomass as measured by dry matter production in ORF54 plants (T1) also increased (FIG. 6a). On an average, the increase in biomass amounts to roughly 66% as compared to the wild-type control (Nipponbare). Once again 12.5% of the ORF54 T1 plant population was seen to produce more dry matter than the highest dry matter yielding wild-type control (Nipponbare) (FIG. 6b). In conclusion down-regulation of ORF54 gene expression, or that of variants of ORF54, in planta by anti-sense or similar technology leads to an increase plant biomass.
Plant Transformation--Ryegrass
[0270] Perennial ryegrass (Lolium perenne L. cv. Impact) was transformed essentially as described in Bajaj et. al. (Plant Cell Reports, 2006, 25: 651-659). Embryogenic callus derived from mersitematic regions of the tillers of selected ryegrass lines and Agrobacterium tumefaciens strain EHA101 carrying a modified binary vector (ORF54, FIG. 4) was used for transformation experiments. Embryogenic calli were immersed with overnight-grown Agrobacterium cultures for 30 minutes with continuous shaking. Calli resistant to hygromycin were selected after sub-culturing them on co-cultivation medium for 4 weeks. After selection, the resistant calli were sub-cultured on regeneration medium every 2 weeks until the plants regenerated. The regenerants that continued to grow after two or three rounds of selection proved to be stable transformants. Each regenerated plant was then multiplied on maintenance medium to produce clonal plantlets and subsequently rooted on MS medium without hormones. A rooted plant from each clone was transferred into contained glasshouse conditions while retaining a clonal counterpart in tissue culture as backup. Eighteen independent transgenic lines (1V1, 1V3, 1V5, 1V8, 1V10, 1V11, 1V20, 2V2, 2V4, 2V5, 2V7, 2V8, 2V9, 3V5, 3V7, 3V10, 3V11, 3V12) and their non-transgenic control plants (T40, T41 and T101, respectively) have been analyzed in a climate-controlled environmental laboratory, where they were assessed for biomass production under fully water condition.
Screening for Increased Biomass in Growth Chamber
[0271] A plant growth system was built using 500 mm long; 90 mm diameter plastic storm-water pipes. The pipes were placed on a mobile tray and supported at the sides by ropes and metal frame. The tubes were plugged at the bottom with rockwool and progressively filled with washed mortar sand using water to achieve uniform packing. At the center of the open end of each tube a clump of perennial ryegrass (5 tillers) was planted. Plants from each event were replicate-planted in three tubes. The plants were arranged at random, one in each of the three replicates, and grown at 70% relative humidity; 16/8 hours day/night cycle and under 650 μmolm-2s-1 light intensity. The plants were irrigated daily once in the morning with 50 mL Hoagland's solution (Hoagland and Arnon, 1938) and again in the afternoon with 50 mL plain water. The plants were acclimated initially for twenty days and then the plants were trimmed back to 15 cm height. All plants were allowed to recover from trimming for the next fourteen days. Plant tiller numbers were recorded after seven and 14 days, respectively from the timed day. After fourteen days, plants were trimmed down to 15 cm height. The harvested samples were dried down at 60° C. for three days and then dry weight recorded. The plants were allowed to grow under fully watered conditions for another fourteen days. Again, the plants were trimmed back to 15 cm height and then the dry weight of the trimmed sample recorded after drying the samples at 60° C. for three days. The plants were allowed to grow for another 13 days and the final data on biomass production (dry matter) recorded by harvesting the plants above 15 cm and drying the samples at 60° C. for three days.
[0272] More than one-fourth of the transgenic events tested produced more biomass than wild type plants in each of the harvest. When cumulative growth was determined over a period of three harvests, one of the transgenic line, 3V7, produced more than 470% biomass; while in four other lines, 2V2; 2V7; and 3V10 the biomass increase ranged from over 35% to 95% (see FIG. 9).
[0273] The above examples illustrate practice of the invention. It will be appreciated by those skilled in the art that numerous variations and modifications may be made without departing from the spirit and scope of the invention.
REFERENCES
[0274] Adams et al. 1991, Science 252:1651-1656. [0275] Chen H, Nelson R S, Sherwood J L. (1994) Biotechniques; 16 (4): 664-8, 670. [0276] Chen et al. 2002, Nucleic Acids Res. 31:101-105 [0277] den Dulk-Ras A, Hooykaas P J. (1995) Methods Mol Biol.; 55: 63-72. [0278] Lee et al. 2003, PNAS 99:12257-12262 [0279] Lee and Lee, 2003 Plant Physiol. 132: 517-529 [0280] Murashige T, Skoog F (1962) Physiol Plant 15: 473-497 [0281] Notredame C., Higgins, D. and Heringa, J. (2000) J. Mol. Biol., 302, 205-217. [0282] Richmond and Somerville 2000, Current Opinion in Plant Biology. 3:108-116 [0283] Ruan et al. 2004, Trends in Biotechnology 22: 23-30. [0284] Schmidt-Eisenlohr H, Domke N, Angerer C, Wanner G, Zambryski P C, Baron C. (1999) J. Bacteriol.; 181 (24): 7485-92. [0285] Sun et al. 2004, BMC Genomics 5: 1.1-1.4 [0286] Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994) CABIOS, 10, 19-29. [0287] Velculescu et al. 1995, Science 270: 484-487
[0288] The above examples illustrate practice of the invention. It will be appreciated by those skilled in the art that numerous variations and modifications may be made without departing from the spirit and scope of the invention.
TABLE-US-00004 Summary of Sequences FULL- LENGTH CODING SEQUENCE SEQ (NUCLEO- ID NO: TYPE SPECIES REFERENCE TIDES) 1 Polypeptide Lolium perenne ORF54 N/A 2 Polypeptide Oryza sativa AK098904.1 N/A 3 Polypeptide Oryza sativa AK065041.1 N/A 4 Polypeptide Oryza sativa AK065992.1 N/A 5 Polypeptide Oryza sativa AK065747.1 N/A 6 Polypeptide Oryza sativa AK062069.1 N/A 7 Polypeptide Oryza sativa XP466543.1 N/A 8 Polypeptide Arabidopsis BAB01075.1 N/A thaliana 9 Polypeptide Arabidopsis AAL15211.1/ N/A thaliana AAK59536.1 10 Polynucleotide Lolium perenne ORF54 Start 302; End 1904 11 Polynucleotide Oryza sativa AK098904.1 Start 326; End 1928 12 Polynucleotide Oryza sativa AK065041.1 Start 326; End 1928 13 Polynucleotide Oryza sativa AK065992.1 Start 136; End 1738 14 Polynucleotide Oryza sativa AK065747.1 Start 486; End 2161 15 Polynucleotide Oryza sativa AK062069.1 Start 2; End 1004 16 Polynucleotide Oryza sativa XP466543.1 Start 184; End 1795 17 Polynucleotide Arabidopsis BAB01075.1 Start 1; thaliana End 1597 18 Polynucleotide Arabidopsis AAL15211.1/ Start 1; thaliana AAK59536.1 End 1297 19 Polynucleotide Vector N/A 20 Polypeptide Consensus, N/A all plants 21 Polypeptide Consensus, N/A dicotyledonous plants
Sequence CWU
1
211534PRTLolium perenne 1Met Asp Ala Thr Thr Ser Ser Gly Ala Ser Ser Ser
Leu Pro Leu His1 5 10
15Leu Ile Val Asp Asp Thr Leu Ser Leu Val Ser Pro Leu Gln Gln Ser
20 25 30Tyr Gln Arg Ser Gln Arg His
Cys Leu Gly Asp Ser Ala Pro Gly Glu 35 40
45Phe Pro Leu Ala Ala Asn Pro Ser Ile Val Leu His Val Leu Thr
Ser 50 55 60Cys Asn Leu Glu Pro Glu
Asp Leu Ala His Leu Glu Ala Thr Cys Lys65 70
75 80Phe Phe Arg Lys Pro Ala Asn Phe Pro Pro Asp
Phe Leu Leu Ser Met 85 90
95Ser Glu Leu Ala Ala Phe Asp Met Cys Gln Asn Arg Ala Ile Phe Lys
100 105 110Pro Met Gly Thr Gln Glu
Lys Glu Met Phe Lys Gln Arg Cys Gly Gly 115 120
125Thr Trp Lys Leu Val Leu Arg Phe Ile Thr Leu Gly Glu Ala
Cys Cys 130 135 140Arg Arg Glu Lys Ser
Gln Ala Ile Ala Gly Pro Gly His Ser Val Ala145 150
155 160Val Thr Ala Ser Gly Ala Ala Tyr Ser Phe
Gly Ser Asn Asn Ser Gly 165 170
175Gln Leu Gly His Asp Arg Leu Glu Glu Glu Trp Arg Pro Arg Pro Ile
180 185 190Arg Ser Leu Gln Gly
Ile Arg Ile Ile Gln Ala Ala Ala Gly Ala Gly 195
200 205Arg Thr Met Leu Val Ser Asp Ala Gly Arg Val Tyr
