CN110373418A - Regulate and control gene and its application of size of plant seed - Google Patents

Regulate and control gene and its application of size of plant seed Download PDF

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CN110373418A
CN110373418A CN201910752694.7A CN201910752694A CN110373418A CN 110373418 A CN110373418 A CN 110373418A CN 201910752694 A CN201910752694 A CN 201910752694A CN 110373418 A CN110373418 A CN 110373418A
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唐晓艳
许纯珏
严维
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Unnamed Prosperous System Crop Design Frontier Laboratory (beijing) Co Ltd
Shenzhen Institute of Molecular Crop Design
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Abstract

The invention discloses a kind of gene of regulation size of plant seed and its applications, belong to molecular biology of plants, biochemistry, science of heredity and field of plant breeding.The present invention obtains a big particle mutant by EMS mutagenesis rice, and utilizes SIMM method positional mutation trait related gene, obtains the gene of a regulation size of plant seedDLZ, for the gene on No. 12 chromosomes, gene locus number is the LOC_Os12g41820(MSU number of logging in).GeneDLZMutation can lead to rice and generate big kernel Characters phenotype, by the seed size for adjusting the controllable plant of expression of the gene, method to obtain big particle mutant plant and Crop Improvement has great theoretical and practical significance the SOYBEAN IN HIGH-YIELD BREEDING work of crop.

Description

Regulate and control gene and its application of size of plant seed
Technical field
The present invention relates to molecular biology of plants, biochemistry, science of heredity and field of plant breeding, in particular to one tune Control seed size gene, relate more specifically to one regulation seed size DLZ gene nucleic acid molecules and its mutant and Its application in breeding.
Technical background
Vegetable seeds is most important food source for the survival of mankind, and rice, corn and wheat are the three big masters in China Grain, grain yield and price national economy.The status of population sustainable growth and the continuous reduction of cultivated area, to China's grain Food production proposes stern challenge.By taking rice as an example, rice yield depends mainly on three elements --- number of productive ear, every fringe Bear fruit grains and grain weight, yield per unit area are also influenced by plant type of rice, these factors are all by polygenes and environment interaction The complex character of control, and there is also correlation between each sub- character, Yao Gailiang yield traits also need to coordinate each composition Relationship between element.According to existing experience, high-yield variety is segmented into four types, i.e., big fringe weighting type, big grain are laid particular stress on Type, more fringe weighting types and synthesis are compatible.The genetic force of panicle number per plant is minimum, it is easy to and it is affected by environment, have to single plant The improvement of effect spike number is not as good as the number of productive ear for adjusting unit area by rational close planting.The genetic force of number of grain per ear is moderate, changes It is good still to have no small difficulty.In contrast, grain is that genetic force is highest in yield forming element again, carries out improvement phase to it To easy (Zhang Qifa, the conception and practice of green super hybridization rice, Science Press, 2009).
Rice grain weight is determined by two factors of seed size, that is, glume volume and endosperm development situation.Glume is usually in water Rice has just been formed before blooming, and the grain of rice size and shape that the post-grouting of fertilization is formed is limited by the volume of glume, therefore, grain husk The volume of shell is the great small prerequisite of grain.Particle shape is commonly referred to as to the description of glume shape and size, usually uses grain length, grain Wide, grain thickness and length-width ratio indicate.It is noted that particle shape is not only important one of yield traits and main appearance Quality trait, there is also certain related between particle shape and the other quality traits such as chalkness ratio of rice, Coarse Rice Rate, polished rice rate Property.Therefore, yield potentiality not only can be improved by the selection to seed size and shape in breeding, can also adjusts indirectly Control rice quality.
Size of plant seed is the quantitative character of controlled by multiple genes, in the research base of early period a large amount of QTL scanning and positioning On plinth, a kind of trend of outburst is presented in the clone of related gene nearly ten years.Base is regulated and controled to the important crops seed size such as rice The excavation and functional study of cause have become a big hot spot of functional genomics research.The discovery and functional analysis of these genes Gradually going deep into for size of plant seed gene regulatory network research is promoted, researcher starts to explore key gene polymerization most Good design provides reason by the yield increasing effect of evaluation favorable genes type polymerization for the breakthrough new varieties of initiative design mode yield By guidance and materials for support.
SIMM(Simultaneous Identification of Multiple Mutations)(Yan et al., Simultaneous identification of multiple causal mutations in rice.Frontiers in Plant Science, 2016) be a kind of positional mutation gene rapidly and efficiently based on two generation sequencing technologies method.With it He compares method, and SIMM can be under the precursor for not needing wild type gene group data, with the mutation in other same or close sources Body is background, while identifying the relevant mutational site of multiple mutant characters, has higher sensitivity and specificity, is facilitated fast Speed positions candidate functional gene, the research of auxiliary rice functional gene and rice design and context.In addition, SIMM can also be used in extreme table Type pond QTL (quantitative trait locus, Quantitative Trait Locus) positioning, effectively reduces candidate section, and auxiliary is fixed Position main effect QTL gene.The method also can be effectively applied to positioning other species EMS mutant candidate's functional gene.
High-resolution melting curve analysis (High-Resolution Melting Curve Analysis, HRM) is one It is kind different based on mononucleotide melting temperature and form the genetic analysis new technology of different shape melting curve, with high quick Perception, can detecte out the difference of single base, and it is at low cost, flux is high, speed is fast, result is accurate, not examined site Limitation.This method divides PCR reaction product using a kind of saturable dye without using sequence-specific probes Analysis.Its principle is: the thermal stability of double-stranded DNA is influenced by its length and base composition, and sequence variation will lead to temperature-rise period The change of double center chain DNA unwinding behavior.Because fluorescent dye used can only be embedded in and be integrated on double-stranded DNA, utilize Real time pcr can be to generate different shape by the variation of fluorescence signal value in real-time detection double-stranded DNA fusion processes The mode of melting curve intuitively shows difference present in PCR product.Meanwhile by means of professional analysis software Genotyping or classification based on different shape melting curve can be realized to test group.
The present invention obtains one by single recessive nuclear gene control by EMS mutagenesis rice rice variety " Huang Huazhan ", screening The big particle mutant of system, and then phenotypic evaluation, genetic analysis and genetic background are carried out to the mutant and identified, and utilizes the side SIMM Method, HRM technology and gene information analysis, successfully position and have cloned a seed size controlling gene DLZ, which is located at 12 On number chromosome, gene locus number is LOC_Os12g41820 (the MSU number of logging in), the mutation of the gene can lead to rice generate it is big Kernel Characters phenotype, so as to be applied to the size of control crop kernel.The present invention helps to improve crop yield and improving quality, To cultivate there is the new rice variety of big grain weight to provide genetic resources and technical support, the raising and high yield to crop economical character Molecular breeding work is of great significance and value.
Summary of the invention
All bibliography being mentioned herein all are incorporated herein by reference.
Unless there are indicating on the contrary, all technical and scientific terms used herein all have common with fields of the present invention The identical meaning that technical staff is generally understood.Unless there are indicating on the contrary, technology that is used herein or mentioning is ability Standard technique well known to the those of ordinary skill of domain.Material, method and example are only used as to illustrate, rather than limit.
The present invention provides the gene DLZ (big grain accounts for, Da Li Zhan) that one regulates and controls seed size, which is located at 12 On number chromosome, gene loci number in rice is LOC_Os12g41820 (the MSU number of logging in, with reference to Rice Genome Annotation Project, http://rice.plantbiology.msu.edu/), can make after the gene mutation containing The seed grain shape of the plant of the mutation becomes larger.
Because often there is single nucleotide polymorphism, the i.e. nucleotide sequence of same gene in the same gene between different cultivars Often there is the difference of Individual base, but there are many rice varieties quantity, inventor can not can be carried out and enumerate, therefore this hair The bright sequence for providing only representative two kinds in long-grained nonglutinous rice and japonica rice.Specifically, the core of the rice DLZ gene Nucleotide sequence is selected from following group of one of sequence:
(a) nucleotide sequence as shown in the gene LOC_Os12g41820 of regulation size of plant seed；
(b) nucleotide sequence as shown in SEQ ID NO:1,2,20 or 21；
(c) its encoding amino acid sequence nucleotides sequence as shown in SEQ ID NO:3,55,56,57,58,59,60 or 61 Column；
It (d) can be with the DNA sequence dna of the DNA hybridization of sequence described in (a)-(c) under high stringency conditions；Or (e) with (a)- (d) sequence has at least 80% (preferably at least 85%) sequence similarity, and has regulation size of plant seed function DNA sequence dna；Or
(f) DNA sequence dna complementary with any sequence of (a)-(e).
The DLZ gene of above-mentioned regulation seed size can be separated from various plants and be obtained.Those skilled in the art should know Dawn, seed size controlling gene of the present invention further include with the nucleotide sequence of DLZ gene or protein sequence very high homology, And the homologous gene with same seed size adjusting function.The homologous gene is included under high stringency conditions can be with this The DNA sequence dna of the nucleotide sequence hybridization of the disclosed DLZ gene of invention." high stringency conditions " used herein be it is well known, Including such as hybridizing in the hybridization solution of NaCl containing 400mM, 40mM PIPES (pH6.4) and 1mM EDTA, the temperature of the hybridization Preferably 53 DEG C -60 DEG C of degree, hybridization time is preferably 12-16 hours, then with the cleaning solution for containing 0.5 × SSC and 0.1%SDS Washing, wash temperature is preferably 62 DEG C -68 DEG C, and wash time is 15-60 minutes.
Homologous gene further include have at least 80% with sequence shown in DLZ gene disclosed in this invention, 85%, 90%, 95%, 98% or 99% sequence similarity, and there is the DNA sequence dna of regulation seed size function, it can divide from any plant From acquisition.Wherein, the percentage of sequence similarity can be obtained by well known bioinformatics, including Myers and Miller algorithm, Needleman-Wunsch overall comparison method, Smith-Waterman Local Alignment method, Pearson and The algorithm of Lipman similarity-searching, Karlin and Altschul, this is well known to the skilled artisan.
Gene order of the present invention can be separated from any plant and be obtained, including but not limited to Btassica, corn, Wheat, sorghum, two section shepherd's purse categories, sinapsis alba, castor bean, sesame, cottonseed, linseed, soybean, Arabidopsis, Phaseolus, peanut, lucerne Mu, oat, rapeseed, barley, oat, rye (Rye), grain, chinese sorghum, triticale, einkorn, Si Peierte wheat (Spelt), emmer, flax, gramagrass (Gramma grass), friction standing grain, false chinese sorghum, fescue grass, perennial ryegrass, sweet Sugarcane, crowberry, papaya, banana, safflower, oil palm, muskmelon, apple, cucumber, dendrobium nobile, gladiolus, chrysanthemum, Liliaceae, cotton, Eucalyptus, sunflower, rape, beet, coffee, ornamental plant and conifer etc..Preferably, plant include corn and soybean, safflower, leaf mustard, Wheat, barley, rye, rice, cotton and sorghum.
The present invention provides a kind of methods of regulation size of plant seed, and the method will be provided by the present invention by influence DLZ gene expression dose, to influence size of plant seed.The influence size of plant seed refers to by reducing DLZ base The expression of cause such as leads to the phenotype of big grain so that the seed size of the plant be made to change.Specifically, it depends on Concrete application demand can influence DLZ gene in the intracorporal expression of plant by a variety of methods, to reach regulation kind The effect of sub- size.More specifically, the expression of regulation DLZ gene can be used many those of ordinary skill in the art and can obtain Tool carry out, such as by mutation, mutagenesis, being transferred to of antisense gene, co-suppression or introducing of hairpin structure etc., be ok For destroying the normal expression of DLZ gene, to obtain the plant that seed becomes larger.
The present invention also provides a kind of method of big particle mutant material for obtaining DLZ gene, the method passes through mutation water The endogenous DLZ gene of rice, or the nucleotide sequence of mutation and the gene of its very high homology, make the work of the plant control seed size It is changed with approach.The nucleotide sequence of the DLZ gene as shown in SEQ ID NO:1,2,20 or 21, the DLZ gene Amino acid sequence is as shown in SEQ ID NO:3,55,56,57,58,59,60 or 61." mutation " includes but is not limited to following Method, such as the gene mutation caused by method physically or chemically, chemical method includes caused by the processing of the mutagens such as EMS Mutagenesis, the mutation can also be point mutation, is also possible to DNA missing or insertion mutation, is also possible to through bases such as RNAi Because of silencing means or by the method for site-directed point mutation, the method for the site-directed point mutation includes but is not limited to ZFN fixed The gene editings methods such as point mutation process, TALEN directed mutagenesis method, and/or CRISPR/Cas9.
The present invention also provides a kind of application methods of the big particle mutant material of DLZ, it is characterised in that the mutant material It is table of the plant caused by the mutation as the nucleotide sequence of DLZ gene, containing saltant type DLZ gene with large seed Type, wherein the nucleotide sequence of the DLZ gene is preferably as shown in SEQ ID NO:1,2,20 or 21.Specifically, institute of the present invention As shown in SEQ ID NO:4, amino acid sequence is mutated as shown in SEQ ID NO:5 in big grain nucleotide sequence after stating mutation In body, the mutation of two bases occurs on the 4th exon of DLZ gene, TTA is specifically sported by AAA, leads to SEQ ID 321st amino acids of NO:3 albumen sport Leu by Lys, and the transcript caused changes with protein product, from And make plant that there is large seed phenotype.Those skilled in the art should know, can be by the nucleotide sequence SEQ ID NO:4 is building up to plant expression vector, Plant Transformation is carried out, to obtain the big particle mutant material of new transgenosis.It is described prominent The application of variant material, the application including but not limited in crossbreeding more specifically refer to including but not limited to cultivate and plant Article kind or strain cultivate plant variety or strain and cultivate plant variety or product that seed sizes become smaller that seed sizes increase System, the big grain kind of identification crop and molecular labeling of granule kind etc. are applied.
The present invention also provides a kind of application of expression cassette in regulation size of plant seed, the expression cassette contains regulation The DNA sequence dna of the DLZ gene of size of plant seed, the nucleotide sequence of the gene of the regulation size of plant seed are selected from following One of the sequence of group:
(a) nucleotide sequence as shown in the gene LOC_Os12g41820 of regulation size of plant seed；
(b) nucleotide sequence as shown in SEQ ID NO:1,2,20 or 21；
(c) its encoding amino acid sequence nucleotides sequence as shown in SEQ ID NO:3,55,56,57,58,59,60 or 61 Column；
It (d) can be with the DNA sequence dna of the DNA hybridization of sequence described in (a)-(c) under high stringency conditions；Or (e) with (a)- (d) sequence has at least 80% (preferably at least 85%) sequence similarity, and has regulation size of plant seed function DNA sequence dna；Or
(f) DNA sequence dna complementary with any sequence of (a)-(e).
Specifically, the seed size controlling gene in above-mentioned expression cassette, which is also operably connected with one, can drive its expression Promoter, the promoter in the construct can be natural promoter or substituted promoter, will drive connected core Expression of the nucleotide sequence in plant.Promoter in expression cassette includes but is not limited to that composition type expression promoter, induction type open Mover, organizing specific expression promoter, space-time specific expression promoter etc..The gene table of constitutive promoter of the present invention Up to not having tissue and temporal, the exogenous gene expression that extraneous factor starts constitutive promoter is almost without shadow It rings.The constitutive promoter includes but is not limited to CaMV35S, FMV35S, rice actin (Actin1) promoter, corn Ubiquitin (Ubiquitin) promoter etc..Tissue-specific promoter of the present invention removes comprising due general promoter member Outside part, also with the characteristic of enhancer and silencer, the advantages of such promoter be can promotor gene in plant specific group The expression for knitting position, avoids the unnecessary expression of foreign gene, to save the overall power consumption of plant.It is of the present invention Inducible promoter refer under certain specific physically or chemically stimulations of signal, turning for gene can be significantly increased Horizontal promoter is recorded, separated inducible promoter includes but is not limited to adverse circumstance inducing expression promoter, photo-induction at present Lead expression promoter, thermal induction expression promoter, wound-inducible expression promoter, fungal induction expression promoter and symbiotic bacteria Inducing expression promoter etc..Tissue-specific promoter of the present invention includes but is not limited to the starting of LTP2 seed specific expression Son, END2 Seeds oil-body-specific promoter, aleurone specific expression promoter etc..
It may also include other components in above-mentioned expression cassette, this depends primarily on the purpose and purposes of vector construction, such as can It further comprise selectable marker gene, targeting or regulating and controlling sequence, critical sequences or boot sequence, introne etc..Above-mentioned construct In may also include other components, this depends primarily on the purpose and purposes of vector construction, such as can further comprise selected marker Gene, targeting or regulating and controlling sequence, critical sequences or boot sequence, introne etc..Expression cassette will also be in desired heterologous nucleotides sequence 3 ' ends of column, which are included in plant, has functional transcription and translation terminator.Terminator can be DLZ base provided by the present invention Itself terminator of cause is also possible to the terminator from external source, such as nopaline synthase or octopine synthase termination area.
It is desirable that guiding the expression product of heterologous nucleotide sequence into specific cells device, such as plastid, amyloplast, Huo Zheyin To endoplasmic reticulum, or in the case where cell surface or cell exocrine, expression cassette also may include the nucleosides for encoding transit peptides Acid sequence.Such transit peptides be it is known in the art, its include but is not limited to the small subunit of Rubisco, plant EPSP synthase, Corn Brittle-1, chloroplast transit peptides etc..
During preparing expression cassette, a variety of DNA fragmentations can be operated, be in proper orientation to provide, or DNA sequence dna in correct reading frame.To reach this purpose, adapter or connector can be used, DNA fragmentation is linked up, or Person further comprises other operations, to provide convenient restriction enzyme site etc..
The above-mentioned expression cassette of the present invention, also further may include a screening-gene, the screening-gene can be used for Plant containing the expression cassette, plant tissue cell or vector selection are come out.The screening-gene includes but is not limited to antibiosis Plain resistant gene anti-herbicide gene or fluorescence protein gene etc..Specifically, the screening-gene includes but unlimited In: chloramphenicol resistance gene, hygromycin gene, streptomycin resistance gene, miramycin resistant gene, sulfamido resistance base Cause, glyphosate gene, glufosinate-resistant gene, bar gene, red fluorescent gene DsRED, mCherry gene, cyan are glimmering Aequorin, yellow fluorescent protein gene, luciferase gene, green fluorescence protein gene etc..
Further, it may also include selectable marker gene in construct provided by the present invention, it is transformed for selecting Cell or tissue.The selectable marker gene includes assigning antibiotic resistance or the gene to Herbicid resistant.Suitable selection Marker gene includes but is not limited to: chloramphenicol resistance gene, hygromycin gene, streptomycin resistance gene, miramycin resistance Gene, sulfamido resistant gene, glyphosate gene, glufosinate-resistant gene.The selectable marker gene can also be red Color fluorogene, cyan fluorescent protein gene, yellow fluorescent protein gene, luciferase gene, green fluorescence protein gene, flower The genes such as green glucoside p1.
Expression cassette or carrier provided by the present invention can be inserted into plasmid, clay, yeast artificial chromosome, bacteria artificial dye Colour solid or other be suitble to be transformed into any carrier in host cell.Preferred host cell is bacterial cell, is especially used In cloning or storage polynucleotides or bacterial cell for converting plant cell, for example, Escherichia coli, Agrobacterium tumdfaciens and Agrobacterium rhizogenes.When host cell is plant cell, expression cassette or carrier can be inserted into the base for the plant cell being converted Because in group.Insertion can be positioning or random insertion.Preferably, such as homologous recombination is inserted through to realize.In addition, table It is positively retained at outside chromosome up to box or carrier.Expression cassette or carrier of the invention may be present in the core, chloroplaset, line of plant cell In plastochondria and/or plastid.Preferably, expression cassette of the invention or carrier are inserted into the chromosomal DNA of plant nucleolus.
The nucleotide sequence and promoter sequence or expression cassette of DLZ gene provided by the present invention can be inserted into carrier, matter Grain, yeast artificial chromosome, bacterial artificial chromosome or other be suitble to be transformed into any carrier in host cell.Preferably Host cell is bacterial cell, in particular for cloning or storing polynucleotides or the bacterial cell for converting plant cell, Such as Escherichia coli, Agrobacterium tumdfaciens and Agrobacterium rhizogenes.When host cell is plant cell, expression cassette or carrier can It is inserted into the genome for the plant cell being converted.Insertion can be positioning or random insertion.
It is of the present invention that nucleotide sequence, carrier or expression cassette are transferred to plant or introduces plant or plant is turned Change, refers both to that nucleotide sequence, carrier or expression cassette are transferred to recipient cell or recipient plant by conventional transgenic method In.Any transgenic method known to plant biotechnology field technical staff can be used to for recombinant expression carrier being transformed into In plant cell, to generate genetically modified plants of the invention.Method for transformation may include method for transformation directly or indirectly.Suitably Direct method include polyethylene glycol induction DNA intake, liposome-mediated conversion, using particle gun importing, electroporation and Microinjection.The method for transformation also includes the methods for plant transformation etc. of mediated by agriculture bacillus.
The present invention provides a kind of production methods of hybrid plant for controlling seed size, which is characterized in that this method packet It includes:
(a) expression cassette provided by the present invention is constructed；
(b) expression cassette for obtaining step (a) imports plant cell；
(c) genetically modified plants are regenerated；With
(d) genetically modified plants are selected；And
(e) optionally, the plant that amplification step (d) obtains is to obtain offspring.
Genetically modified plants of the invention are prepared using method for transformation known to plant biotechnology field technical staff.It is any Method can be used for for recombinant expression carrier being transformed into plant cell, to generate genetically modified plants of the invention.Method for transformation It may include method for transformation directly or indirectly.Suitable direct method includes that the DNA of polyethylene glycol induction takes in, is liposome-mediated Conversion, use particle gun to import, electroporation and microinjection etc..In a specific embodiment of the invention, the present invention makes With the transformation technology based on agrobacterium (reference can be made to Horsch RB etc. (1985) Science 225:1229；White FF, Vectors for Gene Transfer in Higher Plants, Transgenic Plants, volume 1, Engineering and Utilization, Academic Press, 1993, pp.15-38；The .Techniques such as Jenes B For Gene Transfer, Transgenic Plants, volume 1, Engineering and Utilization, Academic Press, 1993, pp.128-143, etc.).Agrobacterium bacterial strain (such as Agrobacterium tumdfaciens or hair root soil bar Bacterium) it include plasmid (Ti or Ri plasmid) and T-DNA element, the plasmid and element are transferred to plant after with Agrobacterium transfection Object, and T-DNA is integrated into the genome of plant cell.T-DNA can be located on Ri- plasmid or Ti- plasmid, or independently wrap It is contained in so-called binary vector.Agrobacterium-mediated method for transformation is described in for example.Agrobacterium-mediated conversion is most It is suitble to dicotyledon, but is also suitble to monocotyledon.Agrobacterium is described in for example the conversion of plant.Conversion can be led Cause instantaneous or stable conversion and expression.Although nucleotide sequence of the invention, which can be inserted into, falls into appointing in these broad varieties In what plant and plant cell, but it is particularly suitable for crop plants cell.
Compared with prior art, the invention has the following beneficial effects:
(1) the present invention provides a rice paddy seed size control gene, the mutation of the gene can make crop (such as rice) Seed become larger, to increase crop yield, provide new genetic resources for the SOYBEAN IN HIGH-YIELD BREEDING of rice.
(2) DLZ gene provided by the invention can be used as control crop kernel size, the base to improve the yield and quality Cause facilitates the new rice variety for selecting high-quality character applied to the improvement of crop varieties.Meanwhile DLZ gene can also be used in Molecular marking technique is the production applications services such as rice big grain SOYBEAN IN HIGH-YIELD BREEDING.
(3) DLZ gene provided by the invention has homologous gene in numerous plants such as corn, sorghum, and DLZ gene is not only It can be used for rice, it can also be used to the rearing new variety of other plants.
Detailed description of the invention
Fig. 1 is the plant forms of wild type Huang Huazhan (HHZ) and big particle mutant (dlz), bar=20cm.
Fig. 2 is the seed (A) and the grain of rice (B) form of Huang Huazhan (HHZ) and big particle mutant (dlz), bar=1cm.
Fig. 3 is that big particle mutant (dlz) with Huang Huazhan (HHZ) hybridizes F2For the mass of 1000 kernel of segregating population.
Fig. 4 is the mutational site that SIMM method positions big particle mutant (dlz), wherein red triangle indicates mutational site institute In position, black letters indicate the base of wild type, and red letters indicate the base of mutant.
Fig. 5 is that big particle mutant (dlz) with Huang Huazhan (HHZ) hybridizes F2For the weight between three candidate genes in segregating population Group single plant and part non-recombinant single plant grain is wide and mass of 1000 kernel, wherein W indicates wild type, and H indicates that heterozygous mutant, M indicate pure Close saltant type.
Fig. 6 is expression quantity of the DLZ gene in wild type Huang Huazhan different tissues organ.DAP1 is indicated after fertilization 1 day.
Fig. 7 is that the DLZ gene for knocking out and spending 11 (ZH11) and long-grained nonglutinous rice Huang Huazhan (HHZ) in japonica rice is pinpointed using CRISPR technology The sequence variations (A) and corresponding transgenic plant seed size (B) of generation, wherein red letters indicate the base of insertion, red Color "-" indicates the base of missing；Z serial number indicates the transgenic plant that 11 backgrounds are spent in japonica rice, and H serial number indicates yellow China Account for the transgenic plant of background.
Fig. 8 is the various important agronomy of wild type Huang Huazhan (W), Heterozygous mutants (H) and dlz Mutants homozygous (M) Shape phenotype and single plant yield compare, and wherein a, b, c difference alphabet registration have significant difference, same word matrix according to through t test Show that difference is not significant.
Fig. 9 is the DLZ base that the DLZ that 11 are spent in japonica rice is carried out to the transgenic plant of gene silencing generation using RNAi technology Because of expression and its wide phenotype of grain, wherein gray bars indicate transgene negative plant.
Figure 10 is to utilize the transgenic plant for spending 11 DLZ gene promoter to generate in CRISPR technical editor japonica rice DLZ gene expression dose, promoter fragment deletion condition and the wide phenotype of grain, wherein gray bars indicate expression quantity variation and grain The wide unconspicuous single plant of character mutation.
Figure 11 is the tetraploid rice of the homologous DLZ albumen of different plants, and wherein Brachypodium di indicates that two fringes are short Handle grass, Hordeum vulgare indicate barley, and Oryza sativa indicates that rice, Oryza brachyant indicate wild rice, Panicum hallii indicates that broomcorn millet, Setaria italica indicate millet, and Zea mays indicates corn, Sorghum bicolor Indicate sorghum.
Specific embodiment
It elaborates below to the embodiment of the present invention, the present embodiment carries out under the premise of the technical scheme of the present invention Implement, the detailed implementation method and specific operation process are given, but protection scope of the present invention is not limited to following implementation Example.
The screening of embodiment 1, rice big grain mutant (dlz)
Using the EMS aqueous solution soaking long-grained nonglutinous rice Huang Huazhan seed (M for containing 0.7% mass concentration0), mutagenic treatment 12 hours, By M0For the solid rear mixed receipts of seed plant, mutant library (M is obtained1).From M1It is used to sieve in seed maturity for the plant of seed Choosing, by Phenotypic Observation, the plant (Fig. 1, Fig. 2A) that acquisition plant development is normal, seed obviously becomes larger.Mutant seeds become larger Increase mainly wide due to grain, and grain-filling degree is unaffected after the increase of glume volume, therefore the width of the grain of rice is also shown It writes and increases (Fig. 2 B), therefore mass of 1000 kernel increases.
The genetic analysis of embodiment 2, rice big grain mutant (dlz)
Dlz mutant is accounted for wild type Huang China and is hybridized, F is obtained1For cenospecies, then take F1The seed of plant plants F2Point Peel off body, and sowing after seed complete ripeness is completely dried, and carries out the measurement of mass of 1000 kernel.The average mass of 1000 kernel of wild type Huang Huazhan is 21 grams or so, and F2The species test of group the result shows that, the single plant of big grain occupies the minority (Fig. 3) in group.Since mass of 1000 kernel is several Character is measured, size is in continuously distributed in group, and with 23 grams for separation, mass of 1000 kernel is set to big kernel Characters greater than 23 grams, It is set to normal character less than or equal to 23 grams, then F2- 1 and F2The normal character of -2 Liang Ge groups and the segregation ratio of big kernel Characters Meet 3:1 (χ2=1.44 < χ2 (0.05, df=1)=3.84) the big kernel Characters for, showing mutant are mutated by a recessive nuclear gene It generates.
The clone of embodiment 3, rice big grain mutant DLZ gene
The gene cloning of mutant takes SIMM method, i.e., constructs F using the raw parents of mutant and open country2Dai Qun Body, the method for carrying out the assignment of genes gene mapping by resurveying sequence.Specifically, dlz mutant is accounted for wild type Huang China and is hybridized, from F2Group The middle mutant plants for choosing 30 extreme phenotypes (25 grams of mass of 1000 kernel or more) extract the genomic DNA and equivalent of blade respectively Mixing builds library standard process according to the sequencing of Illumina Hiseq2000 microarray dataset and carries out building library, and resurveyed by PE101 Sequence.Sequencing data application SIMM method analyzing and positioning mutational site, finally navigates to one of No. 12 end of chromosome of rice The section 450kb is located at LOC_Os12g41220, LOC_Os12g41820 and LOC_ including four single base mutations The code area of Os12g41910 gene, and mutation (Fig. 4) has occurred there are two adjacent base in LOC_Os12g41820 gene.
Separately take three F1The seed of single plant plants F2About 600 to 800 single plants are planted by group, each group, amount to plantation 2100 plants.The genotype in three mutational sites of above-mentioned each single plant is identified using HRM method, and it is single to be screened out from it 80 recombinations Strain, examine recombination single plant and non-recombinant single plant grain is wide and mass of 1000 kernel.As a result, it has been found that in non-recombinant single plant, heterozygous Mass of 1000 kernel shows that dlz gene has semidominant effect: as LOC_Os12g41820 and LOC_ between wild type and saltant type Mass of 1000 kernel when two sites Os12g41910 are all heterozygous, between the wild type and saltant type in the site LOC_Os12g41220 And indifference；When two sites LOC_Os12g41220 and LOC_Os12g41820 are all heterozygous, LOC_Os12g41910 Mass of 1000 kernel between the wild type and saltant type in site also indifference；The only wild type and heterozygosis in the site LOC_Os12g41820 Mass of 1000 kernel significant difference between type, saltant type, and the difference trend is identical as non-recombinant single plant (Fig. 5).Experimental result table Bright LOC_Os12g41820 is DLZ gene.
In wild type Huang Huazhan (long-grained nonglutinous rice), the genomic DNA overall length 6146bp of DLZ gene, sequence such as SEQ ID NO:1 It is shown.The gene has 9 exons, respectively in the 831st to 1151, the 1502nd to 1541, the 2298th of SEQ ID NO:1 To 2427, the 2569th to 3056, the 3148th to 3228, the 3364th to 3462, the 3643rd to 3756, the 3840th to 3911, the 4498th to 4566 nucleotide.Gene coding region (CDS) the overall length 1635bp, sequence such as SEQ ID NO:2 institute Show, CDS encodes 1 protein containing 544 amino acid, and amino acid sequence is as shown in SEQ ID NO:3.In the present invention In provided rice big grain mutant (dlz), the mutation of two bases occurs on the 4th exon of DLZ gene, specially SEQ ID NO:2 the 961st, 962,963 bit bases from AAA sport TTA (see SEQ ID NO:4), so as to cause SEQ ID The 321st amino acids of NO:3 sport leucine (Leu) by lysine (Lys) (see SEQ ID NO:5).By japonica rice variety day This fine (Typical Japonica Genome donor) finds that the nucleotide sequence of DLZ gene is (see SEQ ID compared with rice variety Huang China accounts for NO:20) difference is primarily present in noncoding region, and code area specially exists only on the 6th exon there are a SNP site On the 1170th bit base position of SEQ ID NO:2, Huang Huazhan is G, and OryzasativaLcv.Nipponbare is A, which is located at SEQ ID NO:3 The 390th Lys codon third position (see SEQ ID NO:21), but do not change encoded amino acid (see SEQ ID NO:3), illustrate that DLZ gene difference between japonica rice and long-grained nonglutinous rice is smaller.