Ala Phe Gly Lys 210 215 220Asp Ser Phe
Gly Glu Val Glu Tyr Gly Asn Gln Gly Ser Arg Val Val225
230 235 240Thr Thr Pro Gln Leu Val Glu
Ser Leu Lys Asp Ile Tyr Ile Val Gln 245
250 255Ala Ala Ile Gly Asn Phe Phe Thr Ala Val Leu Ser
Arg Glu Gly Cys 260 265 270Val
Tyr Thr Phe Ser Trp Gly Gly Asp Met Lys Leu Gly His Gln Thr 275
280 285Glu Pro Asn Asp Val Gln Pro His Leu
Leu Ala Gly Pro Leu Glu Asp 290 295
300Ile Pro Val Val Gln Ile Ala Ala Gly Tyr Cys Tyr Leu Leu Leu Leu305
310 315 320Ala Cys Gln Pro
Ser Gly Met Ser Val Tyr Ser Val Gly Cys Gly Leu 325
330 335Gly Gly Lys Leu Gly His Gly Ser Arg Ser
Asp Glu Lys Tyr Pro Arg 340 345
350Leu Ile Glu Gln Phe Gln Thr Leu Asn Ile Gln Pro Val Val Val Ala
355 360 365Ala Gly Ala Trp His Ala Ala
Val Val Gly Lys Asp Gly Arg Val Cys 370 375
380Thr Trp Gly Trp Gly Arg Tyr Gly Cys Leu Gly His Gly Asn Glu
Glu385 390 395 400Cys Glu
Ser Val Pro Lys Val Val Glu Thr Leu Ser Ser Val Lys Ala
405 410 415Val His Val Ala Thr Gly Asp
Tyr Thr Thr Phe Val Val Ser His Lys 420 425
430Gly Asp Val Tyr Ser Phe Gly Cys Gly Glu Ser Ser Ser Leu
Gly His 435 440 445Asn Thr Ala Ile
Glu Gly Asn Asn Arg His Ser Asn Val Leu Ser Pro 450
455 460Glu Leu Val Thr Ser Ser Gln Arg Thr Asp Glu Arg
Val Val His Val465 470 475
480Ser Leu Thr Asn Ser Ile Tyr Trp Asn Ala His Thr Phe Ala Leu Thr
485 490 495Glu Ser Ala Lys Leu
Tyr Ala Phe Gly Ala Gly Asp Lys Gly Gln Leu 500
505 510Gly Thr Glu Leu Val Glu His Arg Ser Glu Arg Gly
Thr Pro Glu Gln 515 520 525Val Asp
Ile Asp Leu Asn 5302534PRTOryza sativa 2Met Asp Ala Thr Thr Ser Ser
Gly Ala Ser Ser Ser Leu Pro Leu His1 5 10
15Leu Ile Ile Asp Asp Ala Leu Ala Leu Val Ser Pro Leu
Gln Gln Ser 20 25 30Phe Gln
Arg Ser Gln Arg His Cys Phe Gly Gly Ser Ala Pro Gly Glu 35
40 45Phe Pro Leu Ala Ala Asn Pro Ser Ile Val
Leu His Val Leu Thr Ser 50 55 60Cys
Asn Leu Glu Pro Asp Asp Leu Ala His Leu Glu Ala Thr Cys Ser65
70 75 80Phe Phe Arg Lys Pro Ala
Asn Phe Pro Pro Asp Phe Gln Leu Ser Met 85
90 95Ser Glu Leu Ala Ala Leu Asp Met Cys Gln Lys Arg
Ala Ile Phe Lys 100 105 110Pro
Met Thr Gln Gln Glu Arg Glu Met Phe Lys Gln Arg Cys Gly Gly 115
120 125Ser Trp Lys Leu Val Leu Arg Phe Ile
Met Ala Gly Glu Ala Cys Cys 130 135
140Arg Arg Glu Lys Ser Gln Ala Ile Ala Gly Pro Gly His Ser Ile Ala145
150 155 160Val Thr Thr Ser
Gly Ala Val Tyr Thr Phe Gly Ser Asn Ser Ser Gly 165
170 175Gln Leu Gly His Gly Ser Leu Glu Glu Glu
Trp Arg Pro Arg Ile Ile 180 185
190Arg Ser Leu Gln Gly Ile Arg Ile Ile Gln Ala Ala Ala Gly Ala Gly
195 200 205Arg Thr Met Leu Val Ser Asp
Ala Gly Arg Val Tyr Ala Phe Gly Lys 210 215
220Asp Ser Phe Gly Glu Val Glu Tyr Ala Ala Gln Gly Ser Arg Val
Val225 230 235 240Thr Thr
Pro Gln Leu Val Glu Ser Leu Lys Asp Ile Tyr Ile Val Gln
245 250 255Ala Ala Ile Gly Asn Phe Phe
Thr Ala Val Leu Ser Arg Glu Gly His 260 265
270Val Tyr Thr Phe Ser Trp Gly Asn Asp Met Lys Leu Gly His
Gln Thr 275 280 285Glu Pro Asn Asp
Val Gln Pro His Leu Leu Ala Gly Pro Leu Glu Asn 290
295 300Ile Pro Val Val Gln Ile Ala Ala Gly Tyr Cys Tyr
Leu Leu Ala Leu305 310 315
320Ala Cys Gln Pro Ser Gly Met Ser Val Tyr Ser Val Gly Cys Gly Leu
325 330 335Gly Gly Lys Leu Gly
His Gly Ser Arg Thr Asp Glu Lys Tyr Pro Arg 340
345 350Leu Ile Glu Gln Phe Gln Ala Leu Asn Ile Gln Pro
Val Val Val Ala 355 360 365Ala Gly
Ala Trp His Ala Ala Val Val Gly Lys Asp Gly Arg Val Cys 370
375 380Thr Trp Gly Trp Gly Arg Tyr Gly Cys Leu Gly
His Gly Asn Glu Glu385 390 395
400Cys Glu Ser Val Pro Lys Val Val Glu Ser Leu Val Asn Val Arg Ala
405 410 415Val His Val Ala
Thr Gly Asp Tyr Thr Thr Phe Val Val Ser Asp Lys 420
425 430Gly Asp Val Tyr Ser Phe Gly Cys Gly Glu Ser
Ser Ser Leu Gly His 435 440 445Asn
Thr Ile Thr Glu Gly Asn Asn Arg His Thr Asn Val Leu Ser Pro 450
455 460Glu Leu Val Thr Ser Leu Lys Arg Thr Asn
Glu Arg Val Ala Gln Ile465 470 475
480Ser Leu Thr Asn Ser Ile Tyr Trp Asn Ala His Thr Phe Ala Leu
Thr 485 490 495Asp Ser Gly
Lys Leu Tyr Ala Phe Gly Ala Gly Asp Lys Gly Gln Leu 500
505 510Gly Thr Glu Leu Val Ala Gln Glu Ser Glu
Arg Gly Thr Pro Glu Arg 515 520
525Val Glu Ile Asp Leu Ser 5303534PRTOryza sativa 3Met Asp Ala Thr Thr
Ser Ser Gly Ala Ser Ser Ser Leu Pro Leu His1 5
10 15Leu Ile Ile Asp Asp Ala Leu Ala Leu Val Ser
Pro Leu Gln Gln Ser 20 25
30Phe Gln Arg Ser Gln Arg His Cys Phe Gly Gly Ser Ala Pro Gly Glu
35 40 45Phe Pro Leu Ala Ala Asn Pro Ser
Ile Val Leu His Val Leu Thr Ser 50 55
60Cys Asn Leu Glu Pro Asp Asp Leu Ala His Leu Glu Ala Thr Cys Ser65
70 75 80Phe Phe Arg Lys Pro
Ala Asn Phe Pro Pro Asp Phe Gln Leu Ser Met 85
90 95Ser Glu Leu Ala Ala Leu Asp Met Cys Gln Lys
Arg Ala Ile Phe Lys 100 105
110Pro Met Thr Gln Gln Glu Arg Glu Met Phe Lys Gln Arg Cys Gly Gly
115 120 125Ser Trp Lys Leu Val Leu Arg
Phe Ile Met Ala Gly Glu Ala Cys Cys 130 135
140Arg Arg Glu Lys Ser Gln Ala Ile Ala Gly Pro Gly His Ser Ile
Ala145 150 155 160Val Thr
Thr Ser Gly Ala Val Tyr Thr Phe Gly Ser Asn Ser Ser Gly
165 170 175Gln Leu Gly His Gly Ser Leu
Glu Glu Glu Trp Arg Pro Arg Ile Ile 180 185
190Arg Ser Leu Gln Gly Ile Arg Ile Ile Gln Ala Ala Ala Gly
Ala Gly 195 200 205Arg Thr Met Leu
Val Ser Asp Ala Gly Arg Val Tyr Ala Phe Gly Lys 210
215 220Asp Ser Phe Gly Glu Val Glu Tyr Ala Ala Gln Gly
Ser Arg Val Val225 230 235
240Thr Thr Pro Gln Leu Val Glu Ser Leu Lys Asp Ile Tyr Ile Val Gln
245 250 255Ala Ala Ile Gly Asn
Phe Phe Thr Ala Val Leu Ser Arg Glu Gly His 260
265 270Val Tyr Thr Phe Ser Trp Gly Asn Asp Met Lys Leu
Gly His Gln Thr 275 280 285Glu Pro
Asn Asp Val Gln Pro His Leu Leu Ala Gly Pro Leu Glu Asn 290
295 300Ile Pro Val Val Gln Ile Ala Ala Gly Tyr Cys
Tyr Leu Leu Ala Leu305 310 315
320Ala Cys Gln Pro Ser Gly Met Ser Val Tyr Ser Val Gly Cys Gly Leu
325 330 335Gly Gly Lys Leu
Gly His Gly Ser Arg Thr Asp Glu Lys Tyr Pro Arg 340
345 350Leu Ile Glu Gln Phe Gln Ala Leu Asn Ile Gln
Pro Val Val Val Ala 355 360 365Ala
Gly Ala Trp His Ala Ala Val Val Gly Lys Asp Gly Arg Val Cys 370
375 380Thr Trp Gly Trp Gly Arg Tyr Gly Cys Leu
Gly His Gly Asn Glu Glu385 390 395
400Cys Glu Ser Val Pro Lys Val Val Glu Ser Leu Val Asn Val Arg
Ala 405 410 415Val His Val
Ala Thr Gly Asp Tyr Thr Thr Phe Val Val Ser Asp Lys 420
425 430Gly Asp Val Tyr Ser Phe Gly Cys Gly Glu
Ser Ser Ser Leu Gly His 435 440
445Asn Thr Ile Thr Glu Gly Asn Asn Arg His Thr Asn Val Leu Ser Pro 450
455 460Glu Leu Val Thr Ser Leu Lys Arg
Thr Asn Glu Arg Val Ala Gln Ile465 470
475 480Ser Leu Thr Asn Ser Ile Tyr Trp Asn Ala His Thr
Phe Ala Leu Thr 485 490
495Asp Ser Gly Lys Leu Tyr Ala Phe Gly Ala Gly Asp Lys Gly Gln Leu
500 505 510Gly Thr Glu Leu Val Ala
Gln Glu Ser Glu Arg Gly Thr Pro Glu Arg 515 520
525Val Glu Ile Asp Leu Ser 5304534PRTOryza sativa 4Met
Asp Ala Thr Thr Ser Ser Gly Ala Ser Ser Ser Leu Pro Leu His1
5 10 15Leu Ile Ile Asp Asp Ala Leu
Ala Leu Val Ser Pro Leu Gln Gln Ser 20 25
30Phe Gln Arg Ser Gln Arg His Cys Phe Gly Gly Ser Ala Pro
Gly Glu 35 40 45Phe Pro Leu Ala
Ala Asn Pro Ser Ile Val Leu His Val Leu Thr Ser 50 55
60Cys Asn Leu Glu Pro Asp Asp Leu Ala His Leu Glu Ala
Thr Cys Ser65 70 75
80Phe Phe Arg Lys Pro Ala Asn Phe Pro Pro Asp Phe Gln Leu Ser Met
85 90 95Ser Glu Leu Ala Ala Leu
Asp Met Cys Gln Lys Arg Ala Ile Phe Lys 100
105 110Pro Met Thr Gln Gln Glu Arg Glu Met Phe Lys Gln
Arg Cys Gly Gly 115 120 125Ser Trp
Lys Leu Val Leu Arg Phe Ile Met Ala Gly Glu Ala Cys Cys 130
135 140Arg Arg Glu Lys Ser Gln Ala Ile Ala Gly Pro
Gly His Ser Ile Ala145 150 155
160Val Thr Thr Ser Gly Ala Val Tyr Thr Phe Gly Ser Asn Ser Ser Gly
165 170 175Gln Leu Gly His
Gly Ser Leu Glu Glu Glu Trp Arg Pro Arg Ile Ile 180
185 190Arg Ser Leu Gln Gly Ile Arg Ile Ile Gln Ala
Ala Ala Gly Ala Gly 195 200 205Arg
Thr Met Leu Val Ser Asp Ala Gly Arg Val Tyr Ala Phe Gly Lys 210
215 220Asp Ser Phe Gly Glu Val Glu Tyr Ala Ala
Gln Gly Ser Arg Val Val225 230 235
240Thr Thr Pro Gln Leu Val Glu Ser Leu Lys Asp Ile Tyr Ile Val
Gln 245 250 255Ala Ala Ile
Gly Asn Phe Phe Thr Ala Val Leu Ser Arg Glu Gly His 260
265 270Val Tyr Thr Phe Ser Trp Gly Asn Asp Met
Lys Leu Gly His Gln Thr 275 280