The expression pattern analysis of embodiment 4, DLZ gene in each histoorgan of rice
According to the cDNA sequence design primer of DLZ gene, upstream primer 820qF:5 '- AGTCCAGGCGTATACAGTGC-3 ' (SEQ ID NO:6), downstream primer 820qR:5 '-TCAGAGCAATCCTGACACCA- 3 ' (SEQ ID NO:7).Simultaneously using rice Ubiquitin gene as internal reference design primer, upstream primer UBqF:5 '- CAACCAGCTGAGGCCCAAGAA-3 ' (SEQ ID NO:8), downstream primer UBqR:5 '- CCAGGGAGATAACAACGGAAGC-3 ' (SEQ ID NO:9).Extract respectively the root of wild type Huang Huazhan, stem, leaf, tiller bud, Lemma, glumelle, gynoecium, anther, different length the total serum IgE of tissues such as 1 to 7 day seed of young fringe, after fertilization and synthesize cDNA Template, the method for taking real-time fluorescence quantitative PCR analyze the expression of DLZ gene.As a result as shown in fig. 6, the gene is in institute There is higher expression in each histoorgan of detection, expression difference is little.
Embodiment 5, fixed point knock out the DLZ gene of long-grained nonglutinous rice and japonica rice
The target site sequence Target 1 of the 1st CRISPR direct mutagenesis is selected on the 1st exon of DLZ gene: 5 '-CCTTCCTGGTCGACCGGCATTGG-3 ' (SEQ ID NO:10) select the 2nd CRISPR fixed on the 3rd exon Target site sequence Target the 2:5 '-GCTGTTCGTGTTGGATCGCTTGG-3 ' (SEQ ID NO:11) of point mutagenesis.Anamorphic zone Adapter-primer U3-Target1-linkerF:5 '-ggcACCTTCCTGGTCGACCGGCAT-3 ' (the SEQ ID of toughness end NO:12), U3-Target1-linkerR:5 '-aaacATGCCGGTCGACCAGGAAGG-3 ' (SEQ ID NO:13), U6a- Target2-linkerF:5 '-gccGCTGTTCGTGTTGGATCGCT-3 ' (SEQ ID NO:14), U6a-Target2- LinkerR:5 '-aaacAGCGATCCAACACGAACAG-3 ' (SEQ ID NO:15).Two butt joint primers are used into ddH respectively2O 10 μM of working solutions are dissolved into, F primer and R primer respectively take 10 μ L to be added to 80 μ L ddH2Mixed diluting is to 1 μM in O, at 90 DEG C 30s is managed, the cooling completion annealing of room temperature is moved to.It is connected into respectively with T4DNA ligase through BsaI linearization for enzyme restriction again PYLsgRNA-U3 or pYLsgRNA-U6a plasmid obtains micro U3::Target1-gRNA and U6a::Target2-gRNA table Up to box.Using above-mentioned expression cassette as template, by two-wheeled PCR amplification and BsaI restriction enzyme site is added, is connected into through BsaI digestion line Property the whole carrier of pYLCRISPR/Cas9-MH (B), finally obtain Pubi::Cas9-U3::Target1-gRNA-U6a:: Target2-gRNA expression cassette.Pcr template combines as shown in table 1 with primer, and universal primer sequence is as shown in table 2, positive colony Identification use respectively SP-L and U3-Target1-linkerR, U3-Target1-linkerF and U6a-Target2-linkerR, Three pairs of primers such as U6a-Target2-linkerF and SP-R carry out bacterium solution PCR detection, and SP-L primer is used for the survey of U3-Target1 Sequence, SP-R are used for the sequencing of U6a-Target2.
Pubi::Cas9-U3::Target1-gRNA-U6a::Target2-gRNA expression cassette is passed through into mediated by agriculture bacillus Rice transformation method is transferred to respectively in the callus for spending 11 and Huang Huazhan Seed inducement to generate in japonica rice variety, by tide The differentiation and regeneration of mycin screening and resistant calli obtains transgenic plant.The base of above-mentioned plant leaf is extracted using CTAB method Because of a group DNA, and transgenic plant T-DNA is with primer pair U3-Target1-linkerF and U6a-Target2-linkerR and is inserted Enter positive detection analysis, the band of the amplifiable 600bp or so out of positive plant.The genomic DNA of positive plant is used into primer again To 820-Target1-F:5 '-CTGACATGGGCGCACATG-3 ' (SEQ ID NO:16) and 820-Target2-R:5 '- CCTCGTATCCTTGCAGCAACTT-3 ' (SEQ ID NO:17) amplifies the region of DNA of the about 1.84kb comprising two target sites Section recycles primer pair 820-T1-SEQ:5 '-GGTGATGCACACGAAGAAGC-3 ' (SEQ ID NO:18) and 820-T2- SEQ:5 '-TCCCTAGTTGCATCCGTTTG-3 ' (SEQ ID NO:19) is respectively sequenced Target1 and Target2.It surveys Sequence result is as shown in Figure 7 A, different base insertion or missing all has occurred on two target sites, and then change DLZ gene Reading frame, and two chromosomes are all mutated, therefore show that the positive plant generated is that DLZ gene function is lost Mutant, it is wide also than spending 11 or Huang Huazhan to greatly increase (Fig. 7 B) in wild type, it was demonstrated that fixed point knocks out japonica rice and long-grained nonglutinous rice DLZ gene leads to dramatically increasing for seed size.
Embodiment 6, dlz mutated gene have significant yield increasing effect under heterozygous state
As shown in figure 8, wide become larger of the grain of big particle mutant (dlz) increases with mass of 1000 kernel, plant height and wild type Huang Hua Zhanxiang Declining than, but stalk is more sturdy, spike number is reduced, and spike length shortens, and grains per panicle also substantially reduces, in summary feature, The decline of Mutants homozygous yield.Big grain mutated gene under heterozygous state plant height, spike number, spike length and grains per panicle all with it is wild Type Huang China accounts for similar, is not significantly different, and the grain of heterozygosis strain it is wide be above wild type Huang Huazhan with mass of 1000 kernel and lower than homozygosis Mutant, stalk is also more sturdy than wild type material, and single plant yield is dramatically increased than wild type Huang Huazhan, shows big grain mutated gene There are the potentiality for increasing rice yield under heterozygous state, can be applied to Cross-incompatibility groups.
Embodiment 7, inhibiting the expression of DLZ gene, can to increase rice grain wide
Based on DLZ gene, primer is separately designed
RNAi-SpeI-1F:
5 '-CACGTGGACCACTAGTATGTGGATGTATGGCTATTTCTGGA-3 ' (SEQ ID NO:22),
RNAi-SpeI-1R:
5 '-GTCCGTACCAACTAGTTCGTATCCTTGCAGCAACTTATTCA-3 ' (SEQ ID NO:23),
RNAi-BamHI-2F:
5 '-TGAATTCGCTGGATCCTCGTATCCTTGCAGCAACTTATTCA-3 ' (SEQ ID NO:24),
RNAi-BamHI-2R:
5 '-GTCGACTGGAGGATCCATGTGGATGTATGGCTATTTCTGGA-3 ' (SEQ ID NO:25).
Using the cDNA of DLZ gene as template, the segment of 214bp is amplified respectively, passes through forward and reverse point of In-Fusion method It is not connected on the restriction enzyme site SpeI and BamHI of expression vector Ubi-intron, is built into RNAi carrier.The carrier is passed through The rice transformation method of mediated by agriculture bacillus, which is transferred in japonica rice, to be spent in 11, to resulting T0Expression quantity point is carried out for transgenic plant Analysis.As a result as shown in figure 9, the endogenous DLZ gene expression dose of transgenic positive plant receives significant inhibition, and grain is wide Also it significantly increases, shows that the expression for inhibiting DLZ gene can increase that rice grain is wide, and the table of the wide size and DLZ gene of grain It is positively correlated up to level.
Embodiment 8 realizes the finely regulating to DLZ gene expression dose by promoter editor
It is arranged 8 in the promoter region (SEQ ID NO:26) of DLZ gene translation initiation site ATG upstream 2087bp CRISPR target site, respectively TP1:5 '-TTTGACAGCTTCCTGATCTT-3 ' (SEQ ID NO:27), TP2:5 '- CAAGTAAGATGCCAAGAATG-3 ' (SEQ ID NO:28), TP3:5 '-TTGTCAACGGGAGAACAAC-3 ' (SEQ ID NO:29), TP4:5 '-TAGGATATTTGAGCTACGG-3 ' (SEQ ID NO:30), TP5:5 '- TAGAAAGAAGTCTGGAGCA-3 ' (SEQ ID NO:31), TP6:5 '-AACGCCAGCTTGAGGGCAG-3 ' (SEQ ID NO: 32), TP7:5 '-TTCTCGTCGTTTCTTGCGTG-3 ' (SEQ ID NO:33), TP8:5 '-GTGTGTGGGTTGACCGAAT- 3 ' (SEQ ID NO:34).It is respectively synthesized the adapter-primer with cohesive end, as shown in table 3, according to the method structure of embodiment 5 Build Pubi::Cas9-U3::TP3-gRNA-U3::TP6-gRNA-U6a::TP1-gRNA-U6a:: TP2-gRNA-U6b::TP4- GRNA-U6b::TP5-gRNA-U6c::TP7-gRNA-U6c::TP8-gRNA expression cassette, pcr template are combined with primer such as 1 institute of table Show, universal primer sequence is as shown in table 2, is transferred in japonica rice and is spent in 11 finally by the rice transformation method of mediated by agriculture bacillus. With primer 820Pro-2087bp-F:5 '-GGAAAGGAAGAAAAGGCTAATATGCTCATC-3 ' (SEQ ID NO:51) and 820Pro-2087bp-R:5 '-ATGTCAGGATGTGCTTCTGGGACAC-3 ' (SEQ ID NO:52) expands T0It is planted for transgenosis The DLZ gene 2087bp promoter region of strain, electrophoresis result is as shown in Figure 10, and the amplified production of many transgenic plants is significantly less than 2087bp shows that different degrees of fragment deletion has occurred in the gene promoter area DLZ in these plant.Amplified production is connected into PEASY-Blunt carrier is sequenced, the promoter sequencing result of plant part such as SEQ ID NO with M13F and M13R universal primer: 53 (8-5 single plants), shown in SEQ ID NO:54 (8-9 single plant).Wherein 8-5 single plant has occurred between TP2 and TP5 The missing of 750bp or so；8-9 single plant has occurred the missing of large fragment between TP2 and TP8, and be inverted insert TP5 and The segment of 350bp or so between TP6.These are the result shows that promoter editor can produce sequence variations abundant.
The DLZ gene expression dose variation of transgenic plant is further detected, the results are shown in Figure 10, a small number of plant DLZ gene expression dose is suppressed to the effect of RNAi gene silencing quite (such as 8-22 single plant), the DLZ of most of plant Gene expression dose has dropped half or so.The wide phenotype of grain of transgenic plant is investigated, discovery DLZ gene expression dose is suppressed Degree is higher, and grain is wide bigger, the plant of expression drop by half or so, and wide size is between wild type and gene knockout It is between plant, i.e., suitable with the phenotype of dlz Heterozygous mutants, show to may be implemented by promoter editing technique to DLZ gene The finely regulating of expression realizes the effect of volume increase.
Homologous gene analysis in embodiment 9, Different Crop
BLASTP search is carried out in the protein sequence input ncbi database that DLZ gene is encoded, it is short to obtain two fringes Handle grass (Brachypodium distachyon) (SEQ ID NO:55), barley (Hordeum vulgare) (SEQ ID NO: 56), wild rice (Oryza brachyantha) (SEQ ID NO:57), broomcorn millet (Panicum hallii) (SEQ ID NO:58), Millet (Setaria italica) (SEQ ID NO:59), corn (Zea mays) (SEQ ID NO:60), sorghum (Sorghum Bicolor) the homologous protein predicted in the crop genes group such as (SEQ ID NO:61).By rice DLZ albumen and these homologous eggs The white amino acid sequence input website ClustalW2 (https: //www.ebi.ac.uk/Tools/msa/clustalw2/) into Row sequence alignment, the homologous protein as the result is shown from different plants all have closely similar conserved sequence, each other together Source property is very high (Figure 11), shows that DLZ albumen function in the seed development of different plants is conservative, plays very important effect.
The DLZ gene of above-mentioned 7 crops is mutated respectively using CRISPR/Cas9 technology, to the transgenosis of acquisition Positive plant carries out the character observation of seed grain size, and transgenic positive plant is that DLZ gene function is lost as the result is shown Mutant, DLZ gene can not normal expression, the wild type of the wide also more corresponding crop of seed grain greatly increases, and it is big that grain is presented Fringe multiple characters, it was demonstrated that the homologous gene of rite-directed mutagenesis different plant species DLZ leads to dramatically increasing for seed size.
The templa-primer combination of gRNA expression cassette is expanded in 1 CRISPR vector construction of table
The universal primer sequence of gRNA expression cassette is expanded in 2 CRISPR vector construction of table
The target spot specific primer sequences of gRNA expression cassette are expanded in 3 promoter editor's CRISPR vector construction of table
Sequence table
<110>Shenzhen Crop Molecular Design Breeding Institute
Unnamed Xingwang System Crop Design Front Laboratory (Beijing) Co., Ltd.
<120>regulate and control gene and its application of size of plant seed
<150> 201910051525.0
<151> 2019-01-21
<160> 61
<170> SIPOSequenceListing 1.0
<210> 1
<211> 6146
<212> DNA
<213> Oryza sativa
<400> 1
cactgcccaa ttgcccatgc tccagacttc tttctactcc tacattccac atatctccat 60
ggacagtaac tcctcccaag ctaccacttc aaccctaatc ccctctctct ctcttccgca 120
gaggtagagt gagagagatg gtcagatagc tagattgata tccctctctc tctctcacag 180
acatctcttt ttgcaagatc tcttcttgtt catcatcttc ttcttttttt ctcccccttt 240
tgcttcacca atccatcttt tgtcacgaga tgtgaccgag ctgaagctag tagtagtgga 300
gcagcgaaag caagtacgcc aagaaaaaaa aaaggaagaa gaaagaagaa agaaagaaag 360
aaaaaaacgc cagcttgagg gcagagggca aaagcggcga cgaggagcag tggccaaagc 420
tcagattctt cccgtgggct atttttacca cccgcatccc ctctctttga gccccttggc 480
cgattcattc accgacgcaa agatccaacc cctcttcagg tgtcggcaga tgccgccttt 540
gtgaggtttc cagtgggggg atttctcgtc gtttcttgcg tgcggttgcg ttcttgatcc 600
agtgagcgca cggatatatc cgccctggtt tagtagagag agagagagag agagagagag 660
agagagagag agagagagag agagggggtt cttgattgag ttccaagtgt tggattgggt 720
tcttggagct gttggattgg gtttttttgg gagagagatg ggggtttgga ggtgtgtggg 780
ttgaccgaat tggatcaaga ttattgcggg aggggggggg gggggttgca atggcggatt 840
tggggctgtg gaagcaaggg tggaggtggg tggtgtccca gaagcacatc ctgacatggg 900
cgcacatggc ggcgagcggc ggcaccgaga ggctggcctt cctggtcgac cggcattggc 960
ccgccgtgtc ccgggcctgc gtgagctccg gccgcctcgc gctcgccgcg ctgcggcaat 1020
ggcgcggctg cgcggcgcgc gggatcctgg agatggctag cctgggccct gcgtccgtgt 1080
tcgtcatcct ctggagcttc ttcgtgtgca tcacctcgcc ggcgtgcgcc ctctacgcgc 1140
tcctgggcat ggtacggcat gcaagtcttg cttgctttgc gctttcgcct tgatgatgta 1200
gtggattatg gataacgatt tgtgcgcgtt ctaaatcttg tcatgtgctc gtctttcttt 1260
ttttcttctt tttatcaagg gtggattgca tgttaggtta cctttctttt cgaaaagtat 1320
agttaaagtg gtaattggtg gtacaaaagt agtatgtcat tacactttca tgagattgat 1380
cagtttgatg tgtttctaga ttcatttatg ctttagttat tgcaagttta tactacactt 1440
cagtaattca cacgtgctgt ttctagatgt tattttggaa ccgttcacag tattttaagc 1500
atcatttgca ttagaaagtt ttatctagtt tgtcttgcta gaggaaggag cacatggaaa 1560
ctaacacttg catatttagg gataagcact actggttcta ttcctatttt gtgtatgtta 1620
gctaatgtgt ttcttgctga gtggttcagt ttcaggttca ttagcagatt atcttattgg 1680
ttgatttatg tcaaaatact taaggtcaat tcgtagtttg cacagtgtac ttcaataaca 1740
tgaatgcaac tcgtttcttt gttcagcctg atatttatga aaaatcttat aatgtgatac 1800
tgtgtttaat atgtatgaac ctgtctagag aattactagc tagtgaaatt ctacttgttt 1860
catttcacac aaagtcaact atgggtagac tggttcatga ccatttattt aggctctggt 1920
acacctgtaa ctactgctgt agttgactat atgacttact tatgctggtt ttctactgtg 1980
gtatgagttt ctccctttgg gataccacct gtgttcagtg gaatgtcagg tagtatctga 2040
ccttttcagc tagattgcac tggataaatt atactgaaat aagcaatagg aatgaattcc 2100
aggactatgt ctcctcttgt tctctccttg caattcctct tattgcaatg cagactgaac 2160
cacttgtttt tactgtcatc tggcatactt gttcagttag taacttctac ttgcgagtaa 2220
ggatgcaaga tttcccatgt aagatggata ctataaatat cattttgtct aattgcttaa 2280
tacctttctt ttttcaggga gctgctgggg cagtcattca ttacatgggc tatacgcctg 2340
gtcttttcat tgtaggatta tttggaatat tgattatgtg gatgtatggc tatttctgga 2400
ttacaggaat gcttctgatt gctggaggtt tgttttatct taatatttaa gtctgttcat 2460
aatgataatt ttgtgttttt gtttgtcaaa tccataaatt tttcttcctc cctagttgca 2520
tccgtttgat tcttttgacc taaaggagga tcctctctgg taatgcaggc tgtatgtgct 2580
ctttgaaaca tgcacgattt gtgatacctg tgttggctat gtatgctgtt tattgtgtgg 2640
ctgttcgtgt tggatcgctt ggtgtcttct tgacattgaa tctttctttc ctgacaaatg 2700
atcttctgaa taagttgctg caaggatacg agggaagcac agaagaaaga cagtttgaag 2760
agccaaaaca ttctgatcct gtcatggatg agttctatcg cagttgtgaa tttccctctg 2820
ctcctgatag tgaacctgag actgtttctt ctgcaaagcc cttttgctca acacccgtcc 2880
aggatgtgtt gcatgtacag aaagaggcat ctcctagcaa agtagtgaaa tcggattctg 2940
tttcattgga tgagatgaag aggatcatgg atggtttgac ccattatgaa gttttgggta 3000
ttcctcggaa tagaagtatt gatcaaaaga ttctgaaaaa ggagtaccac agaatggtaa 3060
taaaccacgg ccttctatac aagggaaaat gagaaattca tgttacaatt acttcatttt 3120
catggtacgt atgctttatt tgtctaggtc ctgcttgtac atcctgataa aaatatggga 3180
aatccactgg cctgtgaatc attcaaaaag cttcagtcag cttatgaggt aaactacaat 3240
ggaagtttat gtcttttctc ttccttgatt atattacagt taaatctggt tgaatatctg 3300
ctcttgatac caaccatggc ttctatacct ggataaaggg taatcattgt agttatgctg 3360
caggtactct cagatttcac aaagaaaaac acttacgacg accaactgag gaaagaagaa 3420
tcacgtaaaa tgactcagag atcacgtgtt gtctctcaac aggtgggttc tagttttcac 3480
aaatttagaa tccacatggt tggattattt ctttaacata tcttatcaat tatccaagca 3540
tacgaatgca gtttattcat gctctcatgt ccttgaccta ctgacctact tgctgttttc 3600
ctttatgggg cccatttgta atttgataaa ctcatcttgc agactggggt agagtttctc 3660
tccgaagagt ccaggcgtat acagtgcaca aagtgtggta attttcatct gtggatatgt 3720
accaagaaaa gcaaagcaaa agcaagatgg tgtcaggttt ggaggccaga attttttttt 3780
caggtacttt taatcgagag tgttcttaca gctaattttg tgggaaccat gtactgtagg 3840
attgctctga ttttcatcca gctaaggatg gagatggatg ggtggaaaat aaattttcgt 3900
catccttcaa ggtaatgttt tataagcaca tcatatgaag agttcacttt attttactta 3960
atgcttgcct tctacagtac tcatagacag agatctagtg tcaatacaat tttaactact 4020
agaaaatgga aattgagtac atattgattt cgaacaaatg gagaatgagg ttttatgaat 4080
ggaagcacaa tgttctgaat gttttgatac aaaattaccg ggcgctgttt cccactgtca 4140
agcttcagtt cctagtactt gttattgcct gaagttagtc atgtgtgttc cgagaccaac 4200
tttggacttg agcaagctca gttttagctg tgtcaagctg atgatctttt atcttctaat 4260
tgtattccac ctaaagaaag catctcattc caagtgttag gtacagtcat tttgttcatt 4320
ccataagcaa cttattctga ctataaggtg agattcagaa attactcagc ttaaaaatgt 4380
gcacacattt tgtagtttcc aactataatg tgtaaattct tcacttctct ttattgaact 4440
ataatgtgta aattcttcag tactctttgt tgaatcaaag tgcattgttc acttcaggaa 4500
atacctcgag cttttgtttg tgcggagagt aaggtatttg atgtgtctga atgggctact 4560
tgccaggtga gtgtctgacg atgttttata tgtttgattt aagttgacat gtatgtgcat 4620
ttgcagcagt gatttttgga tgtctcaatt gatttgatgt catctccata tgcatatttt 4680
tatactcggt tctctgctgt tttcgatgtc ttaactgact atagatatgc ctttggtcaa 4740
ttgactttgt tcagttttgt atttgatgca tattcaaacg tccagattga ctgttttact 4800
ttaaaaattg tttcagttgg taaatgaaaa tttgcttact tcattggaga taggataatt 4860
catgcatgcc atagcccata gccttatttt tctgtgtcaa gtttgtcatg gctataataa 4920
acacaacata ttaatcgcac ccgcatgtca tcccgactga tatctcaatt attgacatac 4980
ctatctaaga gaagagccaa caatgatgaa agtaaaggac taatttggct gtgcaaaatt 5040
ggaccaaaag tttattttac atttcatact tgcttcattc aacataaaca tcaaaatctg 5100
gtacgccaat tttctggcga tacatacagg gtcttgtgat aggttcattt gcatacatta 5160
aaaatgggag cctttctaac tctgttttct ttgcttgatt gtctagggca tggagtgcaa 5220
acctaacact cacggcccat cttttatggt aaacatggtt ggcgcagata ggatgtctca 5280
gagatcctac agttctcgct atccctttag tttgaatgct gagatgatcc ctgaagatga 5340
atttgagcta tggcttcaac aagcattggc atcaggtgtc ttctctgaca gcccgaaacg 5400
caggaaaagc tggagcccct tcaaactacc tcaaaaaggg ataaaaagtt ggcggcgatc 5460
ctcataaggg catagcatta aacagcatgg atctcacctg agtacaacac tgaaaaaggc 5520
tatactcttt gtgaatgtaa atagactgac caacaatttg cctggatgag caactaattt 5580
tgtccaaaaa gagacactga aacaaggggg gtaaaaggaa caaacgctta agacatgact 5640
gcaatgaatc tgactgttga aattagtgtt ctctgcaatg agatcccgcg agttttatcc 5700
gaaaaggtca gatactggga tggcgtgtca ttcatcagtt catcctaaag ctcggaaggg 5760
tatctctgta gcatgttaac ttcagtagtt ttaggggatc ggcatctgag agaatttcaa 5820
aacttcatac ctggttgcca gcataagttc tgcaggtgtt gaaaagttgt tgatcagagt 5880
agcaatttaa ggtctgatgt ttctggggaa cagtaggaga gaaaaaaatg acaaaaaaaa 5940
gagagagttg gttgtaaata catgaaaagt tttcatcaga aattagtatt gtaacattgt 6000
acactgtgat tacatcctgt gcaatactcc cataattcag atctgtgttg taatacacta 6060
catacatcct acaattttct ggtgataata gagatctaat tctcacctat tatcgttatt 6120
tatggttagt cagttactgc tctgta 6146
<210> 2
<211> 1635
<212> DNA
<213> Oryza sativa
<400> 2
atggcggatt tggggctgtg gaagcaaggg tggaggtggg tggtgtccca gaagcacatc 60
ctgacatggg cgcacatggc ggcgagcggc ggcaccgaga ggctggcctt cctggtcgac 120
cggcattggc ccgccgtgtc ccgggcctgc gtgagctccg gccgcctcgc gctcgccgcg 180
ctgcggcaat ggcgcggctg cgcggcgcgc gggatcctgg agatggctag cctgggccct 240
gcgtccgtgt tcgtcatcct ctggagcttc ttcgtgtgca tcacctcgcc ggcgtgcgcc 300
ctctacgcgc tcctgggcat gggagctgct ggggcagtca ttcattacat gggctatacg 360
cctggtcttt tcattgtagg attatttgga atattgatta tgtggatgta tggctatttc 420
tggattacag gaatgcttct gattgctgga ggctgtatgt gctctttgaa acatgcacga 480
tttgtgatac ctgtgttggc tatgtatgct gtttattgtg tggctgttcg tgttggatcg 540
cttggtgtct tcttgacatt gaatctttct ttcctgacaa atgatcttct gaataagttg 600
ctgcaaggat acgagggaag cacagaagaa agacagtttg aagagccaaa acattctgat 660
cctgtcatgg atgagttcta tcgcagttgt gaatttccct ctgctcctga tagtgaacct 720
gagactgttt cttctgcaaa gcccttttgc tcaacacccg tccaggatgt gttgcatgta 780
cagaaagagg catctcctag caaagtagtg aaatcggatt ctgtttcatt ggatgagatg 840
aagaggatca tggatggttt gacccattat gaagttttgg gtattcctcg gaatagaagt 900
attgatcaaa agattctgaa aaaggagtac cacagaatgg tcctgcttgt acatcctgat 960
aaaaatatgg gaaatccact ggcctgtgaa tcattcaaaa agcttcagtc agcttatgag 1020
gtactctcag atttcacaaa gaaaaacact tacgacgacc aactgaggaa agaagaatca 1080
cgtaaaatga ctcagagatc acgtgttgtc tctcaacaga ctggggtaga gtttctctcc 1140
gaagagtcca ggcgtataca gtgcacaaag tgtggtaatt ttcatctgtg gatatgtacc 1200
aagaaaagca aagcaaaagc aagatggtgt caggattgct ctgattttca tccagctaag 1260
gatggagatg gatgggtgga aaataaattt tcgtcatcct tcaaggaaat acctcgagct 1320
tttgtttgtg cggagagtaa ggtatttgat gtgtctgaat gggctacttg ccagggcatg 1380
gagtgcaaac ctaacactca cggcccatct tttatggtaa acatggttgg cgcagatagg 1440
atgtctcaga gatcctacag ttctcgctat ccctttagtt tgaatgctga gatgatccct 1500
gaagatgaat ttgagctatg gcttcaacaa gcattggcat caggtgtctt ctctgacagc 1560
ccgaaacgca ggaaaagctg gagccccttc aaactacctc aaaaagggat aaaaagttgg 1620
cggcgatcct cataa 1635
<210> 3
<211> 544
<212> PRT
<213> Oryza sativa
<400> 3
Met Ala Asp Leu Gly Leu Trp Lys Gln Gly Trp Arg Trp Val Val Ser
1 5 10 15
Gln Lys His Ile Leu Thr Trp Ala His Met Ala Ala Ser Gly Gly Thr
20 25 30
Glu Arg Leu Ala Phe Leu Val Asp Arg His Trp Pro Ala Val Ser Arg
35 40 45
Ala Cys Val Ser Ser Gly Arg Leu Ala Leu Ala Ala Leu Arg Gln Trp
50 55 60
Arg Gly Cys Ala Ala Arg Gly Ile Leu Glu Met Ala Ser Leu Gly Pro
65 70 75 80
Ala Ser Val Phe Val Ile Leu Trp Ser Phe Phe Val Cys Ile Thr Ser
85 90 95
Pro Ala Cys Ala Leu Tyr Ala Leu Leu Gly Met Gly Ala Ala Gly Ala
100 105 110
Val Ile His Tyr Met Gly Tyr Thr Pro Gly Leu Phe Ile Val Gly Leu
115 120 125
Phe Gly Ile Leu Ile Met Trp Met Tyr Gly Tyr Phe Trp Ile Thr Gly
130 135 140
Met Leu Leu Ile Ala Gly Gly Cys Met Cys Ser Leu Lys His Ala Arg
145 150 155 160
Phe Val Ile Pro Val Leu Ala Met Tyr Ala Val Tyr Cys Val Ala Val
165 170 175
Arg Val Gly Ser Leu Gly Val Phe Leu Thr Leu Asn Leu Ser Phe Leu
180 185 190
Thr Asn Asp Leu Leu Asn Lys Leu Leu Gln Gly Tyr Glu Gly Ser Thr
195 200 205
Glu Glu Arg Gln Phe Glu Glu Pro Lys His Ser Asp Pro Val Met Asp
210 215 220
Glu Phe Tyr Arg Ser Cys Glu Phe Pro Ser Ala Pro Asp Ser Glu Pro
225 230 235 240
Glu Thr Val Ser Ser Ala Lys Pro Phe Cys Ser Thr Pro Val Gln Asp