285Glu Pro Asn Asp Val Gln Pro His Leu Leu Ala Gly Pro Leu Glu Asn 290
295 300Ile Pro Val Val Gln Ile Ala Ala
Gly Tyr Cys Tyr Leu Leu Ala Leu305 310
315 320Ala Cys Gln Pro Ser Gly Met Ser Val Tyr Ser Val
Gly Cys Gly Leu 325 330
335Gly Gly Lys Leu Gly His Gly Ser Arg Thr Asp Glu Lys Tyr Pro Arg
340 345 350Leu Ile Glu Gln Phe Gln
Ala Leu Asn Ile Gln Pro Val Val Val Ala 355 360
365Ala Gly Ala Trp His Ala Ala Val Val Gly Lys Asp Gly Arg
Val Cys 370 375 380Thr Trp Gly Trp Gly
Arg Tyr Gly Cys Leu Gly His Gly Asn Glu Glu385 390
395 400Cys Glu Ser Val Pro Lys Val Val Glu Ser
Leu Val Asn Val Arg Ala 405 410
415Val His Val Ala Thr Gly Asp Tyr Thr Thr Phe Val Val Ser Asp Lys
420 425 430Gly Asp Val Tyr Ser
Phe Gly Cys Gly Glu Ser Ser Ser Leu Gly His 435
440 445Asn Thr Ile Thr Glu Gly Asn Asn Arg His Thr Asn
Val Leu Ser Pro 450 455 460Glu Leu Val
Thr Ser Leu Lys Arg Thr Asn Glu Arg Val Ala Gln Ile465
470 475 480Ser Leu Thr Asn Ser Ile Tyr
Trp Asn Ala His Thr Phe Ala Leu Thr 485
490 495Asp Ser Gly Lys Leu Tyr Ala Phe Gly Ala Gly Asp
Lys Gly Gln Leu 500 505 510Gly
Thr Glu Leu Val Ala Gln Glu Ser Glu Arg Gly Thr Pro Glu Arg 515
520 525Val Glu Ile Asp Leu Ser
5305534PRTOryza sativa 5Met Asp Ala Thr Thr Ser Ser Gly Ala Ser Ser Ser
Leu Pro Leu His1 5 10
15Leu Ile Ile Asp Asp Ala Leu Ala Leu Val Ser Pro Leu Gln Gln Ser
20 25 30Phe Gln Arg Ser Gln Arg His
Cys Phe Gly Gly Ser Ala Pro Gly Glu 35 40
45Phe Pro Leu Ala Ala Asn Pro Ser Ile Val Leu His Val Leu Thr
Ser 50 55 60Cys Asn Leu Glu Pro Asp
Asp Leu Ala His Leu Glu Ala Thr Cys Ser65 70
75 80Phe Phe Arg Lys Pro Ala Asn Phe Pro Pro Asp
Phe Gln Leu Ser Met 85 90
95Ser Glu Leu Ala Ala Leu Asp Met Cys Gln Lys Arg Ala Ile Phe Lys
100 105 110Pro Met Thr Gln Gln Glu
Arg Glu Met Phe Lys Gln Arg Cys Gly Gly 115 120
125Ser Trp Lys Leu Val Leu Arg Phe Ile Met Ala Gly Glu Ala
Cys Cys 130 135 140Arg Arg Glu Lys Ser
Gln Ala Ile Ala Gly Pro Gly His Ser Ile Ala145 150
155 160Val Thr Thr Ser Gly Ala Val Tyr Thr Phe
Gly Ser Asn Ser Ser Gly 165 170
175Gln Leu Gly His Gly Ser Leu Glu Glu Glu Trp Arg Pro Arg Ile Ile
180 185 190Arg Ser Leu Gln Gly
Ile Arg Ile Ile Gln Ala Ala Ala Gly Ala Gly 195
200 205Arg Thr Met Leu Val Ser Asp Ala Gly Arg Val Tyr
Ala Phe Gly Lys 210 215 220Asp Ser Phe
Gly Glu Val Glu Tyr Ala Ala Gln Gly Ser Arg Val Val225
230 235 240Thr Thr Pro Gln Leu Val Glu
Ser Leu Lys Asp Ile Tyr Ile Val Gln 245
250 255Ala Ala Ile Gly Asn Phe Phe Thr Ala Val Leu Ser
Arg Glu Gly His 260 265 270Val
Tyr Thr Phe Ser Trp Gly Asn Asp Met Lys Leu Gly His Gln Thr 275
280 285Glu Pro Asn Asp Val Gln Pro His Leu
Leu Ala Gly Pro Leu Glu Asn 290 295
300Ile Pro Val Val Gln Ile Ala Ala Gly Tyr Cys Tyr Leu Leu Ala Leu305
310 315 320Ala Cys Gln Pro
Ser Gly Val Ser Val Tyr Ser Val Gly Cys Gly Leu 325
330 335Gly Gly Lys Leu Gly His Gly Ser Arg Thr
Asp Glu Lys Tyr Pro Arg 340 345
350Leu Ile Glu Gln Phe Gln Ala Leu Asn Ile Gln Pro Val Val Val Ala
355 360 365Ala Gly Ala Trp His Ala Ala
Val Val Gly Lys Asp Gly Arg Val Cys 370 375
380Thr Trp Gly Trp Gly Arg Tyr Gly Cys Leu Gly His Gly Asn Glu
Glu385 390 395 400Cys Glu
Ser Val Pro Lys Val Val Glu Ser Leu Val Asn Val Arg Ala
405 410 415Val His Val Ala Thr Gly Asp
Tyr Thr Thr Phe Val Val Ser Asp Lys 420 425
430Gly Asp Val Tyr Ser Phe Gly Cys Gly Glu Ser Ser Ser Leu
Gly His 435 440 445Asn Thr Ile Thr
Glu Gly Asn Asn Arg His Thr Asn Val Leu Ser Pro 450
455 460Glu Leu Val Thr Ser Leu Lys Arg Thr Asn Glu Arg
Val Ala Gln Ile465 470 475
480Ser Leu Thr Asn Ser Ile Tyr Trp Asn Ala His Thr Phe Ala Leu Thr
485 490 495Asp Ser Gly Lys Leu
Tyr Ala Phe Gly Ala Gly Asp Lys Gly Gln Leu 500
505 510Gly Thr Glu Leu Val Ala Gln Glu Ser Glu Arg Gly
Thr Pro Glu Arg 515 520 525Val Glu
Ile Asp Leu Ser 5306334PRTOryza sativa 6Ile Gln Ala Ala Ala Gly Ala
Gly Arg Thr Met Leu Val Ser Asp Ala1 5 10
15Gly Arg Val Tyr Ala Phe Gly Lys Asp Ser Phe Gly Glu
Val Glu Tyr 20 25 30Ala Ala
Gln Gly Ser Arg Val Val Thr Thr Pro Gln Leu Val Glu Ser 35
40 45Leu Lys Asp Ile Tyr Ile Val Gln Ala Ala
Ile Gly Asn Phe Phe Thr 50 55 60Ala
Val Leu Ser Arg Glu Gly His Val Tyr Thr Phe Ser Trp Gly Asn65
70 75 80Asp Met Lys Leu Gly His
Gln Thr Glu Pro Asn Asp Val Gln Pro His 85
90 95Leu Leu Ala Gly Pro Leu Glu Asn Ile Pro Val Val
Gln Ile Ala Ala 100 105 110Gly
Tyr Cys Tyr Leu Leu Ala Leu Ala Cys Gln Pro Ser Gly Met Ser 115
120 125Val Tyr Ser Val Gly Cys Gly Leu Gly
Gly Lys Leu Gly His Gly Ser 130 135
140Arg Thr Asp Glu Lys Tyr Pro Arg Leu Ile Glu Gln Phe Gln Ala Leu145
150 155 160Asn Ile Gln Pro
Val Val Val Ala Ala Gly Ala Trp His Ala Ala Val 165
170 175Val Gly Lys Asp Gly Arg Val Cys Thr Trp
Gly Trp Gly Arg Tyr Gly 180 185
190Cys Leu Gly His Gly Asn Glu Glu Cys Glu Ser Val Pro Lys Val Val
195 200 205Glu Ser Leu Val Asn Val Arg
Ala Val His Val Ala Thr Gly Asp Tyr 210 215
220Thr Thr Phe Val Val Ser Asp Lys Gly Asp Val Tyr Ser Phe Gly
Cys225 230 235 240Gly Glu
Ser Ser Ser Leu Gly His Asn Thr Ile Thr Glu Gly Asn Asn
245 250 255Arg His Thr Asn Val Leu Ser
Pro Glu Leu Val Thr Ser Leu Lys Arg 260 265
270Thr Asn Glu Arg Val Ala Gln Ile Ser Leu Thr Asn Ser Ile
Tyr Trp 275 280 285Asn Ala His Thr
Phe Ala Leu Thr Asp Ser Gly Lys Leu Tyr Ala Phe 290
295 300Gly Ala Gly Asp Lys Gly Gln Leu Gly Thr Glu Leu
Val Ala Gln Glu305 310 315
320Ser Glu Arg Gly Thr Pro Glu Arg Val Glu Ile Asp Leu Ser
325 3307537PRTOryza sativa 7Met Gln Cys Pro Met Asp Ala
Ala Ala Ser Gly Thr Ser Pro Val Met1 5 10
15Gln Phe His Gly Ile Val Asp Glu Pro Pro Ser His Ser
Ser Pro Leu 20 25 30His Thr
Ala Leu Glu Arg Ser Gln Arg His Cys Tyr Gly His Glu Thr 35
40 45Pro Gly Glu Phe Pro Leu Ala Val Ser Pro
Ser Ile Val Leu His Val 50 55 60Leu
Ser Thr Cys Glu Leu Asp Pro Lys Asp Leu Ala Ala Leu Glu Ala65
70 75 80Thr Cys Thr Phe Phe Ser
Lys Pro Ala Asn Phe Glu Pro Asn Phe Ala 85
90 95Leu Ser Leu Pro Glu Val Ala Ala Phe Asp Met Cys
His Lys Arg Pro 100 105 110Met
Val Lys Leu Met Ala Gln Gln Glu Arg Glu Gln Leu Lys Gln Arg 115
120 125Cys Gly Gly Ser Trp Lys Leu Val Phe
Lys Tyr Ile Val Ala Arg Glu 130 135
140Arg Asn Tyr Ser Arg Ile Val Ala Gly Pro Gly His Ser Ile Val Val145
150 155 160Thr Thr Lys Gly
Asp Ala Tyr Ser Phe Gly Ala Asn Cys Trp Gly Gln 165
170 175Leu Gly Leu Gly Asp Thr Glu Asp Arg Phe
Lys Pro Cys Leu Ile Arg 180 185
190Ser Leu Gln Ser Ile Lys Ile Thr Gln Ala Ala Val Gly Ser Arg Gln
195 200 205Thr Met Leu Val Ser Asp Thr
Gly Ser Val Tyr Ala Phe Gly Lys Gly 210 215
220Ser Phe Val Trp Glu Glu Leu Ser Asp Ala Ala Asp His Ile Thr
Thr225 230 235 240Pro Lys
Ile Val Glu Ser Leu Lys Gly Val Phe Val Val Gln Ala Ala
245 250 255Ile Gly Gly Tyr Phe Ser Ala
Phe Leu Ser Arg Glu Gly Gln Val Tyr 260 265
270Thr Ile Ser Trp Gly Arg Thr Glu Arg Leu Gly His Ser Ser
Asp Pro 275 280 285Ser Asp Val Glu
Pro Arg Leu Leu Ser Gly Pro Leu Glu Gly Val Leu 290
295 300Val Ala Gln Ile Ser Ala Gly Asn Cys Tyr Leu Leu
Met Leu Ala Tyr305 310 315
320Gln Pro Thr Gly Met Ser Val Tyr Ser Val Gly Cys Gly Leu Gly Gly
325 330 335Lys Leu Gly His Gly
Cys Lys Asn Asn Lys Gly Thr Pro Lys Leu Ile 340
345 350Glu His Phe Leu Thr Leu Ser Phe Asn Pro Val Ser
Val Ala Ala Gly 355 360 365Thr Trp
His Ala Ala Ala Leu Gly Asp Asp Gly Arg Val Cys Thr Trp 370
375 380Gly Trp Gly His Thr Gly Cys Leu Gly His Gly
Asp Glu Glu Tyr Arg385 390 395
400Val Leu Pro Thr Val Val Gln Gly Leu Ser Asn Val Lys Ala Val His
405 410 415Val Ser Thr Gly
Glu Tyr Thr Thr Phe Val Val Ser Asp Asn Gly Asp 420
425 430Thr Tyr Ser Phe Gly Ser Ala Glu Ser Leu Asn
Ile Gly Phe Gln Glu 435 440 445Asp
Glu Glu Ala Ala Asp Asp Ala Asp Phe Ser Thr Pro Ser Leu Val 450
455 460Glu Ser Leu Lys Val Leu Asn Asp Lys Ala
Val Gln Ile Ser Thr Thr465 470 475
480Asn Ser Ser Tyr Trp Leu Asn Ser Glu Met Gly Tyr Pro His Thr
Phe 485 490 495Ala Leu Met
Glu Ser Gly Lys Leu Tyr Ala Phe Gly Gly Gly Ile Lys 500
505 510Gly Gln Leu Gly Val Lys Leu Ser Glu Gly
Gln Glu Arg Ala Gln Asn 515 520
525Pro Glu Arg Val Pro Ile Asp Leu Cys 530
5358532PRTArabidopsis thaliana 8Met Asp Ala Thr Ser Gly Thr Pro Ser Leu
Gln Tyr Ile Asn Leu Pro1 5 10