245 250 255
Val Leu His Val Gln Lys Glu Ala Ser Pro Ser Lys Val Val Lys Ser
260 265 270
Asp Ser Val Ser Leu Asp Glu Met Lys Arg Ile Met Asp Gly Leu Thr
275 280 285
His Tyr Glu Val Leu Gly Ile Pro Arg Asn Arg Ser Ile Asp Gln Lys
290 295 300
Ile Leu Lys Lys Glu Tyr His Arg Met Val Leu Leu Val His Pro Asp
305 310 315 320
Lys Asn Met Gly Asn Pro Leu Ala Cys Glu Ser Phe Lys Lys Leu Gln
325 330 335
Ser Ala Tyr Glu Val Leu Ser Asp Phe Thr Lys Lys Asn Thr Tyr Asp
340 345 350
Asp Gln Leu Arg Lys Glu Glu Ser Arg Lys Met Thr Gln Arg Ser Arg
355 360 365
Val Val Ser Gln Gln Thr Gly Val Glu Phe Leu Ser Glu Glu Ser Arg
370 375 380
Arg Ile Gln Cys Thr Lys Cys Gly Asn Phe His Leu Trp Ile Cys Thr
385 390 395 400
Lys Lys Ser Lys Ala Lys Ala Arg Trp Cys Gln Asp Cys Ser Asp Phe
405 410 415
His Pro Ala Lys Asp Gly Asp Gly Trp Val Glu Asn Lys Phe Ser Ser
420 425 430
Ser Phe Lys Glu Ile Pro Arg Ala Phe Val Cys Ala Glu Ser Lys Val
435 440 445
Phe Asp Val Ser Glu Trp Ala Thr Cys Gln Gly Met Glu Cys Lys Pro
450 455 460
Asn Thr His Gly Pro Ser Phe Met Val Asn Met Val Gly Ala Asp Arg
465 470 475 480
Met Ser Gln Arg Ser Tyr Ser Ser Arg Tyr Pro Phe Ser Leu Asn Ala
485 490 495
Glu Met Ile Pro Glu Asp Glu Phe Glu Leu Trp Leu Gln Gln Ala Leu
500 505 510
Ala Ser Gly Val Phe Ser Asp Ser Pro Lys Arg Arg Lys Ser Trp Ser
515 520 525
Pro Phe Lys Leu Pro Gln Lys Gly Ile Lys Ser Trp Arg Arg Ser Ser
530 535 540
<210> 4
<211> 6146
<212> DNA
<213> Oryza sativa
<400> 4
cactgcccaa ttgcccatgc tccagacttc tttctactcc tacattccac atatctccat 60
ggacagtaac tcctcccaag ctaccacttc aaccctaatc ccctctctct ctcttccgca 120
gaggtagagt gagagagatg gtcagatagc tagattgata tccctctctc tctctcacag 180
acatctcttt ttgcaagatc tcttcttgtt catcatcttc ttcttttttt ctcccccttt 240
tgcttcacca atccatcttt tgtcacgaga tgtgaccgag ctgaagctag tagtagtgga 300
gcagcgaaag caagtacgcc aagaaaaaaa aaaggaagaa gaaagaagaa agaaagaaag 360
aaaaaaacgc cagcttgagg gcagagggca aaagcggcga cgaggagcag tggccaaagc 420
tcagattctt cccgtgggct atttttacca cccgcatccc ctctctttga gccccttggc 480
cgattcattc accgacgcaa agatccaacc cctcttcagg tgtcggcaga tgccgccttt 540
gtgaggtttc cagtgggggg atttctcgtc gtttcttgcg tgcggttgcg ttcttgatcc 600
agtgagcgca cggatatatc cgccctggtt tagtagagag agagagagag agagagagag 660
agagagagag agagagagag agagggggtt cttgattgag ttccaagtgt tggattgggt 720
tcttggagct gttggattgg gtttttttgg gagagagatg ggggtttgga ggtgtgtggg 780
ttgaccgaat tggatcaaga ttattgcggg aggggggggg gggggttgca atggcggatt 840
tggggctgtg gaagcaaggg tggaggtggg tggtgtccca gaagcacatc ctgacatggg 900
cgcacatggc ggcgagcggc ggcaccgaga ggctggcctt cctggtcgac cggcattggc 960
ccgccgtgtc ccgggcctgc gtgagctccg gccgcctcgc gctcgccgcg ctgcggcaat 1020
ggcgcggctg cgcggcgcgc gggatcctgg agatggctag cctgggccct gcgtccgtgt 1080
tcgtcatcct ctggagcttc ttcgtgtgca tcacctcgcc ggcgtgcgcc ctctacgcgc 1140
tcctgggcat ggtacggcat gcaagtcttg cttgctttgc gctttcgcct tgatgatgta 1200
gtggattatg gataacgatt tgtgcgcgtt ctaaatcttg tcatgtgctc gtctttcttt 1260
ttttcttctt tttatcaagg gtggattgca tgttaggtta cctttctttt cgaaaagtat 1320
agttaaagtg gtaattggtg gtacaaaagt agtatgtcat tacactttca tgagattgat 1380
cagtttgatg tgtttctaga ttcatttatg ctttagttat tgcaagttta tactacactt 1440
cagtaattca cacgtgctgt ttctagatgt tattttggaa ccgttcacag tattttaagc 1500
atcatttgca ttagaaagtt ttatctagtt tgtcttgcta gaggaaggag cacatggaaa 1560
ctaacacttg catatttagg gataagcact actggttcta ttcctatttt gtgtatgtta 1620
gctaatgtgt ttcttgctga gtggttcagt ttcaggttca ttagcagatt atcttattgg 1680
ttgatttatg tcaaaatact taaggtcaat tcgtagtttg cacagtgtac ttcaataaca 1740
tgaatgcaac tcgtttcttt gttcagcctg atatttatga aaaatcttat aatgtgatac 1800
tgtgtttaat atgtatgaac ctgtctagag aattactagc tagtgaaatt ctacttgttt 1860
catttcacac aaagtcaact atgggtagac tggttcatga ccatttattt aggctctggt 1920
acacctgtaa ctactgctgt agttgactat atgacttact tatgctggtt ttctactgtg 1980
gtatgagttt ctccctttgg gataccacct gtgttcagtg gaatgtcagg tagtatctga 2040
ccttttcagc tagattgcac tggataaatt atactgaaat aagcaatagg aatgaattcc 2100
aggactatgt ctcctcttgt tctctccttg caattcctct tattgcaatg cagactgaac 2160
cacttgtttt tactgtcatc tggcatactt gttcagttag taacttctac ttgcgagtaa 2220
ggatgcaaga tttcccatgt aagatggata ctataaatat cattttgtct aattgcttaa 2280
tacctttctt ttttcaggga gctgctgggg cagtcattca ttacatgggc tatacgcctg 2340
gtcttttcat tgtaggatta tttggaatat tgattatgtg gatgtatggc tatttctgga 2400
ttacaggaat gcttctgatt gctggaggtt tgttttatct taatatttaa gtctgttcat 2460
aatgataatt ttgtgttttt gtttgtcaaa tccataaatt tttcttcctc cctagttgca 2520
tccgtttgat tcttttgacc taaaggagga tcctctctgg taatgcaggc tgtatgtgct 2580
ctttgaaaca tgcacgattt gtgatacctg tgttggctat gtatgctgtt tattgtgtgg 2640
ctgttcgtgt tggatcgctt ggtgtcttct tgacattgaa tctttctttc ctgacaaatg 2700
atcttctgaa taagttgctg caaggatacg agggaagcac agaagaaaga cagtttgaag 2760
agccaaaaca ttctgatcct gtcatggatg agttctatcg cagttgtgaa tttccctctg 2820
ctcctgatag tgaacctgag actgtttctt ctgcaaagcc cttttgctca acacccgtcc 2880
aggatgtgtt gcatgtacag aaagaggcat ctcctagcaa agtagtgaaa tcggattctg 2940
tttcattgga tgagatgaag aggatcatgg atggtttgac ccattatgaa gttttgggta 3000
ttcctcggaa tagaagtatt gatcaaaaga ttctgaaaaa ggagtaccac agaatggtaa 3060
taaaccacgg ccttctatac aagggaaaat gagaaattca tgttacaatt acttcatttt 3120
catggtacgt atgctttatt tgtctaggtc ctgcttgtac atcctgattt aaatatggga 3180
aatccactgg cctgtgaatc attcaaaaag cttcagtcag cttatgaggt aaactacaat 3240
ggaagtttat gtcttttctc ttccttgatt atattacagt taaatctggt tgaatatctg 3300
ctcttgatac caaccatggc ttctatacct ggataaaggg taatcattgt agttatgctg 3360
caggtactct cagatttcac aaagaaaaac acttacgacg accaactgag gaaagaagaa 3420
tcacgtaaaa tgactcagag atcacgtgtt gtctctcaac aggtgggttc tagttttcac 3480
aaatttagaa tccacatggt tggattattt ctttaacata tcttatcaat tatccaagca 3540
tacgaatgca gtttattcat gctctcatgt ccttgaccta ctgacctact tgctgttttc 3600
ctttatgggg cccatttgta atttgataaa ctcatcttgc agactggggt agagtttctc 3660
tccgaagagt ccaggcgtat acagtgcaca aagtgtggta attttcatct gtggatatgt 3720
accaagaaaa gcaaagcaaa agcaagatgg tgtcaggttt ggaggccaga attttttttt 3780
caggtacttt taatcgagag tgttcttaca gctaattttg tgggaaccat gtactgtagg 3840
attgctctga ttttcatcca gctaaggatg gagatggatg ggtggaaaat aaattttcgt 3900
catccttcaa ggtaatgttt tataagcaca tcatatgaag agttcacttt attttactta 3960
atgcttgcct tctacagtac tcatagacag agatctagtg tcaatacaat tttaactact 4020
agaaaatgga aattgagtac atattgattt cgaacaaatg gagaatgagg ttttatgaat 4080
ggaagcacaa tgttctgaat gttttgatac aaaattaccg ggcgctgttt cccactgtca 4140
agcttcagtt cctagtactt gttattgcct gaagttagtc atgtgtgttc cgagaccaac 4200
tttggacttg agcaagctca gttttagctg tgtcaagctg atgatctttt atcttctaat 4260
tgtattccac ctaaagaaag catctcattc caagtgttag gtacagtcat tttgttcatt 4320
ccataagcaa cttattctga ctataaggtg agattcagaa attactcagc ttaaaaatgt 4380
gcacacattt tgtagtttcc aactataatg tgtaaattct tcacttctct ttattgaact 4440
ataatgtgta aattcttcag tactctttgt tgaatcaaag tgcattgttc acttcaggaa 4500
atacctcgag cttttgtttg tgcggagagt aaggtatttg atgtgtctga atgggctact 4560
tgccaggtga gtgtctgacg atgttttata tgtttgattt aagttgacat gtatgtgcat 4620
ttgcagcagt gatttttgga tgtctcaatt gatttgatgt catctccata tgcatatttt 4680
tatactcggt tctctgctgt tttcgatgtc ttaactgact atagatatgc ctttggtcaa 4740
ttgactttgt tcagttttgt atttgatgca tattcaaacg tccagattga ctgttttact 4800
ttaaaaattg tttcagttgg taaatgaaaa tttgcttact tcattggaga taggataatt 4860
catgcatgcc atagcccata gccttatttt tctgtgtcaa gtttgtcatg gctataataa 4920
acacaacata ttaatcgcac ccgcatgtca tcccgactga tatctcaatt attgacatac 4980
ctatctaaga gaagagccaa caatgatgaa agtaaaggac taatttggct gtgcaaaatt 5040
ggaccaaaag tttattttac atttcatact tgcttcattc aacataaaca tcaaaatctg 5100
gtacgccaat tttctggcga tacatacagg gtcttgtgat aggttcattt gcatacatta 5160
aaaatgggag cctttctaac tctgttttct ttgcttgatt gtctagggca tggagtgcaa 5220
acctaacact cacggcccat cttttatggt aaacatggtt ggcgcagata ggatgtctca 5280
gagatcctac agttctcgct atccctttag tttgaatgct gagatgatcc ctgaagatga 5340
atttgagcta tggcttcaac aagcattggc atcaggtgtc ttctctgaca gcccgaaacg 5400
caggaaaagc tggagcccct tcaaactacc tcaaaaaggg ataaaaagtt ggcggcgatc 5460
ctcataaggg catagcatta aacagcatgg atctcacctg agtacaacac tgaaaaaggc 5520
tatactcttt gtgaatgtaa atagactgac caacaatttg cctggatgag caactaattt 5580
tgtccaaaaa gagacactga aacaaggggg gtaaaaggaa caaacgctta agacatgact 5640
gcaatgaatc tgactgttga aattagtgtt ctctgcaatg agatcccgcg agttttatcc 5700
gaaaaggtca gatactggga tggcgtgtca ttcatcagtt catcctaaag ctcggaaggg 5760
tatctctgta gcatgttaac ttcagtagtt ttaggggatc ggcatctgag agaatttcaa 5820
aacttcatac ctggttgcca gcataagttc tgcaggtgtt gaaaagttgt tgatcagagt 5880
agcaatttaa ggtctgatgt ttctggggaa cagtaggaga gaaaaaaatg acaaaaaaaa 5940
gagagagttg gttgtaaata catgaaaagt tttcatcaga aattagtatt gtaacattgt 6000
acactgtgat tacatcctgt gcaatactcc cataattcag atctgtgttg taatacacta 6060
catacatcct acaattttct ggtgataata gagatctaat tctcacctat tatcgttatt 6120
tatggttagt cagttactgc tctgta 6146
<210> 5
<211> 544
<212> PRT
<213> Oryza sativa
<400> 5
Met Ala Asp Leu Gly Leu Trp Lys Gln Gly Trp Arg Trp Val Val Ser
1 5 10 15
Gln Lys His Ile Leu Thr Trp Ala His Met Ala Ala Ser Gly Gly Thr
20 25 30
Glu Arg Leu Ala Phe Leu Val Asp Arg His Trp Pro Ala Val Ser Arg
35 40 45
Ala Cys Val Ser Ser Gly Arg Leu Ala Leu Ala Ala Leu Arg Gln Trp
50 55 60
Arg Gly Cys Ala Ala Arg Gly Ile Leu Glu Met Ala Ser Leu Gly Pro
65 70 75 80
Ala Ser Val Phe Val Ile Leu Trp Ser Phe Phe Val Cys Ile Thr Ser
85 90 95
Pro Ala Cys Ala Leu Tyr Ala Leu Leu Gly Met Gly Ala Ala Gly Ala
100 105 110
Val Ile His Tyr Met Gly Tyr Thr Pro Gly Leu Phe Ile Val Gly Leu
115 120 125
Phe Gly Ile Leu Ile Met Trp Met Tyr Gly Tyr Phe Trp Ile Thr Gly
130 135 140
Met Leu Leu Ile Ala Gly Gly Cys Met Cys Ser Leu Lys His Ala Arg
145 150 155 160
Phe Val Ile Pro Val Leu Ala Met Tyr Ala Val Tyr Cys Val Ala Val
165 170 175
Arg Val Gly Ser Leu Gly Val Phe Leu Thr Leu Asn Leu Ser Phe Leu
180 185 190
Thr Asn Asp Leu Leu Asn Lys Leu Leu Gln Gly Tyr Glu Gly Ser Thr
195 200 205
Glu Glu Arg Gln Phe Glu Glu Pro Lys His Ser Asp Pro Val Met Asp
210 215 220
Glu Phe Tyr Arg Ser Cys Glu Phe Pro Ser Ala Pro Asp Ser Glu Pro
225 230 235 240
Glu Thr Val Ser Ser Ala Lys Pro Phe Cys Ser Thr Pro Val Gln Asp
245 250 255
Val Leu His Val Gln Lys Glu Ala Ser Pro Ser Lys Val Val Lys Ser
260 265 270
Asp Ser Val Ser Leu Asp Glu Met Lys Arg Ile Met Asp Gly Leu Thr
275 280 285
His Tyr Glu Val Leu Gly Ile Pro Arg Asn Arg Ser Ile Asp Gln Lys
290 295 300
Ile Leu Lys Lys Glu Tyr His Arg Met Val Leu Leu Val His Pro Asp
305 310 315 320
Leu Asn Met Gly Asn Pro Leu Ala Cys Glu Ser Phe Lys Lys Leu Gln
325 330 335
Ser Ala Tyr Glu Val Leu Ser Asp Phe Thr Lys Lys Asn Thr Tyr Asp
340 345 350
Asp Gln Leu Arg Lys Glu Glu Ser Arg Lys Met Thr Gln Arg Ser Arg
355 360 365
Val Val Ser Gln Gln Thr Gly Val Glu Phe Leu Ser Glu Glu Ser Arg
370 375 380
Arg Ile Gln Cys Thr Lys Cys Gly Asn Phe His Leu Trp Ile Cys Thr
385 390 395 400
Lys Lys Ser Lys Ala Lys Ala Arg Trp Cys Gln Asp Cys Ser Asp Phe