15Glu Gln Ser Val Ser Thr Thr Ser Pro Pro Val Ser Pro Phe Gln Arg
20 25 30Pro Lys Arg His Cys Phe
Gly Asp Thr Thr Pro Gly Glu Phe Pro Leu 35 40
45Ala Ala Asn Pro Ser Ile Val Leu His Val Leu Thr Glu Cys
Arg Leu 50 55 60Asp Pro Arg Asp Leu
Ala Asn Leu Glu Ala Thr Cys Ser Phe Phe Ser65 70
75 80Gln Pro Ala Asn Phe Ala Pro Asp Ile Asn
Leu Ser Leu Ser Glu Leu 85 90
95Ala Ala Leu Asp Met Cys Asn Lys Arg Val Ile Phe Lys Pro Met Asn
100 105 110Glu Glu Glu Arg Gln
Glu Met Lys Arg Arg Cys Gly Gly Ser Trp Lys 115
120 125Leu Val Leu Arg Phe Leu Leu Ala Gly Glu Ala Cys
Cys Arg Arg Glu 130 135 140Lys Ser Gln
Ala Val Ala Gly Pro Gly His Ser Val Ala Val Thr Ser145
150 155 160Lys Gly Glu Val Tyr Thr Phe
Gly Tyr Asn Asn Ser Gly Gln Leu Gly 165
170 175His Gly His Thr Glu Asp Glu Ala Arg Ile Gln Pro
Val Arg Ser Leu 180 185 190Gln
Gly Val Arg Ile Ile Gln Ala Ala Ala Gly Ala Ala Arg Thr Met 195
200 205Leu Ile Ser Asp Asp Gly Lys Val Tyr
Ala Cys Gly Lys Glu Ser Phe 210 215
220Gly Glu Ala Glu Tyr Gly Gly Gln Gly Thr Lys Pro Val Thr Thr Pro225
230 235 240Gln Leu Val Thr
Ser Leu Lys Asn Ile Phe Val Val Gln Ala Ala Ile 245
250 255Gly Asn Tyr Phe Thr Ala Val Leu Ser Arg
Glu Gly Lys Val Tyr Thr 260 265
270Phe Ser Trp Gly Asn Asp Gly Arg Leu Gly His Gln Thr Glu Ala Ala
275 280 285Asp Val Glu Pro Arg Pro Leu
Leu Gly Pro Leu Glu Asn Val Pro Val 290 295
300Val Gln Ile Ala Ala Gly Tyr Cys Tyr Leu Leu Ala Leu Ala Cys
Gln305 310 315 320Pro Asn
Gly Met Ser Val Tyr Ser Val Gly Cys Gly Leu Gly Gly Lys
325 330 335Leu Gly His Gly Ser Arg Thr
Asp Glu Lys Tyr Pro Arg Val Ile Glu 340 345
350Gln Phe Gln Ile Leu Asn Leu Gln Pro Arg Val Val Ala Ala
Gly Ala 355 360 365Trp His Ala Ala
Val Val Gly Gln Asp Gly Arg Val Cys Thr Trp Gly 370
375 380Trp Gly Arg Tyr Gly Cys Leu Gly His Gly Asn Glu
Glu Cys Glu Ser385 390 395
400Val Pro Lys Val Val Glu Gly Leu Ser His Val Lys Ala Val His Val
405 410 415Ala Thr Gly Asp Tyr
Thr Thr Phe Val Val Ser Asp Asp Gly Asp Val 420
425 430Tyr Ser Phe Gly Cys Gly Glu Ser Ala Ser Leu Gly
His His Pro Ser 435 440 445Phe Asp
Glu Gln Gly Asn Arg His Ala Asn Val Leu Ser Pro Thr Val 450
455 460Val Thr Ser Leu Lys Gln Val Asn Glu Arg Met
Val Gln Ile Ser Leu465 470 475
480Thr Asn Ser Ile Tyr Trp Asn Ala His Thr Phe Ala Leu Thr Glu Ser
485 490 495Gly Lys Leu Phe
Ala Phe Gly Ala Gly Asp Gln Gly Gln Leu Gly Thr 500
505 510Glu Leu Gly Lys Asn Gln Lys Glu Arg Cys Val
Pro Glu Lys Val Asp 515 520 525Ile
Asp Leu Ser 5309432PRTArabidopsis thaliana 9Met Cys Asn Lys Arg Val
Ile Phe Lys Pro Met Asn Glu Glu Glu Arg1 5
10 15Gln Glu Met Lys Arg Arg Cys Gly Gly Ser Trp Lys
Leu Val Leu Arg 20 25 30Phe
Leu Leu Ala Gly Glu Ala Cys Cys Arg Arg Glu Lys Ser Gln Ala 35
40 45Val Ala Gly Pro Gly His Ser Val Ala
Val Thr Ser Lys Gly Glu Val 50 55
60Tyr Thr Phe Gly Tyr Asn Asn Ser Gly Gln Leu Gly His Gly His Thr65
70 75 80Glu Asp Glu Ala Arg
Ile Gln Pro Val Arg Ser Leu Gln Gly Val Arg 85
90 95Ile Ile Gln Ala Ala Ala Gly Ala Ala Arg Thr
Met Leu Ile Ser Asp 100 105
110Asp Gly Lys Val Tyr Ala Cys Gly Lys Glu Ser Phe Gly Glu Ala Glu
115 120 125Tyr Gly Gly Gln Gly Thr Lys
Pro Val Thr Thr Pro Gln Leu Val Thr 130 135
140Ser Leu Lys Asn Ile Phe Val Val Gln Ala Ala Ile Gly Asn Tyr
Phe145 150 155 160Thr Ala
Val Leu Ser Arg Glu Gly Lys Val Tyr Thr Phe Ser Trp Gly
165 170 175Asn Asp Gly Arg Leu Gly His
Gln Thr Glu Ala Ala Asp Val Glu Pro 180 185
190Arg Pro Leu Leu Gly Pro Leu Glu Asn Val Pro Val Val Gln
Ile Ala 195 200 205Ala Gly Tyr Cys
Tyr Leu Leu Ala Leu Ala Cys Gln Pro Asn Gly Met 210
215 220Ser Val Tyr Ser Val Gly Cys Gly Leu Gly Gly Lys
Leu Gly His Gly225 230 235
240Ser Arg Thr Asp Glu Lys Tyr Pro Arg Val Ile Glu Gln Phe Gln Ile
245 250 255Leu Asn Leu Gln Pro
Arg Val Val Ala Ala Gly Ala Trp His Ala Ala 260
265 270Val Val Gly Gln Asp Gly Arg Val Cys Thr Trp Gly
Trp Gly Arg Tyr 275 280 285Gly Cys
Leu Gly His Gly Asn Glu Glu Cys Glu Ser Val Pro Lys Val 290
295 300Val Glu Gly Leu Ser His Val Lys Ala Val His
Val Ala Thr Gly Asp305 310 315
320Tyr Thr Thr Phe Val Val Ser Asp Asp Gly Asp Val Tyr Ser Phe Gly
325 330 335Cys Gly Glu Ser
Ala Ser Leu Gly His His Pro Ser Phe Asp Glu Gln 340
345 350Gly Asn Arg His Ala Asn Val Leu Ser Pro Thr
Val Val Thr Ser Leu 355 360 365Lys
Gln Val Asn Glu Arg Met Val Gln Ile Ser Leu Thr Asn Ser Ile 370
375 380Tyr Trp Asn Ala His Thr Phe Ala Leu Thr
Glu Ser Gly Lys Leu Phe385 390 395
400Ala Phe Gly Ala Gly Asp Gln Gly Gln Leu Gly Thr Glu Leu Gly
Lys 405 410 415Asn Gln Lys
Glu Arg Cys Val Pro Glu Lys Val Asp Ile Asp Leu Ser 420
425 430102175DNALolium perenne 10gaagcccaaa
gcatcccaca caaccaagag gagagagacc ttatcaaaaa aaaagaggag 60agagacgaca
aatccgctcc ccacccccac catcgttcct tcccagctgg tcgatcgatg 120accttgttca
tcctcatcac gctcggagct caattcgtct cctgactccg ccaagaggga 180ggtggattat
cttgagggga acggtcatgt acttcagtgc actctggtgt tgaggcctca 240agtcaggaac
accccaagtt cgagttgaaa gcatatccac tgcaagtcag agctgtcgca 300tatggatgcc
acaacgagca gcggagcttc ctcttctctt cccctccatc tcattgtgga 360tgatacacta
tccctcgttt ctccactgca gcaatcgtac caacgatcgc agcgtcattg 420ccttggtgat
tctgctcctg gggagtttcc gttggctgca aacccatcaa tagtcctcca 480tgtcctcaca
tcatgcaatc tagaacccga ggacctcgct cacttggagg caacatgcaa 540attcttcagg
aagcctgcca atttccctcc tgacttccta ttgtcaatgt cggaacttgc 600ggctttcgac
atgtgccaga atcgtgctat atttaagcct atgggtacac aagaaaaaga 660aatgtttaag
cagcgctgcg gcggtacctg gaagctagtg cttaggttca taactctagg 720tgaagcatgt
tgtcggcgag aaaaatctca ggcaattgct ggacctggcc acagcgtcgc 780tgtgacagca
agtggcgctg cttactcttt tgggtccaac aactccggcc aacttggcca 840tgaccgttta
gaagaggagt ggagaccacg tcccatcaga tcattgcagg gtattcgaat 900tattcaggca
gcagcaggag cagggcgtac tatgctcgtt agtgatgctg gtagggtgta 960tgcatttggg
aaggattcct ttggagaggt agaatatggg aatcaaggtt caagggttgt 1020gactacgcca
cagttggtgg aatcattgaa ggacatatac attgtacagg ctgcaatagg 1080gaacttcttt
actgctgtgt tatctcggga gggatgcgta tatacatttt cttggggtgg 1140cgacatgaaa
cttggtcacc aaacagagcc aaacgatgta cagcctcatc ttctcgcagg 1200ccctcttgag
gacattccag tagtgcagat agctgcaggc tactgctatc tccttcttct 1260ggcatgccaa
ccaagtggca tgtctgttta ttctgttggt tgtggtttag gagggaagct 1320tggccatggc
tcgcgaagtg atgagaaata ccctaggttg attgagcagt tccagaccct 1380gaatatacag
ccagtggtgg ttgctgcggg tgcttggcat gctgctgttg tgggcaagga 1440tgggcgagtt
tgtacttggg gatgggggcg ttatggctgc ttggggcatg gtaatgagga 1500atgtgagtct
gttcccaagg tagttgagac cttgagcagt gtgaaggctg tccatgtagc 1560aaccggagat
tacaccacat ttgttgtgtc acataaaggt gatgtttact cgtttggatg 1620tggtgaatca
tcaagccttg gccacaatac tgcgattgag ggtaataaca ggcacagcaa 1680tgtccttagc
cctgagctgg tgacctcttc gcagagaacc gatgaaaggg tggtgcatgt 1740cagcctaacg
aattccatat actggaatgc acatacattt gcactgacag agtcagcaaa 1800attgtatgca
ttcggcgcag gggacaaagg acagctaggc actgaacttg tcgaacaccg 1860aagcgagagg
ggtaccccgg agcaggtcga tattgacctc aattaggttc agttgcagca 1920caatgcctcc
ctttcgccct tttgcttcag ttgcacactt ctaaccatca cttttctaac 1980tcaccactct
ttgcattgca tgctcctagt ctgtaccgcg ttgatccttg tcaatattgt 2040tagatttgtt
agccagcaaa acaaggaatt tgtttttcat atgattgatt ctctttagaa 2100agcttgtgta
tatatttgtg attgtaaata taacaagcag gtcttcttgt cagttccttc 2160aaacatgagc
cgctg
2175112237DNAOryza sativa 11ggcttttctc tctctcctct cctctctctc tctctctctc
tctcccttct ccacgcacgc 60acatcatcat tattgggagg ctcgtccctg tccgtcgctc
gcctgctctc atcctctccc 120tctctctctc ctcccccgtt tctcggttag attcacgcct
ctccaacctc ctcccccaac 180cccaagaatt cccgatcgat tgattgactg gccgacccgc
ccgttgaatg gtcctgtact 240taattcactc tggacttctg gagagccagg aacaccacaa
gtttgagata aaagcataag 300gcagtgcaag tcgcagccgt ggcagatgga cgccacgacg
agcagtggag cttcctcctc 360tctacccctc caccttatca tagatgatgc tcttgccctt
gtttctccgt tgcagcagtc 420gtttcagagg tcacagcgcc attgctttgg tggctctgct
cctggagaat tccccttggc 480tgcaaaccca tcaattgtcc tccatgtcct cacatcatgc
aatctggaac ctgatgacct 540cgctcacttg gaggcaacat gctcgttttt ccggaagcct
gccaatttcc ctcccgattt 600tcagttgtca atgtcagaac tcgcagcgtt ggatatgtgc
cagaaacggg cgatatttaa 660acctatgact caacaagaaa gagaaatgtt taagcaacgt
tgcggcggga gttggaagct 720ggttcttagg tttataatgg caggtgaagc atgttgccgg
agggaaaaat ctcaggcaat 780cgctggacct ggtcacagca tcgctgtgac aacaagcggt
gcagtgtata cttttgggtc 840caacagctct ggtcaacttg gccatggtag tttagaagag
gagtggaggc cacggattat 900cagatcattg cagggtatta gaattattca agcggcagca
ggagcaggac gcacaatgct 960tgttagtgat gctggtaggg tctatgcatt tggaaaggat
tcatttggag aagtggaata 1020tgcagcccaa ggttctaggg ttgtcaccac accacagctg
gtggaatcat tgaaggacat 1080atacattgtc caggcagcaa tcgggaactt ctttactgca
gttttatctc gggaaggtca 1140tgtgtataca ttttcttggg ggaatgacat gaaacttggt
catcagacag agccaaatga 1200tgttcagcct catcttctag caggccctct tgagaacatt
ccagttgtgc agattgccgc 1260aggctactgc tatctcctgg ctctggcatg ccaaccaagt
ggcatgtctg tttattctgt 1320tggttgtggg ttaggtggga aacttggcca tggttctcga
accgatgaga aataccctag 1380gttaatcgag cagttccaag ctttgaatat acaaccagta
gtggttgctg ctggtgcttg 1440gcatgctgct gttgtaggca aggatgggcg tgtttgcact
tggggatggg ggcggtatgg 1500ctgcttgggt cacggtaatg aggaatgtga gtctgttcct
aaggttgttg agtccttagt 1560caatgtgagg gctgtccatg tagcaactgg agattacacc
acatttgttg tatctgataa 1620aggtgatgtt tactcgtttg gatgtggtga atcatcaagt
cttggccaca acactataac 1680tgagggtaat aataggcaca ctaatgtcct tagcccggag
ttggtgactt ctttgaagag 1740aacaaatgaa agggttgctc agatcagcct cactaactcc
atttactgga atgcacatac 1800atttgcactg acagattcag gaaaactcta tgcgtttggt
gcaggggaca aagggcagct 1860aggtaccgaa ctcgtcgcgc aggaaagcga gagggggaca
ccggagcgtg ttgaaattga 1920cctcagttag gtccaaattg caacgccact tcatctcctt
ttctctccag atgcactctt 1980ctaacgttaa ctttcaaatt gattgcattg cgcgcccttt
agcttgttgg ctgttcatca 2040gcctcatcct gctctgcagc taatccttgt gaaaatagtt
accatcaatt aaacagtctg 2100ttgttcatat gattggttcg gtttagaaac tttgtatata
tgattatcat gtaaatataa 2160cagtcaggtc tcattgccag ttcctttaaa acatgagtag
ctggctttta acatcctgtg 2220aaatttacct taactct
2237122237DNAOryza sativa 12ggcttttctc tctctcctct
cctctctctc tctctctctc tctcccttct ccacgcacgc 60acatcatcat tattgggagg
ctcgtccctg tccgtcgctc gcctgctctc atcctctccc 120tctctctctc ctcccccgtt
tctcggttag attcacgcct ctccaacctc ctcccccaac 180cccaagaatt cccgatcgat
tgattgactg gccgacccgc ccgttgaatg gtcctgtact 240taattcactc tggacttctg
gagagccagg aacaccacaa gtttgagata aaagcataag 300gcagtgcaag tcgcagccgt
ggcagatgga cgccacgacg agcagtggag cttcctcctc 360tctacccctc caccttatca
tagatgatgc tcttgccctt gtttctccgt tgcagcagtc 420gtttcagagg tcacagcgcc
attgctttgg tggctctgct cctggagaat tccccttggc 480tgcaaaccca tcaattgtcc
tccatgtcct cacatcatgc aatctggaac ctgatgacct 540cgctcacttg gaggcaacat
gctcgttttt ccggaagcct gccaatttcc ctcccgattt 600tcagttgtca atgtcagaac
tcgcagcgtt ggatatgtgc cagaaacggg cgatatttaa 660acctatgact caacaagaaa
gagaaatgtt taagcaacgt tgcggcggga gttggaagct 720ggttcttagg tttataatgg
caggtgaagc atgttgccgg agggaaaaat ctcaggcaat 780cgctggacct ggtcacagca
tcgctgtgac aacaagcggt gcagtgtata cttttgggtc 840caacagctct ggtcaacttg
gccatggtag tttagaagag gagtggaggc cacggattat 900cagatcattg cagggtatta
gaattattca agcggcagca ggagcaggac gcacaatgct 960tgttagtgat gctggtaggg
tctatgcatt tggaaaggat tcatttggag aagtggaata 1020tgcagcccaa ggttctaggg
ttgtcaccac accacagctg gtggaatcat tgaaggacat 1080atacattgtc caggcagcaa
tcgggaactt ctttactgca gttttatctc gggaaggtca 1140tgtgtataca ttttcttggg
ggaatgacat gaaacttggt catcagacag agccaaatga 1200tgttcagcct catcttctag
caggccctct tgagaacatt ccagttgtgc agattgccgc 1260aggctactgc tatctcctgg
ctctggcatg ccaaccaagt ggcatgtctg tttattctgt 1320tggttgtggg ttaggtggga
aacttggcca tggttctcga accgatgaga aataccctag 1380gttaatcgag cagttccaag
ctttgaatat acaaccagta gtggttgctg ctggtgcttg 1440gcatgctgct gttgtaggca
aggatgggcg tgtttgcact tggggatggg ggcggtatgg 1500ctgcttgggt cacggtaatg
aggaatgtga gtctgttcct aaggttgttg agtccttagt 1560caatgtgagg gctgtccatg
tagcaactgg agattacacc acatttgttg tatctgataa 1620aggtgatgtt tactcgtttg
gatgtggtga atcatcaagt cttggccaca acactataac 1680tgagggtaat aataggcaca
ctaatgtcct tagcccggag ttggtgactt ctttgaagag 1740aacaaatgaa agggttgctc
agatcagcct cactaactcc atttactgga atgcacatac 1800atttgcactg acagattcag
gaaaactcta tgcgtttggt gcaggggaca aagggcagct 1860aggtaccgaa ctcgtcgcgc
aggaaagcga gagggggaca ccggagcgtg ttgaaattga 1920cctcagttag gtccaaattg
caacgccact tcatctcctt ttctctccag atgcactctt 1980ctaacgttaa ctttcaaatt
gattgcattg cgcgcccttt agcttgttgg ctgttcatca 2040gcctcatcct gctctgcagc
taatccttgt gaaaatagtt accatcaatt aaacagtctg 2100ttgttcatat gattggttcg
gtttagaaac tttgtatata tgattatcat gtaaatataa 2160cagtcaggtc tcattgccag
ttcctttaaa acatgagtag ctggctttta acatcctgtg 2220aaatttacct taactct
2237132145DNAOryza sativa
13gattgattct ccgacgattt tgtccaacgc tacgagaatg gtcctgtact taattcactc
60tggacttctg gagagccagg aacaccacaa gtttgagata aaagcataag gcagtgcaag
120tcgcagccgt ggcagatgga cgccacgacg agcagtggag cttcctcctc tctacccctc
180caccttatca tagatgatgc tcttgccctt gtttctccgt tgcagcagtc gtttcagagg
240tcacagcgcc attgctttgg tggctctgct cctggagaat tccccttggc tgcaaaccca
300tcaattgtcc tccatgtcct cacatcatgc aatctggaac ctgatgacct cgctcacttg
360gaggcaacat gctcgttttt ccggaagcct gccaatttcc ctcccgattt tcagttgtca
420atgtcagaac tcgcagcgtt ggatatgtgc cagaaacggg cgatatttaa acctatgact
480caacaagaaa gagaaatgtt taagcaacgt tgcggcggga gttggaagct ggttcttagg
540tttataatgg caggtgaagc atgttgccgg agggaaaaat ctcaggcaat cgctggacct
600ggtcacagca tcgctgtgac aacaagcggt gcagtgtata cttttgggtc caacagctct
660ggtcaacttg gccatggtag tttagaagag gagtggaggc cacggattat cagatcattg
720cagggtatta gaattattca agcggcagca ggagcaggac gcacaatgct tgttagtgat
780gctggtaggg tctatgcatt tggaaaggat tcatttggag aagtggaata tgcagcccaa
840ggttctaggg ttgtcaccac accacagctg gtggaatcat tgaaggacat atacattgtc
900caggcagcaa tcgggaactt ctttactgca gttttatctc gggaaggtca tgtgtataca
960ttttcttggg ggaatgacat gaaacttggt catcagacag agccaaatga tgttcagcct
1020catcttctag caggccctct tgagaacatt ccagttgtgc agattgccgc aggctactgc
1080tatctcctgg ctctggcatg ccaaccaagt ggcatgtctg tttattctgt tggttgtggg
1140ttaggtggga aacttggcca tggttctcga accgatgaga aataccctag gttaatcgag
1200cagttccaag ctttgaatat acaaccagta gtggttgctg ctggtgcttg gcatgctgct
1260gttgtaggca aggatgggcg tgtttgcact tggggatggg ggcggtatgg ctgcttgggt
1320cacggtaatg aggaatgtga gtctgttcct aaggttgttg agtccttagt caatgtgagg
1380gctgtccatg tagcaactgg agattacacc acatttgttg tatctgataa aggtgatgtt
1440tactcgtttg gatgtggtga atcatcaagt cttggccaca acactataac tgagggtaat
1500aataggcaca ctaatgtcct tagcccggag ttggtgactt ctttgaaaag aacaaatgaa
1560agggttgctc agatcagcct cactaactcc atttactgga atgcacatac atttgcactg
1620acagattcag gaaaactcta tgcgtttggt gcaggggaca aagggcagct aggtaccgaa
1680ctcgtcgcgc aggaaagcga gagggggaca ccggagcgtg ttgaaattga cctcagttag
1740gtccaaattg caacgccact tcatctcctt ttctctccag atgcactctt ctaacgttaa
1800ctttcaaatt gattgcattg cgcgcccttt agcttgttgg ctgttcatca gcctcatcct
1860gctctgcagc taatccttgt gaaaatagtt accatcaatt aaacagtctg ttgttcatat
1920gattggttcg gtttagaaac tttgtatata tgattatcat gtaaatataa cagtcaggtc
1980tcattgccag ttcctttaaa acatgagtag ctggctttta acatcctgtg aaatttacct
2040taactctaca tctgcaccat ttatatttct tctaaacagg ggtgtgtgtg tgtgtgagag
2100agagatataa taatatatat ttatatattt atttctagtt gattg
2145142405DNAOryza sativa 14tctgggtttg ggggcactgt tttgagattt cagttgtttt
cttcctttgt ttccttgatt 60tttggaggtt gttttgcatc tgatttagtg tctgtttgcc
atccagaaga atatggcatc 120tctatatgtt tggtcagggt gatatgctct gtttgattgc
attgactttg ggaatgcttt 180caattattga tatgctctga ttgtttgttt acaagtgttg
gatggattct ttctcatcca 240cttttctgat tcggatgaat aatataacat attattgttt
aattgtgtta aactaaggtg 300tgctgatggg cttgttagag aagttactta acaattaatg
attgtagtta catgtcttga 360ttacattaat gcattgcaca ttacagaatg gtcctgtact
taattcactc tggacttctg 420gagagccagg aacaccacaa gtttgagata aaagcataag
gcagtgcaag tcgcagccgt 480ggcagatgga cgccacgacg agcagtggag cttcctcctc
tctacccctc caccttatca 540tagatgatgc tcttgccctt gtttctccgt tgcagcagtc
gtttcagagg tcacagcgcc 600attgctttgg tggctctgct cctggagaat tccccttggc
tgcaaaccca tcaattgtcc 660tccatgtcct cacatcatgc aatctggaac ctgatgacct
cgctcacttg gaggcaagta 720gtgttttgtt cttcctctcc aatgtagaat tgatattgcc
actgatgaaa tttgtgcttg 780cgataggcaa catgctcgtt tttccggaag cctgccaatt
tccctcccga ttttcagttg 840tcaatgtcag aactcgcagc gttggatatg tgccagaaac