405 410 415
His Pro Ala Lys Asp Gly Asp Gly Trp Val Glu Asn Lys Phe Ser Ser
420 425 430
Ser Phe Lys Glu Ile Pro Arg Ala Phe Val Cys Ala Glu Ser Lys Val
435 440 445
Phe Asp Val Ser Glu Trp Ala Thr Cys Gln Gly Met Glu Cys Lys Pro
450 455 460
Asn Thr His Gly Pro Ser Phe Met Val Asn Met Val Gly Ala Asp Arg
465 470 475 480
Met Ser Gln Arg Ser Tyr Ser Ser Arg Tyr Pro Phe Ser Leu Asn Ala
485 490 495
Glu Met Ile Pro Glu Asp Glu Phe Glu Leu Trp Leu Gln Gln Ala Leu
500 505 510
Ala Ser Gly Val Phe Ser Asp Ser Pro Lys Arg Arg Lys Ser Trp Ser
515 520 525
Pro Phe Lys Leu Pro Gln Lys Gly Ile Lys Ser Trp Arg Arg Ser Ser
530 535 540
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 6
agtccaggcg tatacagtgc 20
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 7
tcagagcaat cctgacacca 20
<210> 8
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 8
caaccagctg aggcccaaga a 21
<210> 9
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 9
ccagggagat aacaacggaa gc 22
<210> 10
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 10
ccttcctggt cgaccggcat tgg 23
<210> 11
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 11
gctgttcgtg ttggatcgct tgg 23
<210> 12
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 12
ggcaccttcc tggtcgaccg gcat 24
<210> 13
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 13
aaacatgccg gtcgaccagg aagg 24
<210> 14
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 14
gccgctgttc gtgttggatc gct 23
<210> 15
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 15
aaacagcgat ccaacacgaa cag 23
<210> 16
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 16
ctgacatggg cgcacatg 18
<210> 17
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 17
cctcgtatcc ttgcagcaac tt 22
<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 18
ggtgatgcac acgaagaagc 20
<210> 19
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 19
tccctagttg catccgtttg 20
<210> 20
<211> 6118
<212> DNA
<213> Oryza sativa
<400> 20
cactgcccaa ttgcccatgc tccagacttc tttctactcc tacattccac atatctccat 60
ggacagtaac tcctcccaag ctaccacttc aaccctaatc ccctctctct ctcttccgca 120
gaggtagagt gagagagatg gtcagatagc tagattgata tccctctctc tctctcacac 180
acatctcttt ttgcaagatc tcttcttgtt catcatcttc ttcttttttt ctcccccttt 240
tgcttcacca atccatcttt tgtcacgaga tgtggccgag ctgaagctag tagtagtgga 300
gcagcgaaag caagtacgcc aagaaaaaaa aaaggaagaa gaaagaagaa agaaagaaag 360
aaaaaaacgc cagcttgagg gcagagggca aaagcggcga cgaggagcag tggccaaagc 420
tcagattctt cccgtgggct atttttacca cccgcatccc ctctctttga gccccttggc 480
cgattcattc accgacgcaa agatccaacc cctcttcagg tgtcggcaga tgccgccttt 540
gtgaggtttc cagtgggggg atttctcgtc gtttcttgcg tgcggttgcg ttcttgatcc 600
agtgagcgca cggatatatc cgccctggtt tagtagagag agagagagag agagagagag 660
agagagagag aggttcttga ttgagttcca agtgttggat tgggttcttg gagctgttgg 720
attgggtttt tttgggagag agatgggggt ttggaggtgt gtgggttgac cgaattggat 780
caagattatt gcgggagggg gggggggggg ttgcaatggc ggatttgggg ctgtggaagc 840
aagggtggag gtgggtggtg tcccagaagc acatcctgac atgggcgcac atggcggcga 900
gcggcggcac cgagaggctg gccttcctgg tcgaccggca ttggcccgcc gtgtcccggg 960
cctgcgtgag ctccggccgc ctcgcgctcg ccgcgctgcg gcaatggcgc ggctgcgcgg 1020
cgcgcgggat cctggagatg gctagcctgg gccctgcgtc cgtgttcgtc atcctctgga 1080
gcttcttcgt gtgcatcacc tcgccggcgt gcgccctcta cgcgctcctg ggcatggtac 1140
ggcatgcaag tcttgcttgc tttgcgcttt cgccttgatg atgtagtgga ttatggataa 1200
cgatttgtgc gcgttctaaa tcttgtcatg tgctcgtctt tctttttttc ttctttttat 1260
caagggtgga ttgcatgtta ggttaccttt cttttcgaaa agtatagtta aagtggtaat 1320
tggtggtaca aaagtagtat gtcattacac tttcatgaga ttgatcagtt tgatgtgttt 1380
ctagattcat ttatgcttta gttattgcaa gtttatacta cacttcagta attcacacgt 1440
gctgtttcta gatgttattt tggaaccgtt cacagtattt taagcagaga aagttttatc 1500
tagtttgtct tgctagagga aggagcacat ggaaactaac acttgcatat ttagggataa 1560
gcactactgg ttctattcct attttgtgta tgttagctaa tgtgtttctt gctgagtggt 1620
tcagtttcag gttcattagc agattatctt attggttgat ttatgtcaaa atacttaagg 1680
tcaattcgta gtttgcacag tgtacttcaa taacatgaat gcaactcgtt tctttgttca 1740
gcctgatatt tatgaaaaat cttataatgt gatactgtgt ttaatatgta tgaacctgtc 1800
tagagaatta ctagctagtg aaattctact tgtttcattt cacacaaagt caactatggg 1860
tagactggtt catgaccatt tatttaggct ctggtacacc tgtaactact gctgtagttg 1920
actatatgac ttacttctgc tggttttcta ctgtggtatg agtttctccc tttgggatac 1980
cacctgtgtt cagtggaatg tcaggtagta tctgaccttt tcagctagat tgcactggat 2040
aaattatact gaaataagca ataggaatga attccaggac tatgtctcct cttgttctct 2100
ccttgcaatt cctcttattg caatgcagac tgaaccactt gtttttacta tcatctggca 2160
tacttgttca gttagtaact tctacttgcg agtaaggatg caagatttcc catgtaagat 2220
ggatactata aatatcattt tgtctaattg cttaatacct ttcttttttc agggagctgc 2280
tggggcagtc attcattaca tgggctatac gcctggtctt ttcattgtag gattatttgg 2340
aatattgatt atgtggatgt atggctattt ctggattaca ggaatgcttc tgattgctgg 2400
aggtttgttt tatcttaata tttaagtctg ttcataatga taattttgtg tttttgtttg 2460
tcaaatccat aaatttttct tcctccctag ttgcatccgt ttgattcttt tgacctaaag 2520
gaggatcctc tctggtaatg caggctgtat gtgctctttg aaacatgcac gatttgtgat 2580
acctgtgttg gctatgtatg ctgtttattg tgtggctgtt cgtgttggat cgcttggtgt 2640
cttcttgaca ttgaatcttt ctttcctgac aaatgatctt ctgaataagt tgctgcaagg 2700
atacgaggga agcacagaag aaagacagtt tgaagagcca aaacattctg atcctgtcat 2760
ggatgagttc tatcgcagtt gtgaatttcc ctctgctcct gatagtgaac ctgagactgt 2820
ttcttctgca aagccctttt gctcaacacc cgtccaggat gtgttgcatg tacagaaaga 2880
ggcatctcct agcaaagtag tgaaatcgga ttctgtttca ttggatgaga tgaagaggat 2940
catggatggt ttgacccatt atgaagtttt gggtattcct cggaatagaa gtattgatca 3000
aaagattctg aaaaaggagt accacagaat ggtaataaac cacggccttc tatacaaggg 3060
aaaatgagaa attcatgtta caattacttc attttcatgg tacgtatgct ttatttgtct 3120
aggtcctgct tgtacatcct gataaaaata tgggaaatcc actggcctgt gaatcattca 3180
aaaagcttca gtcagcttat gaggtaaact acaatggaag tttatgtctt ttctcttcct 3240
tgattatatt acagttaaat ctggttgaat atctgctctt gataccaacc atggcttcta 3300
tacctggata aagggtaatc attgtagtta tgctgcaggt actctcagat ttcacaaaga 3360
aaaacactta cgacgaccaa ctgaggaaag aagaatcacg taaaatgact cagagatcac 3420
gtgttgtctc tcaacaggtg ggttctagtt ttcacaaatt tagaatccac atggttggat 3480
tatttcttta acatatctta tcaattatcc aagcatacga atgcagttta ttcatgctct 3540
catgtccttg acctactgac ctacttgctg ttttccttta tggggcccat ttgtaatttg 3600
ataaactcat cttgcagact ggggtagagt ttctctccga agagtccagg cgtatacagt 3660
gcacaaaatg tggtaatttt catctgtgga tatgtaccaa gaaaagcaaa gcaaaagcaa 3720
gatggtgtca ggtttggagg ccagaatttt tttttcaggt acttttaatc gagagtgttc 3780
ttacagctaa ttttgtggga accatgtact gtaggattgc tctgattttc atccagctaa 3840
ggatggagat ggatgggtgg aaaataaatt ttcgtcatcc ttcaaggtaa tgttttataa 3900
gcacatcata tgaagagttc actttatttt acttaatgct tgccttctac agtactcata 3960
gacagagatc tagtgtcaat agaattttaa ctactagaaa atggaaattg agtacatgtt 4020
gatttcgaac aaatggagaa tgaggtttta tgaatggaag cacaatgttc tgaatgtttt 4080
gatacaaaat taccgggcgc tgtttcccac tgtcaagctt cagttcctag tacttgttat 4140
tgcctgaagt tagtcatgtg tgttccgaga ccaactttgg acttgagcaa gctcagtttt 4200
agctgtgtca agctgatgat cttttatctt ctaattgtat tccacctaaa gaaagcatct 4260
cattccaagt gttaggtaca gtcattttgt tcattccata agcaacttat tctgactata 4320
gggtgagatt cagaaattac tcagcttaaa aatgtgcaca cattttgtag tttccaacta 4380
taatgtgtaa attcttcact tctctttatt gaactataat gtgtaaattc ttcagtactc 4440
tttgttgaat caaagtgcat tgttcacttc aggaaatacc tcgagctttt gtttgtgcgg 4500
agagtaaggt atttgatgtg tctgaatggg ctacttgcca ggtgagtgtc tgacgatgtt 4560
ttatatgttt gatttaagtt gacatgtatg tgcatttgca gcagtgattt ttggatgtct 4620
caattgattt gatgtcatct ccatatgcat atttttatac tcggttctct gctgttttcg 4680
atgtcttaac tgactataga tatgcctttg gtcaattgac tttgttcagt tttgtatttg 4740
atgcatattc aaacgtccag attgactgtt ttactttaaa aattgtttca gttggtaaat 4800
gaaaatttgc ttacttcatt ggagatagga taattcatgc atgccatagc ccatagcctt 4860
atttttctgt gtcaagtttg tcatggctat aataaacaca acatattaat tgcacctgca 4920
tgtcatcccg actgatatct caattattga catacctatc taagagaaga gccaacaatg 4980
atgaaagtaa aggactaatt tggctgtgca aaattggacc ataagtttat tttacatttc 5040
atacttgctt cattcaacat aaacatcaaa atctggtacg ccaattttct ggcgatacat 5100
acagggtctt gtgataggtt catttgcata cattaaaaat gggagccttt ctaactctgt 5160
tttctttgct tgattgtcta gggcatggag tgcaaaccta acactcacgg cccatctttt 5220
atggtaaaca tggttggcgc agataggatg tctcagagat cctacagttc tcgctatccc 5280
tttagtttga atgctgagat gatccctgaa gatgaatttg agctatggct tcaacaagca 5340
ttggcatcag gtgtcttctc tgacagcccg aaacgcagga aaagctggag ccccttcaaa 5400
ctacctcaaa aagggataaa aagttggcgg cgatcctcat aagggcatag cattgaacag 5460
catggatctc acctgagtac aacactgaaa aaggctatac tctttgtgaa tgtaaataga 5520
ctgaccaaca atttgcctgg atgagcaact aattttgtcc aaaaagagac tgaaacaagg 5580
ggggtaaaag gaacaaacgc ttaagacatg actgcaataa atctgactgt tgaaattagt 5640
gttctctgca atgagatccc gcgagtttta tccgaaaagg tcagacactg ggatggtgtg 5700
tcattcatca gttcatccta agctcggaag ggtatctctg tagcatgtta acttcagtag 5760
ttttagggga tcggcatctg agagaatttc aaaacttcat acctggttgc cagcataagt 5820
tctgcaggtg ttgaaaagtt gttgatcaga gtagcaattt aaggtctgat gtttctgggg 5880
aacagtagga gagaaaaaaa tgacaaaaaa aagagagagt aggttgtaaa tacatgaaaa 5940
gttttcatca gaaattagtg ttgtaacatt gtacactgtg attacatcct gtgcaatact 6000
cccataattc agatctgtgt tgtaatacac tacatacatc ctacaatttt ctggtgataa 6060
tagagatcta attctcacct attatcgtta tttatggtta gtcagttact gctctgta 6118
<210> 21
<211> 1635
<212> DNA
<213> Oryza sativa
<400> 21
atggcggatt tggggctgtg gaagcaaggg tggaggtggg tggtgtccca gaagcacatc 60
ctgacatggg cgcacatggc ggcgagcggc ggcaccgaga ggctggcctt cctggtcgac 120
cggcattggc ccgccgtgtc ccgggcctgc gtgagctccg gccgcctcgc gctcgccgcg 180
ctgcggcaat ggcgcggctg cgcggcgcgc gggatcctgg agatggctag cctgggccct 240
gcgtccgtgt tcgtcatcct ctggagcttc ttcgtgtgca tcacctcgcc ggcgtgcgcc 300
ctctacgcgc tcctgggcat gggagctgct ggggcagtca ttcattacat gggctatacg 360
cctggtcttt tcattgtagg attatttgga atattgatta tgtggatgta tggctatttc 420
tggattacag gaatgcttct gattgctgga ggctgtatgt gctctttgaa acatgcacga 480
tttgtgatac ctgtgttggc tatgtatgct gtttattgtg tggctgttcg tgttggatcg 540
cttggtgtct tcttgacatt gaatctttct ttcctgacaa atgatcttct gaataagttg 600
ctgcaaggat acgagggaag cacagaagaa agacagtttg aagagccaaa acattctgat 660
cctgtcatgg atgagttcta tcgcagttgt gaatttccct ctgctcctga tagtgaacct 720
gagactgttt cttctgcaaa gcccttttgc tcaacacccg tccaggatgt gttgcatgta 780
cagaaagagg catctcctag caaagtagtg aaatcggatt ctgtttcatt ggatgagatg 840
aagaggatca tggatggttt gacccattat gaagttttgg gtattcctcg gaatagaagt 900
attgatcaaa agattctgaa aaaggagtac cacagaatgg tcctgcttgt acatcctgat 960
aaaaatatgg gaaatccact ggcctgtgaa tcattcaaaa agcttcagtc agcttatgag 1020
gtactctcag atttcacaaa gaaaaacact tacgacgacc aactgaggaa agaagaatca 1080
cgtaaaatga ctcagagatc acgtgttgtc tctcaacaga ctggggtaga gtttctctcc 1140
gaagagtcca ggcgtataca gtgcacaaaa tgtggtaatt ttcatctgtg gatatgtacc 1200
aagaaaagca aagcaaaagc aagatggtgt caggattgct ctgattttca tccagctaag 1260