gggcgatatt taaacctatg 900actcaacaag aaagagaaat gtttaagcaa cgttgcggcg
ggagttggaa gctggttctt 960aggtttataa tggcaggtga agcatgttgc cggagggaaa
aatctcaggc aatcgctgga 1020cctggtcaca gcatcgctgt gacaacaagc ggtgcagtgt
atacttttgg gtccaacagc 1080tctggtcaac ttggccatgg tagtttagaa gaggagtgga
ggccacggat tatcagatca 1140ttgcagggta ttagaattat tcaagcggca gcaggagcag
gacgcacaat gcttgttagt 1200gatgctggta gggtctatgc atttggaaag gattcatttg
gagaagtgga atatgcagcc 1260caaggttcta gggttgtcac cacaccacag ctggtggaat
cattgaagga catatacatt 1320gtccaggcag caatcgggaa cttctttact gcagttttat
ctcgggaagg tcatgtgtat 1380acattttctt gggggaatga catgaaactt ggtcatcaga
cagagccaaa tgatgttcag 1440cctcatcttc tagcaggccc tcttgagaac attccagttg
tgcagattgc cgcaggctac 1500tgctatctcc tggctctggc atgccaacca agtggcgtgt
ctgtttattc tgttggttgt 1560gggttaggtg ggaaacttgg ccatggttct cgaaccgatg
agaaataccc taggttaatc 1620gagcagttcc aagctttgaa tatacaacca gtagtggttg
ctgctggtgc ttggcatgct 1680gctgttgtag gcaaggatgg gcgtgtttgc acttggggat
gggggcggta tggctgcttg 1740ggtcacggta atgaggaatg tgagtctgtt cctaaggttg
ttgagtcctt agtcaatgtg 1800agggctgtcc atgtagcaac tggagattac accacatttg
ttgtatctga taaaggtgat 1860gtttactcgt ttggatgtgg tgaatcatca agtcttggcc
acaacactat aactgagggt 1920aataataggc acactaatgt ccttagcccg gagttggtga
cttctttgaa aagaacaaat 1980gaaagggttg ctcagatcag cctcactaac tccatttact
ggaatgcaca tacatttgca 2040ctgacagatt caggaaaact ctatgcgttt ggtgcagggg
acaaagggca gctaggtacc 2100gaactcgtcg cgcaggaaag cgagaggggg acaccggagc
gtgttgaaat tgacctcagt 2160taggtccaaa ttgcaacgcc acttcatctc cttttctctc
cagatgcact cttctaacgt 2220taactttcaa attgattgca ttgcgcgccc tttagcttgt
tggctgttca tcagcctcat 2280cctgctctgc agctaatcct tgtgaaaata gttaccatca
attaaacagt ctgttgttca 2340tatgattggt tcggtttaga aactttgtat atatgattat
catgtaaata taacagtcag 2400gtccc
2405151313DNAOryza sativa 15tattcaagcg gcagcaggag
caggacgcac aatgcttgtt agtgatgctg gtagggtcta 60tgcatttgga aaggattcat
ttggagaagt ggaatatgca gcccaaggtt ctagggttgt 120caccacacca cagctggtgg
aatcattgaa ggacatatac attgtccagg cagcaatcgg 180gaacttcttt actgcagttt
tatctcggga aggtcatgtg tatacatttt cttgggggaa 240tgacatgaaa cttggtcatc
agacagagcc aaatgatgtt cagcctcatc ttctagcagg 300ccctcttgag aacattccag
ttgtgcagat tgccgcaggc tactgctatc tcctggctct 360ggcatgccaa ccaagtggca
tgtctgttta ttctgttggt tgtgggttag gtgggaaact 420tggccatggt tctcgaaccg
atgagaaata ccctaggtta atcgagcagt tccaagcttt 480gaatatacaa ccagtagtgg
ttgctgctgg tgcttggcat gctgctgttg taggcaagga 540tgggcgtgtt tgcacttggg
gatgggggcg gtatggctgc ttgggtcacg gtaatgagga 600atgtgagtct gttcctaagg
ttgttgagtc cttagtcaat gtgagggctg tccatgtagc 660aactggagat tacaccacat
ttgttgtatc tgataaaggt gatgtttact cgtttggatg 720tggtgaatca tcaagtcttg
gccacaacac tataactgag ggtaataata ggcacactaa 780tgtccttagc ccggagttgg
tgacttcttt gaaaagaaca aatgaaaggg ttgctcagat 840cagcctcact aactccattt
actggaatgc acatacattt gcactgacag attcaggaaa 900actctatgcg tttggtgcag
gggacaaagg gcagctaggt accgaactcg tcgcgcagga 960aagcgagagg gggacaccgg
agcgtgttga aattgacctc agttaggtcc aaattgcaac 1020gccacttcat ctccttttct
ctccagatgc actcttctaa cgttaacttt caaattgatt 1080gcattgcgcg ccctttagct
tgttggctgt tcatcagcct catcctgctc tgcagctaat 1140ccttgtgaaa atagttacca
tcaattaaac agtctgttgt tcatatgatt ggttcggttt 1200agaaactttg tatatatgat
tatcatgtaa atataacagt caggtctcat tgccagttcc 1260tttaaaacat gagtagctgg
cttttaacat cctgtgaaat ttaccttaac tct 1313162012DNAOryza sativa
16ctcttttctc ctccaaggtg accacctcct ccccaccgcc ccccacatcc ggttccccga
60ggttgcagcc tctgcagcct tgcgaggatc agatgaaccc atcgtcacca gacgaattct
120ccaactgagt actctgctcc cataatccta tttatccaca cagcgagggg cactgcaaga
180agcatgcagt gcccaatgga cgcagctgca agtggaactt cgcctgtgat gcagttccat
240ggcattgttg atgagccacc ctcccattca tctccgctgc acacggcgct ggaacgttcg
300cagcgccatt gctatggtca tgaaacccca ggagaattcc cccttgctgt gagcccctcc
360attgtgctac atgtgctctc cacctgcgag ctagatccta aagatctcgc tgcactggag
420gctacatgta cattcttcag taaacctgca aatttcgagc caaactttgc tctatcgctt
480ccagaggttg cggcatttga tatgtgccat aaaagaccca tggttaagct aatggcacag
540caggaacggg agcaactgaa gcagaggtgt ggtggatctt ggaagcttgt tttcaagtat
600attgtggcta gagaaaggaa ttactctcgg attgtcgccg ggccgggcca tagtattgtt
660gtcaccacaa agggagatgc atactcattt ggggctaatt gctggggcca gcttggcctt
720ggggatactg aagatcggtt caagccatgc cttattaggt ctttgcaaag catcaaaatc
780acacaggctg cagttggatc aaggcagaca atgcttgtga gtgacacagg aagtgtctat
840gcatttggga agggtagctt tgtgtgggaa gagctttctg atgcagctga tcacattacc
900actcctaaga tagtggagtc gctaaagggt gtgtttgtag ttcaagcagc cattggtggt
960tacttctctg cgtttctatc tagagagggt caggtttaca cgatctcgtg ggggcgaacc
1020gagaggcttg gccatagttc ggatccttca gatgttgagc ctcgtcttct ctctggacca
1080cttgagggtg ttcttgttgc acagatttct gctgggaatt gctatctcct tatgttggcc
1140taccagccaa ctggaatgtc agtgtattct gtaggctgtg gtttaggagg caagcttggt
1200cacggatgca aaaacaataa gggcaccccc aagttgattg aacatttcct gacattgagc
1260tttaatccgg tttcagttgc ggctggcact tggcatgctg cagctctagg tgacgatggg
1320cgtgtctgca cctggggttg gggccatact ggttgtttgg gacatggcga tgaggagtac
1380agggttctcc ccactgtggt tcaaggattg agcaatgtga aggctgtgca tgtctccacc
1440ggtgaataca ccacctttgt tgtctccgat aacggcgata catactcctt tggatccgct
1500gaatccctga atataggttt ccaggaggat gaggaagcag cagatgatgc agatttttct
1560accccaagct tggtagaatc actgaaggtg ttgaatgata aggctgtaca gattagcaca
1620acaaattctt catattggct caactcagaa atgggatacc cgcatacgtt cgcgctcatg
1680gaatccggta aactgtatgc ctttggggga gggatcaaag gccagcttgg tgtcaagctc
1740tctgagggtc aagaaagagc tcagaaccca gagcgagtcc cgatcgatct ctgctaattt
1800caaccagcat tctggtgacc atttgcaatg acatattctg tgatctgtgg ttagcatgcc
1860ctcctgaatt tcataggagg aactcaaatg ttagcatcga tgtaaatact aggggcactt
1920acctttggtt cttctctgaa gtaacagtgt ggttgttggt agttcttgca ttttgaattg
1980ttggtgcagc caagtcctga ccgagtcttc tg
2012171599DNAArabidopsis thaliana 17atggatgcta cgagtggaac tccgagttta
cagtatatta acttgccgga acaatctgtt 60tcgactactt ctcctcctgt gtcaccattt
cagaggccaa aacgacattg ctttggtgac 120acaactccag gagagtttcc tttagcagct
aacccttcca ttgtcctaca tgttctcact 180gaatgtagat tggatcctcg tgacctcgct
aatctcgagg caacatgctc gttctttagc 240cagccagcaa actttgcccc ggacattaac
ctatcactat cggagctcgc tgctctcgac 300atgtgtaata aaagggtgat tttcaagccg
atgaatgaag aagaacgtca agagatgaaa 360cgtaggtgcg gaggatcatg gaaattagtc
cttcggtttt tgctggctgg tgaagcgtgt 420tgtcgaagag agaaatctca agctgttgct
ggtcctggtc atagtgtagc agtcacatcg 480aaaggagaag tttatacttt cggatataat
aactctggac agctaggaca tggtcatacc 540gaggacgaag ctcgaattca acctgttaga
tcattgcagg gagttcgaat catccaagca 600gctgctggtg ctgctcggac aatgctaata
agcgatgacg gaaaagttta tgcgtgtgga 660aaagaatcct tcggggaagc tgaatacgga
gggcaaggga ctaaaccagt tacaactcct 720cagcttgtaa catctttaaa aaacatattt
gtagtgcaag cagctattgg gaattacttt 780accgctgttc tctcccgaga aggaaaggtt
tatacattct cgtggggcaa tgacggtaga 840ctaggacacc aaactgaggc tgcggatgtc
gagcctcgtc ctttgttagg cccactcgag 900aatgtacccg ttgtgcagat tgctgctggt
tattgctacc ttcttgcctt agcctgtcaa 960ccaaatggca tgtctgttta ctcagttggt
tgcggtttgg gaggcaaact tggtcatggg 1020tcaagaacag atgagaagta tcctcgggtc
atcgagcagt ttcagatatt gaatcttcaa 1080cctagggtag ttgcagcggg tgcttggcat
gccgcggtgg taggtcagga tggaagagtg 1140tgcacttggg gttggggaag atatggatgt
ttaggtcacg gtaacgagga gtgtgaatca 1200gtccctaagg ttgttgaagg tctaagccat
gtcaaagcag ttcatgtcgc aacaggagac 1260tacactactt ttgtggtctc agacgatggt
gatgtttact cgtttggctg cggcgaatcc 1320gctagtctcg gtcaccatcc atcctttgat
gaacagggta atcgacatgc aaacgtgcta 1380agtccaacgg tagtaacatc gctaaaacaa
gtgaacgagc ggatggtcca gataagtcta 1440acgaactcca tatactggaa cgctcataca
tttgcgctca cggaatcggg gaagctattc 1500gcgtttggtg caggcgatca gggtcagctt
ggaacagagc ttggtaagaa ccaaaaagaa 1560aggtgtgtac cggaaaaagt ggatatcgat
ctcagctag 1599181330DNAArabidopsis thaliana
18atgtgtaata aaagggtgat tttcaagccg atgaatgaag aagaacgtca agagatgaaa
60cgtaggtgcg gaggatcatg gaaattagtc cttcggtttt tgctggctgg tgaagcgtgt
120tgtcgaagag agaaatctca agctgttgct ggtcctggtc atagtgtagc agtcacatcg
180aaaggagaag tttatacttt cggatataat aactctggac agctaggaca tggtcatacc
240gaggacgaag ctcgaattca acctgttaga tcattgcagg gagttcgaat catccaagca
300gctgctggtg ctgctcggac aatgctaata agcgatgacg gaaaagttta tgcgtgtgga
360aaagaatcct tcggggaagc tgaatacgga gggcaaggga ctaaaccagt tacaactcct
420cagcttgtaa catctttaaa aaacatattt gtagtgcaag cagctattgg gaattacttt
480accgctgttc tctcccgaga aggaaaggtt tatacattct cgtggggcaa tgacggtaga
540ctaggacacc aaactgaggc tgcggatgtc gagcctcgtc ctttgttagg cccactcgag
600aatgtacccg ttgtgcagat tgctgctggt tattgctacc ttcttgcctt agcctgtcaa
660ccaaatggca tgtctgttta ctcagttggt tgcggtttgg gaggcaaact tggtcatggg
720tcaagaacag atgagaagta tcctcgggtc atcgagcagt ttcagatatt gaatcttcaa
780cctagggtag ttgcagcggg tgcttggcat gccgcggtgg taggtcagga tggaagagtg
840tgcacttggg gttggggaag atatggatgt ttaggtcacg gtaacgagga gtgtgaatca
900gtccctaagg ttgttgaagg tctaagccat gtcaaagcag ttcatgtcgc aacaggagac
960tacactactt ttgtggtctc agacgatggt gatgtttact cgtttggctg cggcgaatcc
1020gctagtctcg gtcaccatcc atcctttgat gaacagggta atcgacatgc aaacgtgcta
1080agtccaacgg tagtaacatc gctaaaacaa gtgaacgagc ggatggtcca gataagtcta
1140acgaactcca tatactggaa cgctcataca tttgcgctca cggaatcggg gaagctattc
1200gcgtttggtg caggcgatca gggtcagctt ggaacagagc ttggtaagaa ccaaaaagaa
1260aggtgtgtac cggaaaaagt ggatatcgat ctcagctagc tagctgactt atgtggtttg
1320gttggtaaga
13301912159DNAArtificialVector 19ggaattcgat atcaagcttg gcactggccg
tcgttttaca acgtcgtgac tgggaaaacc 60ctggcgttac ccaacttaat cgccttgcag
cacatccccc tttcgccagc tggcgtaata 120gcgaagaggc ccgcaccgat cgcccttccc
aacagttgcg cagcctgaat ggcgaatgct 180agagcagctt gagcttggat cagattgtcg
tttcccgcct tcagtttaaa ctatcagtgt 240ttgacaggat atattggcgg gtaaacctaa
gagaaaagag cgtttattag aataacggat 300atttaaaagg gcgtgaaaag gtttatccgt
tcgtccattt gtatgtgcat gccaaccaca 360gggttcccct cgggatcaaa gtactttgat
ccaacccctc cgctgctata gtgcagtcgg 420cttctgacgt tcagtgcagc cgtcttctga
aaacgacatg tcgcacaagt cctaagttac 480gcgacaggct gccgccctgc ccttttcctg
gcgttttctt gtcgcgtgtt ttagtcgcat 540aaagtagaat acttgcgact agaaccggag
acattacgcc atgaacaaga gcgccgccgc 600tggcctgctg ggctatgccc gcgtcagcac
cgacgaccag gacttgacca accaacgggc 660cgaactgcac gcggccggct gcaccaagct
gttttccgag aagatcaccg gcaccaggcg 720cgaccgcccg gagctggcca ggatgcttga
ccacctacgc cctggcgacg ttgtgacagt 780gaccaggcta gaccgcctgg cccgcagcac
ccgcgaccta ctggacattg ccgagcgcat 840ccaggaggcc ggcgcgggcc tgcgtagcct
ggcagagccg tgggccgaca ccaccacgcc 900ggccggccgc atggtgttga ccgtgttcgc
cggcattgcc gagttcgagc gttccctaat 960catcgaccgc acccggagcg ggcgcgaggc
cgccaaggcc cgaggcgtga agtttggccc 1020ccgccctacc ctcaccccgg cacagatcgc
gcacgcccgc gagctgatcg accaggaagg 1080ccgcaccgtg aaagaggcgg ctgcactgct
tggcgtgcat cgctcgaccc tgtaccgcgc 1140acttgagcgc agcgaggaag tgacgcccac
cgaggccagg cggcgcggtg ccttccgtga 1200ggacgcattg accgaggccg acgccctggc
ggccgccgag aatgaacgcc aagaggaaca 1260agcatgaaac cgcaccagga cggccaggac
gaaccgtttt tcattaccga agagatcgag 1320gcggagatga tcgcggccgg gtacgtgttc
gagccgcccg cgcacgtctc aaccgtgcgg 1380ctgcatgaaa tcctggccgg tttgtctgat
gccaagctgg cggcctggcc ggccagcttg 1440gccgctgaag aaaccgagcg ccgccgtcta
aaaaggtgat gtgtatttga gtaaaacagc 1500ttgcgtcatg cggtcgctgc gtatatgatg
cgatgagtaa ataaacaaat acgcaagggg 1560aacgcatgaa ggttatcgct gtacttaacc
agaaaggcgg gtcaggcaag acgaccatcg 1620caacccatct agcccgcgcc ctgcaactcg
ccggggccga tgttctgtta gtcgattccg 1680atccccaggg cagtgcccgc gattgggcgg
ccgtgcggga agatcaaccg ctaaccgttg 1740tcggcatcga ccgcccgacg attgaccgcg
acgtgaaggc catcggccgg cgcgacttcg 1800tagtgatcga cggagcgccc caggcggcgg
acttggctgt gtccgcgatc aaggcagccg 1860acttcgtgct gattccggtg cagccaagcc
cttacgacat atgggccacc gccgacctgg 1920tggagctggt taagcagcgc attgaggtca
cggatggaag gctacaagcg gcctttgtcg 1980tgtcgcgggc gatcaaaggc acgcgcatcg
gcggtgaggt tgccgaggcg ctggccgggt 2040acgagctgcc cattcttgag tcccgtatca
cgcagcgcgt gagctaccca ggcactgccg 2100ccgccggcac aaccgttctt gaatcagaac
ccgagggcga cgctgcccgc gaggtccagg 2160cgctggccgc tgaaattaaa tcaaaactca
tttgagttaa tgaggtaaag agaaaatgag 2220caaaagcaca aacacgctaa gtgccggccg
tccgagcgca cgcagcagca aggctgcaac 2280gttggccagc ctggcagaca cgccagccat
gaagcgggtc aactttcagt tgccggcgga 2340ggatcacacc aagctgaaga tgtacgcggt
acgccaaggc aagaccatta ccgagctgct 2400atctgaatac atcgcgcagc taccagagta
aatgagcaaa tgaataaatg agtagatgaa 2460ttttagcggc taaaggaggc ggcatggaaa
atcaagaaca accaggcacc gacgccgtgg 2520aatgccccat gtgtggagga acgggcggtt
ggccaggcgt aagcggctgg gttgtctgcc 2580ggccctgcaa tggcactgga acccccaagc
ccgaggaatc ggcgtgacgg tcgcaaacca 2640tccggcccgg tacaaatcgg cgcggcgctg
ggtgatgacc tggtggagaa gttgaaggcc 2700gcgcaggccg cccagcggca acgcatcgag
gcagaagcac gccccggtga atcgtggcaa 2760gcggccgctg atcgaatccg caaagaatcc
cggcaaccgc cggcagccgg tgcgccgtcg 2820attaggaagc cgcccaaggg cgacgagcaa
ccagattttt tcgttccgat gctctatgac 2880gtgggcaccc gcgatagtcg cagcatcatg
gacgtggccg ttttccgtct gtcgaagcgt 2940gaccgacgag ctggcgaggt gatccgctac
gagcttccag acgggcacgt agaggtttcc 3000gcagggccgg ccggcatggc cagtgtgtgg
gattacgacc tggtactgat ggcggtttcc 3060catctaaccg aatccatgaa ccgataccgg
gaagggaagg gagacaagcc cggccgcgtg 3120ttccgtccac acgttgcgga cgtactcaag
ttctgccggc gagccgatgg cggaaagcag 3180aaagacgacc tggtagaaac ctgcattcgg
ttaaacacca cgcacgttgc catgcagcgt 3240acgaagaagg ccaagaacgg ccgcctggtg
acggtatccg agggtgaagc cttgattagc 3300cgctacaaga tcgtaaagag cgaaaccggg
cggccggagt acatcgagat cgagctagct 3360gattggatgt accgcgagat cacagaaggc
aagaacccgg acgtgctgac ggttcacccc 3420gattactttt tgatcgatcc cggcatcggc
cgttttctct accgcctggc acgccgcgcc 3480gcaggcaagg cagaagccag atggttgttc
aagacgatct acgaacgcag tggcagcgcc 3540ggagagttca agaagttctg tttcaccgtg
cgcaagctga tcgggtcaaa tgacctgccg 3600gagtacgatt tgaaggagga ggcggggcag
gctggcccga tcctagtcat gcgctaccgc 3660aacctgatcg agggcgaagc atccgccggt
tcctaatgta cggagcagat gctagggcaa 3720attgccctag caggggaaaa aggtcgaaaa
ggtctctttc ctgtggatag cacgtacatt 3780gggaacccaa agccgtacat tgggaaccgg
aacccgtaca ttgggaaccc aaagccgtac 3840attgggaacc ggtcacacat gtaagtgact
gatataaaag agaaaaaagg cgatttttcc 3900gcctaaaact ctttaaaact tattaaaact
cttaaaaccc gcctggcctg tgcataactg 3960tctggccagc gcacagccga agagctgcaa
aaagcgccta cccttcggtc gctgcgctcc 4020ctacgccccg ccgcttcgcg tcggcctatc
gcggccgctg gccgctcaaa aatggctggc 4080ctacggccag gcaatctacc agggcgcgga
caagccgcgc cgtcgccact cgaccgccgg 4140cgcccacatc aaggcaccct gcctcgcgcg
tttcggtgat gacggtgaaa acctctgaca 4200catgcagctc ccggagacgg tcacagcttg
tctgtaagcg gatgccggga gcagacaagc 4260ccgtcagggc gcgtcagcgg gtgttggcgg
gtgtcggggc gcagccatga cccagtcacg 4320tagcgatagc ggagtgtata ctggcttaac
tatgcggcat cagagcagat tgtactgaga 4380gtgcaccata tgcggtgtga aataccgcac
agatgcgtaa ggagaaaata ccgcatcagg 4440cgctcttccg cttcctcgct cactgactcg
ctgcgctcgg tcgttcggct gcggcgagcg 4500gtatcagctc actcaaaggc ggtaatacgg
ttatccacag aatcagggga taacgcagga 4560aagaacatgt gagcaaaagg ccagcaaaag
gccaggaacc gtaaaaaggc cgcgttgctg 4620gcgtttttcc ataggctccg cccccctgac
gagcatcaca aaaatcgacg ctcaagtcag 4680aggtggcgaa acccgacagg actataaaga
taccaggcgt ttccccctgg aagctccctc 4740gtgcgctctc ctgttccgac cctgccgctt
accggatacc tgtccgcctt tctcccttcg 4800ggaagcgtgg cgctttctca tagctcacgc
tgtaggtatc tcagttcggt gtaggtcgtt 4860cgctccaagc tgggctgtgt gcacgaaccc
cccgttcagc ccgaccgctg cgccttatcc 4920ggtaactatc gtcttgagtc caacccggta
agacacgact tatcgccact ggcagcagcc 4980actggtaaca ggattagcag agcgaggtat
gtaggcggtg ctacagagtt cttgaagtgg 5040tggcctaact acggctacac tagaaggaca
gtatttggta tctgcgctct gctgaagcca 5100gttaccttcg gaaaaagagt tggtagctct
tgatccggca aacaaaccac cgctggtagc 5160ggtggttttt ttgtttgcaa gcagcagatt
acgcgcagaa aaaaaggatc tcaagaagat 5220cctttgatct tttctacggg gtctgacgct
cagtggaacg aaaactcacg ttaagggatt 5280ttggtcatgc attctaggta ctaaaacaat
tcatccagta aaatataata ttttattttc 5340tcccaatcag gcttgatccc cagtaagtca
aaaaatagct cgacatactg ttcttccccg 5400atatcctccc tgatcgaccg gacgcagaag
gcaatgtcat accacttgtc cgccctgccg 5460cttctcccaa gatcaataaa gccacttact
ttgccatctt tcacaaagat gttgctgtct 5520cccaggtcgc cgtgggaaaa gacaagttcc
tcttcgggct tttccgtctt taaaaaatca 5580tacagctcgc gcggatcttt aaatggagtg
tcttcttccc agttttcgca atccacatcg 5640gccagatcgt tattcagtaa gtaatccaat