gatggagatg gatgggtgga aaataaattt tcgtcatcct tcaaggaaat acctcgagct 1320
tttgtttgtg cggagagtaa ggtatttgat gtgtctgaat gggctacttg ccagggcatg 1380
gagtgcaaac ctaacactca cggcccatct tttatggtaa acatggttgg cgcagatagg 1440
atgtctcaga gatcctacag ttctcgctat ccctttagtt tgaatgctga gatgatccct 1500
gaagatgaat ttgagctatg gcttcaacaa gcattggcat caggtgtctt ctctgacagc 1560
ccgaaacgca ggaaaagctg gagccccttc aaactacctc aaaaagggat aaaaagttgg 1620
cggcgatcct cataa 1635
<210> 22
<211> 41
<212> DNA
<213> Artificial Sequence
<400> 22
cacgtggacc actagtatgt ggatgtatgg ctatttctgg a 41
<210> 23
<211> 40
<212> DNA
<213> Artificial Sequence
<400> 23
gtccgtacca actagttcgt atccttgcag caacttattc 40
<210> 24
<211> 41
<212> DNA
<213> Artificial Sequence
<400> 24
tgaattcgct ggatcctcgt atccttgcag caacttattc a 41
<210> 25
<211> 41
<212> DNA
<213> Artificial Sequence
<400> 25
gtcgactgga ggatccatgt ggatgtatgg ctatttctgg a 41
<210> 26
<211> 2087
<212> DNA
<213> Oryza sativa
<400> 26
ggaaaggaag aaaaggctaa tatgctcatc ttttttcata gattatactc catatcagta 60
gtatattgtg tcataaagaa taaaagagat atcagactgc cccccctctc tctctcccct 120
ttctcttgca cacaatatca tgatcacact atatttttag tcataggaag agatatttga 180
gaattttgac agcttcctga tcttaggttt ctttattgaa ctgatcttct tttatcagtg 240
ggataaaatg ttgccctata gctatattta caaggacaaa ccaaatgttt tagtatatac 300
cagaatcaaa catgcaacaa ttaattaagt attatagaac taaaaccact ttgttaaaag 360
caaggtctaa attatctgga gaaagtaaga agcaacatgt gatatattat aatattgtct 420
agtttttgta ctaaggtgtg tgttgcaatt gatgcaagtg gggtgtagca taatccatac 480
aagtaagatg ccaagaatgg ggaggagaga ctgtgattat ggcaggaaca tgctcttaat 540
cagtatacag aagtactact actaactact tgcaattact ccaatctctc tcttttctca 600
ttaactgcaa tgcataatcc gtactatccc gtgcaagtaa ctcaaaactt aaggcctcgg 660
ttagggctac taaatgaact atctgcaaat cccgttgttc tcccgttgac aatcatatac 720
ttagcatatt actcattgct tgtttgttag cttatcaagc acatcaaaaa aataaaattt 780
ttaaacttag ttttaagtta tcttgaatca tcgtttattg tcaatattat ctttttgaac 840
cgtcaataaa aaatataaaa aattatctat gaactttcct ttttctgctt cattcttttt 900
tatggcttat cagccatagt tcaaacgatc caccgtagct caaatatcct actactaatt 960
atttttcagc taaaaaagtt agcttccatt ttccaacctt acaatcaagc taacacagtc 1020
actgtcatat aaatagtata ctcaccctaa tcaagctaaa tcttttattt tcctaatgac 1080
tgaactccga aataatatta aattagaaat ctaatgatct agaagatgaa aaccacctct 1140
tttctaatca agctctcttt tgtaaccacc caccaccaca gccatcaaca ccaccaacag 1200
tccaacactg cccaattgcc catgctccag acttctttct actcctacat tccacatatc 1260
tccatggaca gtaactcctc ccaagctacc acttcaaccc taatcccctc tctctctctt 1320
ccgcagaggt agagtgagag agatggtcag atagctagat tgatatccct ctctctctct 1380
cacacacatc tctttttgca agatctcttc ttgttcatca tcttcttctt tttttctccc 1440
ccttttgctt caccaatcca tcttttgtca cgagatgtgg ccgagctgaa gctagtagta 1500
gtggagcagc gaaagcaagt acgccaagaa aaaaaaaagg aagaagaaag aagaaagaaa 1560
gaaagaaaaa aacgccagct tgagggcaga gggcaaaagc ggcgacgagg agcagtggcc 1620
aaagctcaga ttcttcccgt gggctatttt taccacccgc atcccctctc tttgagcccc 1680
ttggccgatt cattcaccga cgcaaagatc caacccctct tcaggtgtcg gcagatgccg 1740
cctttgtgag gtttccagtg gggggatttc tcgtcgtttc ttgcgtgcgg ttgcgttctt 1800
gatccagtga gcgcacggat atatccgccc tggtttagta gagagagaga gagagagaga 1860
gagagagaga gagagaggtt cttgattgag ttccaagtgt tggattgggt tcttggagct 1920
gttggattgg gtttttttgg gagagagatg ggggtttgga ggtgtgtggg ttgaccgaat 1980
tggatcaaga ttattgcggg aggggggggg gggggttgca atggcggatt tggggctgtg 2040
gaagcaaggg tggaggtggg tggtgtccca gaagcacatc ctgacat 2087
<210> 27
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 27
tttgacagct tcctgatctt 20
<210> 28
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 28
caagtaagat gccaagaatg 20
<210> 29
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 29
ttgtcaacgg gagaacaac 19
<210> 30
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 30
taggatattt gagctacgg 19
<210> 31
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 31
tagaaagaag tctggagca 19
<210> 32
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 32
aacgccagct tgagggcag 19
<210> 33
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 33
ttctcgtcgt ttcttgcgtg 20
<210> 34
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 34
gtgtgtgggt tgaccgaat 19
<210> 35
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 35
ggcattgtca acgggagaac aac 23
<210> 36
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 36
aaacgttgtt ctcccgttga caa 23
<210> 37
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 37
ggcaaacgcc agcttgaggg cag 23
<210> 38
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 38
aaacctgccc tcaagctggc gtt 23
<210> 39
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 39
gccgtttgac agcttcctga tctt 24
<210> 40
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 40
aaacaagatc aggaagctgt caaa 24
<210> 41
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 41
gccgcaagta agatgccaag aatg 24
<210> 42
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 42
aaaccattct tggcatctta ctt 23
<210> 43
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 43
gttgtaggat atttgagcta cgg 23
<210> 44
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 44
aaacccgtag ctcaaatatc cta 23
<210> 45
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 45
gttgtagaaa gaagtctgga gca 23
<210> 46
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 46
aaactgctcc agacttcttt cta 23
<210> 47
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 47
tcagttctcg tcgtttcttg cgtg 24
<210> 48
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 48
aaaccacgca agaaacgacg agaa 24
<210> 49
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 49
tcaggtgtgt gggttgaccg aat 23
<210> 50
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 50
aaacattcgg tcaacccaca cac 23
<210> 51
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 51
ggaaaggaag aaaaggctaa tatgctcatc 30
<210> 52
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 52
atgtcaggat gtgcttctgg gacac 25
<210> 53
<211> 1344
<212> DNA
<213> Oryza sativa
<400> 53
ggaaaggaag aaaaggctaa tatgctcatc ttttttcata gattatactc catatcagta 60
gtatattgtg tcataaagaa taaaagagat atcagactgc cccccctctc tctctcccct 120
ttctcttgca cacaatatca tgatcacact atatttttag tcataggaag agatatttga 180
gaattttgac agcttcctga tggtttcttt attgaactga tcttctttta tcagtgggat 240
aaaatgttgc cctatagcta tatttacaag gacaaaccaa atgttttagt atataccaga 300
atcaaacatg caacaattaa ttaagtatta tagaactaaa accactttgt taaaagcaag 360
gtctaaatta tctggagaaa gtaagaagca acatgtgata tattataata ttgtctagtt 420
tttgtactaa ggtgtgtgtt gcaattgatg caagtggggt gtagcataat ccatacaagt 480
aagatgccaa gatccagact tctttctact cctacattcc acatatctcc atggacagta 540
actcctccca agctaccact tcaaccctaa tcccctctct ctctcttccg cagaggtaga 600
gtgagagaga tggtcagata gctagattga tatccctctc tctctctcac acacatctct 660
ttttgcaaga tctcttcttg ttcatcatct tcttcttttt ttctccccct tttgcttcac 720
caatccatct tttgtcacga gatgtggccg agctgaagct agtagtagtg gagcagcgaa 780
agcaagtacg ccaagaaaaa aaaaggaaga agaaagaaga aagaaagaaa gaaaaaaacg 840
ccagcttgag ggacagaggg caaaagcggc gacgaggagc agtggccaaa gctcagatcc 900
ttcccgtggg ctatttttac cacccgcatc ccctctcttt gagccccttg gccgattcat 960
tcaccgacgc aaagatccaa cccctcttca ggtgtcggca gatgccgcct ttgtgaggtt 1020
tccagtgggg ggatttctcg tcgtttcttg cgtgcggttg cgttcttgat ccagtgagcg 1080
cacggatata tccgccctgg tttagtagag agagagagag agagagagag agagaggttc 1140
ttgattgagt tccaagtgtt ggattgggtt cttggagctg ttggattggg tttttttggg 1200
agagagatgg gggtttggag gtgtgtgggt tgaccaattg gatcaagatt attgcgggag 1260
ggggggggga ggttgcaatg gcggatttgg ggctgtggaa gcaagggtgg aggtgggtgg 1320
tgtcccagaa gcacatcctg acat 1344
<210> 54
<211> 981
<212> DNA
<213> Oryza sativa
<400> 54
ggaaaggaag aaaaggctaa tatgctcatc ttttttcata gattatactc catatcagta 60
gtatattgtg tcataaagaa taaaagagat atcagactgc cccccctctc tctctcccct 120
ttctcttgca cacaatatca tgatcacact atatttttag tcataggaag agatatttga 180
gaattttgac agcttcctga tattgtgtga tcatgatatc aggtttcttt attgaactga 240
tcttctttta tcagtgggat aaaatgttgc cctatagcta tatttacaag gacaaaccaa 300
atgttttagt atataccaga atcaaacatg caacaattaa ttaagtatta tagaactaaa 360
accactttgt taaaagcaag gtctaaatta tctggagaaa gtaagaagca acatgtgata 420
tattataata ttgtctagtt tttgtactaa ggtgtgtgtt gcaattgatg caagtggggt 480
gtagcataat ccatacaagt aagatgccaa gaccctcaag ctggcgtttt tttctttctt 540
tctttcttct ttcttcttcc tttttttttc ttggcgtact tgctttcgct gctccactac 600
tactagcttc agctcggcca catctcgtga caaaagatgg attggtgaag caaaaggggg 660
agaaaaaaag aagaagatga tgaacaagaa gagatcttgc aaaaagagat gtgtgtgaga 720
gagagagagg gatatcaatc tagctatctg accatctctc tcactctacc tctgcggaag 780
agagagagag gggattaggg ttgaagtggt agcttgggag gagttactgt ccatggagat 840
atgtggaatg taggagtaga aagaagtctg gagaattgga tcaagattat tgcgggaggg 900
gggggggggt tgcaatggcg gatttggggc tgtggaagca agggtggagg tgggtggtgt 960
cccagaagca catcctgaca t 981
<210> 55
<211> 528
<212> PRT
<213> Brachypodium_distachyon
<400> 55
Met Asp Ile Met Thr Trp Ala His Met Ala Ala Gly Cys Gly Arg Glu
1 5 10 15
Arg Val Ala Ser Leu Val Asp Arg His Trp Pro Ala Val Ser Arg Ala
20 25 30
Cys Val Cys Ser Ser Cys Phe Val Leu Ala Ala Leu Arg Gln Trp Gln
35 40 45
Gly Cys Thr Ala Arg Gly Phe Leu Gly Leu Ala Ser Leu Gly Pro Ala
50 55 60
Ala Val Phe Val Ile Leu Trp Ser Phe Phe Val Cys Met Thr Ser Pro
65 70 75 80
Val Cys Ala Leu Tyr Ala Leu Leu Ile Leu Gly Ala Thr Gly Ala Val
85 90 95
Ile His Tyr Met Gly Tyr Thr Pro Gly Leu Leu Ile Val Gly Leu Phe
100 105 110
Gly Ile Leu Ile Met Trp Met Tyr Gly Tyr Phe Trp Ile Thr Gly Met
115 120 125
Leu Leu Val Ala Gly Gly Ser Met Cys Ser Leu Lys His Ala Arg Phe
130 135 140
Val Ile Pro Val Leu Ala Val Tyr Ala Val Tyr Cys Val Ala Val Arg
145 150 155 160
Val Gly Trp Leu Gly Val Phe Leu Thr Leu Asn Leu Ser Phe Leu Thr
165 170 175
Asn Asp Leu Leu Asn Lys Leu Leu Gln Gly Tyr Glu Gly Ser Thr Glu
180 185 190
Glu Met Glu Phe Glu Glu Met Lys Asp Pro His Pro Gly Met Asp Glu
195 200 205
Phe Tyr Pro Ser Tyr Glu Tyr Pro Pro Ala Pro Asp Ser Glu Pro Glu
210 215 220
Thr Val Ser Ser Ala Lys Pro Phe Cys Ala Ser Pro Thr Gln Asp Val
225 230 235 240
Leu His Val Gln Lys Glu Ala Ser Pro Ser Lys Ile Val Lys Ser Asp
245 250 255
Ser Thr Ala Leu Asp Glu Met Lys Arg Ile Met Asp Gly Ser Thr Tyr
260 265 270
Tyr Glu Ile Phe Gly Ile Pro Arg Asn Arg Ser Ala Asp Leu Lys Ile
275 280 285
Leu Lys Gly Glu Tyr Arg Arg Met Ala Met Leu Val His Pro Asp Lys
290 295 300
Asn Met Gly Asn Ser Leu Ala Cys Glu Ser Phe Lys Lys Leu Gln Ser
305 310 315 320
Ala Tyr Glu Val Leu Ser Asp Leu Thr Lys Lys Asn Ser Tyr Asp Glu
325 330 335
Gln Leu Arg Lys Glu Glu Ser Arg Gln Met Thr Gln Arg Ser Arg Val
340 345 350
Val Ser Gln Gln Ser Gly Val Glu Phe Leu Ser Glu Glu Ser Arg Arg
355 360 365
Ile Gln Cys Thr Lys Cys Gly Asn Phe His Leu Trp Ile Cys Thr Lys
370 375 380
Arg Ser Lys Ala Lys Ala Arg Trp Cys Gln Asp Cys Ser Gln His His
385 390 395 400