tcggctaagc ggctgtctaa gctattcgta 5700tagggacaat ccgatatgtc gatggagtga
aagagcctga tgcactccgc atacagctcg 5760ataatctttt cagggctttg ttcatcttca
tactcttccg agcaaaggac gccatcggcc 5820tcactcatga gcagattgct ccagccatca
tgccgttcaa agtgcaggac ctttggaaca 5880ggcagctttc cttccagcca tagcatcatg
tccttttccc gttccacatc ataggtggtc 5940cctttatacc ggctgtccgt catttttaaa
tataggtttt cattttctcc caccagctta 6000tataccttag caggagacat tccttccgta
tcttttacgc agcggtattt ttcgatcagt 6060tttttcaatt ccggtgatat tctcatttta
gccatttatt atttccttcc tcttttctac 6120agtatttaaa gataccccaa gaagctaatt
ataacaagac gaactccaat tcactgttcc 6180ttgcattcta aaaccttaaa taccagaaaa
cagctttttc aaagttgttt tcaaagttgg 6240cgtataacat agtatcgacg gagccgattt
tgaaaccgcg gtgatcacag gcagcaacgc 6300tctgtcatcg ttacaatcaa catgctaccc
tccgcgagat catccgtgtt tcaaacccgg 6360cagcttagtt gccgttcttc cgaatagcat
cggtaacatg agcaaagtct gccgccttac 6420aacggctctc ccgctgacgc cgtcccggac
tgatgggctg cctgtatcga gtggtgattt 6480tgtgccgagc tgccggtcgg ggagctgttg
gctggctggt ggcaggatat attgtggtgt 6540aaacaaattg acgcttagac aacttaataa
cacattgcgg acgtttttaa tgtactgaat 6600taacgccgaa ttaattcggg ggatctggat
tttagtactg gattttggtt ttaggaatta 6660gaaattttat tgatagaagt attttacaaa
tacaaataca tactaagggt ttcttatatg 6720ctcaacacat gagcgaaacc ctataggaac
cctaattccc ttatctggga actactcaca 6780cattattatg gagaaactcg agcttgtcga
tcgacagatc cggtcggcat ctactctatt 6840tctttgccct cggacgagtg ctggggcgtc
ggtttccact atcggcgagt acttctacac 6900agccatcggt ccagacggcc gcgcttctgc
gggcgatttg tgtacgcccg acagtcccgg 6960ctccggatcg gacgattgcg tcgcatcgac
cctgcgccca agctgcatca tcgaaattgc 7020cgtcaaccaa gctctgatag agttggtcaa
gaccaatgcg gagcatatac gcccggagtc 7080gtggcgatcc tgcaagctcc ggatgcctcc
gctcgaagta gcgcgtctgc tgctccatac 7140aagccaacca cggcctccag aagaagatgt
tggcgacctc gtattgggaa tccccgaaca 7200tcgcctcgct ccagtcaatg accgctgtta
tgcggccatt gtccgtcagg acattgttgg 7260agccgaaatc cgcgtgcacg aggtgccgga
cttcggggca gtcctcggcc caaagcatca 7320gctcatcgag agcctgcgcg acggacgcac
tgacggtgtc gtccatcaca gtttgccagt 7380gatacacatg gggatcagca atcgcgcata
tgaaatcacg ccatgtagtg tattgaccga 7440ttccttgcgg tccgaatggg ccgaacccgc
tcgtctggct aagatcggcc gcagcgatcg 7500catccatagc ctccgcgacc ggttgtagaa
cagcgggcag ttcggtttca ggcaggtctt 7560gcaacgtgac accctgtgca cggcgggaga
tgcaataggt caggctctcg ctaaactccc 7620caatgtcaag cacttccgga atcgggagcg
cggccgatgc aaagtgccga taaacataac 7680gatctttgta gaaaccatcg gcgcagctat
ttacccgcag gacatatcca cgccctccta 7740catcgaagct gaaagcacga gattcttcgc
cctccgagag ctgcatcagg tcggagacgc 7800tgtcgaactt ttcgatcaga aacttctcga
cagacgtcgc ggtgagttca ggctttttca 7860tatctcattg cccccccgga tctgcgaaag
ctcgagagag atagatttgt agagagagac 7920tggtgatttc agcgtgtcct ctccaaatga
aatgaacttc cttatataga ggaaggtctt 7980gcgaaggata gtgggattgt gcgtcatccc
ttacgtcagt ggagatatca catcaatcca 8040cttgctttga agacgtggtt ggaacgtctt
ctttttccac gatgctcctc gtgggtgggg 8100gtccatcttt gggaccactg tcggcagagg
catcttgaac gatagccttt cctttatcgc 8160aatgatggca tttgtaggtg ccaccttcct
tttctactgt ccttttgatg aagtgacaga 8220tagctgggca atggaatccg aggaggtttc
ccgatattac cctttgttga aaagtctcaa 8280tagccctttg gtcttctgag actgtatctt
tgatattctt ggagtagacg agagtgtcgt 8340gctccaccat gttatcacat caatccactt
gctttgaaga cgtggttgga acgtcttctt 8400tttccacgat gctcctcgtg ggtgggggtc
catctttggg accactgtcg gcagaggcat 8460cttgaacgat agcctttcct ttatcgcaat
gatggcattt gtaggtgcca ccttcctttt 8520ctactgtcct tttgatgaag tgacagatag
ctgggcaatg gaatccgagg aggtttcccg 8580atattaccct ttgttgaaaa gtctcaatag
ccctttggtc ttctgagact gtatctttga 8640tattcttgga gtagacgaga gtgtcgtgct
ccaccatgtt ggcaagctgc tctagccaat 8700acgcaaaccg cctctccccg cgcgttggcc
gattcattaa tgcagctggc acgacaggtt 8760tcccgactgg aaagcgggca gtgagcgcaa
cgcaattaat gtgagttagc tcactcatta 8820ggcaccccag gctttacact ttatgcttcc
ggctcgtatg ttgtgtggaa ttgtgagcgg 8880ataacaattt cacacaggaa acagctatga
ccatgattac gaattccctt aattaaggcg 8940cgccgatact gaattaacgc cgaattaatt
cgggggatct ggattttagt actggatttt 9000ggttttagga attagaaatt ttattgatag
aagtatttta caaatacaaa tacatactaa 9060gggtttctta tatgctcaac acatgagcga
aaccctatag gaaccctaat tcccttatct 9120gggaactact cacacattat tatggagaaa
ccaggccgaa gcccaaagca tcccacacaa 9180ccaagaggag agagacctta tcaaaaaaaa
agaggagaga gacgacaaat ccgctcccca 9240cccccaccat cgttccttcc cagctggtcg
atcgatgacc ttgttcatcc tcatcacgct 9300cggagctcaa ttcgtctcct gactccgcca
agagggaggt ggattatctt gaggggaacg 9360gtcatgtact tcagtgcact ctggtgttga
ggcctcaagt caggaacacc ccaagttcga 9420gttgaaagca tatccactgc aagtcagagc
tgtcgcatat ggatgccaca acgagcagcg 9480gagcttcctc ttctcttccc ctccatctca
ttgtggatga tacactatcc ctcgtttctc 9540cactgcagca atcgtaccaa cgatcgcagc
gtcattgcct tggtgattct gctcctgggg 9600agtttccgtt ggctgcaaac ccatcaatag
tcctccatgt cctcacatca tgcaatctag 9660aacccgagga cctcgctcac ttggaggcaa
catgcaaatt cttcaggaag cctgccaatt 9720tccctcctga cttcctattg tcaatgtcgg
aacttgcggc tttcgacatg tgccagaatc 9780gtgctatatt taagcctatg ggtacacaag
aaaaagaaat gtttaagcag cgctgcggcg 9840gtacctggaa gctagtgctt aggttcataa
ctctaggtga agcatgttgt cggcgagaaa 9900aatctcaggc aattgctgga cctggccaca
gcgtcgctgt gacagcaagt ggcgctgctt 9960actcttttgg gtccaacaac tccggccaac
ttggccatga ccgtttagaa gaggagtgga 10020gaccacgtcc catcagatca ttgcagggta
ttcgaattat tcaggcagca gcaggagcag 10080ggcgtactat gctcgttagt gatgctggta
gggtgtatgc atttgggaag gattcctttg 10140gagaggtaga atatgggaat caaggttcaa
gggttgtgac tacgccacag ttggtggaat 10200cattgaagga catatacatt gtacaggctg
caatagggaa cttctttact gctgtgttat 10260ctcgggaggg atgcgtatat acattttctt
ggggtggcga catgaaactt ggtcaccaaa 10320cagagccaaa cgatgtacag cctcatcttc
tcgcaggccc tcttgaggac attccagtag 10380tgcagatagc tgcaggctac tgctatctcc
ttcttctggc atgccaacca agtggcatgt 10440ctgtttattc tgttggttgt ggtttaggag
ggaagcttgg ccatggctcg cgaagtgatg 10500agaaataccc taggttgatt gagcagttcc
agaccctgaa tatacagcca gtggtggttg 10560ctgcgggtgc ttggcatgct gctgttgtgg
gcaaggatgg gcgagtttgt acttggggat 10620gggggcgtta tggctgcttg gggcatggta
atgaggaatg tgagtctgtt cccaaggtag 10680ttgagacctt gagcagtgtg aaggctgtcc
atgtagcaac cggagattac accacatttg 10740ttgtgtcaca taaaggtgat gtttactcgt
ttggatgtgg tgaatcatca agccttggcc 10800acaatactgc gattgagggt aataacaggc
acagcaatgt ccttagccct gagctggtga 10860cctcttcgca gagaaccgat gaaagggtgg
tgcatgtcag cctaacgaat tccatatact 10920ggaatgcaca tacatttgca ctgacagagt
cagcaaaatt gtatgcattc ggcgcagggg 10980acaaaggaca gctaggcact gaacttgtcg
aacaccgaag cgagaggggt accccggagc 11040aggtcgatat tgacctcaat taggttcagt
tgcagcacaa tgcctccctt tcgccctttt 11100gcttcagttg cacacttcta accatcactt
ttctaactca ccactctttg cattgcatgc 11160tcctagtctg taccgcgttg atccttgtca
atattgttag atttgttagc cagcaaaaca 11220aggaatttgt ttttcatatg attgattctc
tttagaaagc ttgtgtatat atttgtgatt 11280gtaaatataa caagcaggtc ttcttgtcag
ttccttcaaa catgagccgc tgctaatgga 11340gagagataga tttgtagaga gagactggtg
atttcagcgt gtcctctcca aatgaaatga 11400acttccttat atagaggaag ggtcttgcga
aggatagtgg gattgtgcgt catcccttac 11460gtcagtggag atatcacatc aatccacttg
ctttgaagac gtggttggaa cgtcttcttt 11520ttccacgatg ctcctcgtgg gtgggggtcc
atctttggga ccactgtcgg cagagcatct 11580tgaacgatag cctttccttt atcgcaatga
tggcatttgt aggtgccacc ttccttttct 11640actgtccttt tgatgaagtg acagatagct
gggcaatgga atccgaggag gtttcccgat 11700attacccttt gttgaaaagt ctcaatagcc
ctttggcctt ctgagactgt atctttgata 11760ttcttggagt agacgagagt gtcgtgctcc
accatgttca catcaatcca cttgctttga 11820agacgtggtt ggaacgtctt ctttttccac
gatgctcctc gtgggtgggg gtccatcttt 11880gggaccactg tcggcagagg catcttgaac
gatagccttt cctttatcgc aatgatggca 11940tttgtaggtg ccaccttcct tttctactgt
ccttttgatg aagtgacaga tagctgggca 12000atggaatccg aggaggtttc ccgatattac
cctttgttga aaagtctcaa tagccctttg 12060gtcttctgag actgtatctt tgatattctt
ggagtagacg agagtgtcgt gctccaccat 12120gttggcaagc tgctcttatt aattaaggcg
cgccctgca 121592016PRTArtificialConsensus, all
plants 20Ser Val Tyr Ser Val Gly Cys Gly Leu Gly Gly Lys Leu Gly His Gly1
5 10
152120PRTArtificialConsensus, dicotyledonous plants 21Gly Met Ser Val Tyr
Ser Val Gly Cys Gly Leu Gly Gly Lys Leu Gly1 5
10 15His Gly Ser Arg 20
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