Val Ala Lys Asp Gly Asp Gly Trp Val Glu Asn Gly Tyr Ser Thr Ser
405 410 415
Leu Lys Ile Glu Ile Pro Arg Ala Phe Val Cys Ala Glu Ser Lys Ile
420 425 430
Phe Asp Val Ser Glu Trp Ala Thr Cys Gln Gly Met Glu Cys Lys Pro
435 440 445
Asn Thr His Gly Pro Thr Phe Met Val Asn Met Val Gly Ala Asp Arg
450 455 460
Met Pro Gln Arg Ser Tyr Ser Ser Arg Tyr Pro Phe Ser Leu Asp Ala
465 470 475 480
Glu Met Ile Pro Glu Asp Glu Phe Asp Leu Trp Leu Gln Gln Ala Leu
485 490 495
Ala Thr Gly Val Phe Ser Asp Ser Pro Lys Arg Arg Lys Ser Trp Ser
500 505 510
Pro Phe Lys Leu Thr Gln Lys Gly Val Arg Ser Trp Arg Arg Ser Ser
515 520 525
<210> 56
<211> 545
<212> PRT
<213> Hordeum_vulgare
<400> 56
Met Ala Gly Leu Gly Leu Trp Asn Gln Gly Trp Thr Trp Val Leu Ser
1 5 10 15
Gln Lys His Val Val Ala Trp Ala His Ala Ala Ala Gly Cys Gly Arg
20 25 30
Asp Arg Leu Ala Phe Leu Val Asp Arg His Trp Pro Ala Val Ser Arg
35 40 45
Ala Cys Ala Thr Ser Ser Arg Leu Val Leu Glu Ala Leu Arg Gln Trp
50 55 60
Arg Gly Cys Thr Ala Arg Gly Leu Leu Ala Leu Ala Ser Leu Gly Pro
65 70 75 80
Ala Ala Val Phe Val Ile Leu Trp Ser Cys Phe Val Cys Met Thr Ser
85 90 95
Ser Ala Cys Ala Leu Tyr Ala Leu Leu Ala Leu Gly Ala Val Gly Ala
100 105 110
Val Ile His Tyr Met Gly Tyr Thr Pro Gly Leu Leu Ile Val Gly Leu
115 120 125
Phe Gly Ile Met Ile Met Trp Met Tyr Gly Tyr Phe Trp Ile Thr Gly
130 135 140
Met Leu Leu Val Ala Gly Gly Cys Met Cys Ser Leu Lys His Ala Arg
145 150 155 160
Phe Val Ile Pro Val Leu Ala Met Tyr Ala Val Tyr Cys Val Ala Val
165 170 175
Arg Val Gly Trp Leu Gly Val Phe Phe Met Leu Asn Leu Ser Phe Leu
180 185 190
Thr Asn Asp Leu Leu Asn Lys Leu Leu Gln Gly Tyr Glu Gly Ser Thr
195 200 205
Glu Glu Arg Pro Phe Glu Glu Met Lys Asp Ser Asp Pro Ala Thr Asp
210 215 220
Ala Phe Phe Arg Gly Cys Glu Tyr Pro Pro Ala Pro Glu Ser Glu Pro
225 230 235 240
Glu Thr Val Ser Ser Ala Lys Pro Phe Cys Ala Ala Pro Thr Gln Asp
245 250 255
Val Leu His Val Gln Lys Glu Pro Ser Pro Thr Lys Ile Val Lys Ser
260 265 270
Asn Ser Thr Ser Leu Asp Glu Met Lys Arg Ile Met Asp Gly Ser Thr
275 280 285
Tyr Tyr Glu Val Leu Gly Ile Pro Arg Ser Lys Ser Ile Asn Gln Ile
290 295 300
Glu Leu Lys Lys Glu Tyr Arg Lys Leu Ala Val Leu Val His Pro Asp
305 310 315 320
Lys Asn Met Gly Asn Pro Leu Ala Cys Glu Ser Phe Lys Lys Leu Gln
325 330 335
Ser Ala Phe Glu Val Leu Ser Asp Leu Thr Lys Lys Asn Gly Tyr Asp
340 345 350
Glu Gln Leu Arg Lys Glu Glu Ser Arg Gln Met Thr Gln Arg Ser Arg
355 360 365
Val Val Ser Gln Pro Ser Gly Val Glu Phe Leu Ser Glu Glu Ser Arg
370 375 380
Arg Ile Gln Cys Thr Lys Cys Gly Asn Phe His Leu Trp Ile Cys Thr
385 390 395 400
Lys Arg Ser Lys Ala Lys Ala Arg Trp Cys Gln Glu Cys Ser Gln Tyr
405 410 415
His Val Ala Lys Asp Gly Asp Gly Trp Val Glu Asn Arg Tyr Ser Thr
420 425 430
Ser Leu Lys Ile Glu Ile Pro Arg Ala Phe Val Cys Ala Glu Ser Lys
435 440 445
Ile Phe Asp Val Ser Glu Trp Ala Thr Cys Gln Gly Met Glu Cys Lys
450 455 460
Pro Asn Thr His Gly Pro Thr Phe Met Val Asn Met Val Gly Ala Asp
465 470 475 480
Arg Met Pro Gln Arg Ser His Ser Ser Arg Tyr Pro Phe Ser Leu Asp
485 490 495
Ala Glu Met Ile Pro Glu Asp Glu Phe Glu Leu Trp Leu Gln Gln Ala
500 505 510
Leu Ala Thr Gly Val Phe Ser Asp Ser Pro Lys Arg Arg Lys Ser Trp
515 520 525
Ser Pro Phe Lys Leu Pro Gln Lys Gly Ile Arg Ser Trp Arg Arg Ser
530 535 540
Ser
545
<210> 57
<211> 545
<212> PRT
<213> Oryza_brachyantha
<400> 57
Met Ala Asp Leu Gly Leu Trp Lys Gln Gly Trp Arg Trp Val Leu Ser
1 5 10 15
Gln Lys His Ile Leu Thr Trp Ala His Met Ala Ala Ser Gly Gly Thr
20 25 30
Glu Arg Leu Ala Phe Leu Val Asp Arg His Trp Pro Ala Val Ser Arg
35 40 45
Thr Cys Val Ser Ser Gly Arg Leu Ala Leu Ala Ala Leu Arg Gln Trp
50 55 60
Arg Gly Cys Ala Ala Arg Gly Ile Leu Glu Met Ala Ser Leu Gly Pro
65 70 75 80
Ala Ser Val Phe Val Ile Leu Trp Ser Cys Phe Val Cys Met Thr Ser
85 90 95
Pro Ala Cys Ala Leu Tyr Ala Leu Leu Ser Leu Gly Ala Ala Gly Ala
100 105 110
Val Ile His Tyr Met Gly Tyr Thr Pro Gly Leu Phe Ile Val Gly Leu
115 120 125
Phe Gly Ile Leu Ile Met Trp Met Tyr Gly Tyr Phe Trp Ile Thr Gly
130 135 140
Met Leu Leu Ile Ser Gly Gly Cys Met Cys Ser Leu Lys His Ala Arg
145 150 155 160
Phe Val Ile Pro Val Leu Ala Met Tyr Ala Val Tyr Cys Val Ala Val
165 170 175
Arg Val Gly Leu Leu Gly Val Phe Leu Thr Leu Asn Leu Ser Phe Leu
180 185 190
Thr Asn Asp Leu Met Asn Lys Leu Leu Gln Gly Tyr Glu Gly Ser Thr
195 200 205
Glu Glu Arg Gln Phe Glu Glu Thr Lys His Ser Asp Pro Val Met Asp
210 215 220
Glu Phe Tyr Arg Ser Cys Glu Tyr Pro Thr Ala Pro Asp Ser Glu Pro
225 230 235 240
Glu Thr Val Ser Ser Ala Lys Pro Phe Cys Ser Thr Pro Val Gln Asp
245 250 255
Val Leu His Val Gln Lys Glu Ala Ser Pro Ser Lys Val Val Lys Ser
260 265 270
Asp Ser Val Ser Leu Asp Glu Met Lys Arg Ile Met Asp Gly Leu Thr
275 280 285
His Tyr Glu Val Leu Gly Ile Pro Arg Asn Arg Ser Ile Asp Gln Lys
290 295 300
Thr Leu Lys Lys Glu Tyr His Arg Met Val Leu Leu Val His Pro Asp
305 310 315 320
Lys Asn Met Gly Asn Pro Leu Ala Cys Glu Ser Phe Lys Lys Leu Gln
325 330 335
Ser Ala Tyr Glu Val Leu Ser Asp Phe Thr Lys Lys Asn Thr Tyr Asp
340 345 350
Asp Gln Leu Arg Lys Glu Glu Ser Arg Lys Met Thr Gln Arg Ser Arg
355 360 365
Val Val Ser Gln Gln Thr Gly Val Glu Phe Leu Ser Glu Glu Ser Arg
370 375 380
Arg Ile Gln Cys Thr Lys Cys Gly Asn Phe His Leu Trp Ile Cys Thr
385 390 395 400
Lys Lys Ser Lys Ala Lys Ala Arg Trp Cys Gln Asp Cys Ser Asp Phe
405 410 415
His Pro Ala Lys Asp Gly Asp Gly Trp Val Glu Asn Lys Phe Ser Ala
420 425 430
Ser Phe Lys Met Glu Ile Pro Arg Ala Phe Val Cys Ala Glu Ser Lys
435 440 445
Ile Phe Asp Val Ser Glu Trp Ala Thr Cys Gln Gly Met Glu Cys Lys
450 455 460
Pro Asn Thr His Gly Pro Ser Phe Met Val Asn Met Val Gly Ala Asp
465 470 475 480
Arg Met Ser Gln Arg Ser Tyr Ser Ser Arg Tyr Pro Phe Ser Leu Asn
485 490 495
Ala Glu Met Val Pro Glu Asp Glu Phe Glu Leu Trp Leu Gln Gln Ala
500 505 510
Leu Ala Ser Gly Val Phe Ala Asp Ser Pro Lys Arg Arg Lys Ser Trp
515 520 525
Ser Pro Phe Lys Leu Pro Gln Lys Gly Ile Lys Ser Trp Arg Arg Ser
530 535 540
Ser
545
<210> 58
<211> 545
<212> PRT
<213> Panicum_hallii
<400> 58
Met Ala Asp Leu Gly Leu Trp Lys Gln Ala Trp Arg Trp Val Leu Ser
1 5 10 15
Gln Lys His Ile Leu Ala Trp Ala His Thr Ala Ala Cys Gly Ser Arg
20 25 30
Glu Arg Leu Ala Phe Leu Val Asp Arg His Trp Pro Ala Val Ser Arg
35 40 45
Ala Cys Ala Thr Ser Ser Arg Leu Ala Leu Ala Ala Leu Arg Gln Trp
50 55 60
Arg Gly Cys Met Ala Arg Gly Val Leu Ala Val Ala Ser Leu Gly Pro
65 70 75 80
Ala Ala Val Phe Val Ile Leu Trp Ser Phe Phe Val Cys Met Thr Ser
85 90 95
Pro Ala Trp Ala Leu Phe Ala Leu Leu Ser Leu Gly Ala Ala Gly Ala
100 105 110
Val Val His Tyr Met Gly Tyr Thr Pro Gly Leu Phe Ile Val Gly Leu
115 120 125
Phe Gly Ile Leu Ile Met Trp Met Tyr Gly Tyr Phe Trp Ile Thr Gly
130 135 140
Met Leu Leu Val Ala Gly Gly Cys Met Cys Ser Leu Lys His Ala Arg
145 150 155 160
Tyr Val Ile Pro Ile Leu Thr Thr Tyr Ala Ile Tyr Cys Val Ala Val
165 170 175
Arg Val Gly Trp Leu Gly Val Phe Leu Thr Leu Asn Leu Ser Phe Leu
180 185 190
Thr Asn Asp Leu Leu Asn Lys Leu Leu Gln Gly Tyr Glu Gly Cys Thr
195 200 205
Glu Glu Glu Gln Phe Glu Asp Met Lys Asp Ser Asp Pro Val Met Asp
210 215 220
Glu Phe Tyr Arg Ser Cys Glu Phe Pro Pro Thr Pro Asp Ser Glu Pro
225 230 235 240
Glu Thr Val Ser Ser Ala Lys Pro Tyr Cys Ser Ala Pro Thr Gln Asp
245 250 255
Val Leu His Val Gln Lys Glu Glu Pro Pro Ser Lys Val Val Lys Ser
260 265 270
Asp Ser Ser Ser Leu Asp Glu Ile Lys Arg Ile Met Asp Gly Ser Asn
275 280 285
Tyr Tyr Glu Val Leu Gly Ile Pro Arg Asn Arg Ser Ile Asp Gln Lys
290 295 300
Ser Leu Lys Lys Glu Tyr His Arg Met Val Leu Leu Val His Pro Asp
305 310 315 320
Lys Asn Met Gly Asn Pro Leu Ala Cys Glu Ser Phe Lys Lys Leu Gln
325 330 335
Ser Ala Tyr Glu Val Leu Ser Asp Phe Thr Lys Lys Asn Ser Tyr Asp
340 345 350
Glu Gln Leu Arg Lys Glu Glu Ser Gln Asn Met Thr Pro Arg Ser Arg
355 360 365
Val Val Ser Gln Gln Ser Gly Val Glu Phe Leu Ser Glu Glu Ser Arg
370 375 380
Arg Ile Gln Cys Thr Lys Cys Gly Asn Phe His Ile Trp Ile Cys Thr
385 390 395 400
Lys Arg Ser Lys Thr Lys Ala Arg Phe Cys Gln Gly Cys Asp Gln Phe
405 410 415
His Gln Ala Lys Asp Gly Asp Gly Trp Val Glu Thr Arg Phe Ser Thr
420 425 430
Ser Val Lys Met Glu Ile Pro Arg Ala Phe Val Cys Ala Glu Ser Lys
435 440 445
Ile Phe Asp Val Ser Glu Trp Ala Thr Cys Gln Gly Met Glu Cys Lys
450 455 460
Pro Asn Thr His Gly Pro Thr Phe Met Val Asn Met Val Gly Ala Asp
465 470 475 480
Arg Met Pro Gln Arg Ser Tyr Ser Ser Arg Tyr Pro Phe Ser Leu Asp
485 490 495
Ala Glu Met Ile Pro Glu Asp Glu Phe Glu Leu Trp Leu Gln Gln Ala
500 505 510
Leu Ala Ser Gly Val Phe Ala Asp Ser Pro Lys Arg Arg Lys Ser Trp
515 520 525
Ser Pro Phe Lys Leu Pro Gln Lys Gly Ile Lys Ser Trp Arg Arg Ser
530 535 540
Ser
545
<210> 59
<211> 544
<212> PRT
<213> Setaria_italica
<400> 59
Met Ala Asp Leu Gly Leu Trp Lys Gln Ala Trp Arg Trp Val Leu Ser
1 5 10 15
Gln Lys His Ile Leu Ala Trp Ala His Thr Ala Ala Cys Gly Ser Arg
20 25 30
Glu Arg Leu Ala Phe Leu Val Asp Arg His Trp Pro Ala Val Ser Arg
35 40 45
Ala Cys Ala Thr Ser Ser Arg Leu Ala Leu Ala Ala Leu Leu Gln Trp
50 55 60
Arg Gly Cys Met Ala Arg Gly Val Leu Ala Val Ala Ser Leu Gly Pro
65 70 75 80
Ala Ala Val Phe Val Ile Leu Trp Ser Phe Phe Val Cys Met Thr Ser
85 90 95
Pro Ala Trp Ala Leu Phe Ala Leu Leu Leu Leu Gly Ala Ala Gly Ala
100 105 110
Val Val His Tyr Met Gly Tyr Thr Pro Gly Leu Phe Ile Val Gly Leu
115 120 125
Phe Gly Ile Leu Ile Met Trp Met Tyr Gly Tyr Phe Trp Ile Thr Gly
130 135 140
Met Leu Leu Val Ala Gly Gly Cys Met Cys Ser Leu Lys His Ala Arg
145 150 155 160
Tyr Val Ile Pro Ile Leu Thr Thr Tyr Ala Ile Tyr Cys Val Ala Ile
165 170 175
Arg Val Gly Trp Leu Gly Val Phe Leu Thr Leu Asn Leu Ser Phe Leu
180 185 190
Ala Asn Asp Leu Leu Asn Lys Leu Leu Gln Gly Tyr Glu Glu Ser Thr
195 200 205
Glu Glu Lys Phe Glu Asp Met Lys Asp Ser Asp Pro Val Met Asp Glu
210 215 220
Phe Tyr Arg Ser Cys Glu Phe Pro Pro Ala Pro Asp Ser Glu Pro Glu
225 230 235 240
Thr Val Ser Ser Ala Lys Pro Tyr Cys Ser Ser Pro Thr Gln Asp Val
245 250 255
Leu His Val Gln Lys Glu Glu Pro Pro Ser Lys Val Val Lys Ser Asp
260 265 270
Ser Ser Ser Leu Asp Glu Ile Lys Arg Ile Met Asp Gly Ser Asn His
275 280 285
Tyr Glu Val Leu Gly Ile Pro Arg Asn Arg Ser Ile Asp Gln Lys Ser
290 295 300
Leu Lys Lys Glu Tyr His Arg Met Val Leu Leu Val His Pro Asp Lys
305 310 315 320
Asn Met Gly Asn Pro Leu Ala Cys Glu Ser Phe Lys Lys Leu Gln Ser
325 330 335
Ala Tyr Glu Val Leu Ser Asp Phe Thr Lys Arg Asn Ser Tyr Asp Glu
340 345 350
Gln Leu Arg Lys Glu Glu Ser Gln Lys Met Thr Pro Arg Ser Arg Val
355 360 365
Val Ser Gln Gln Gly Gly Val Glu Phe Leu Ser Glu Glu Ser Arg Arg
370 375 380
Ile Gln Cys Thr Lys Cys Gly Asn Phe His Ile Trp Ile Cys Thr Lys
385 390 395 400
Arg Ser Lys Thr Lys Ala Arg Phe Cys Gln Gly Cys Asp Gln Tyr His
405 410 415
Gln Ala Lys Asp Gly Asp Gly Trp Val Glu Thr Arg Phe Ser Thr Ser
420 425 430
Tyr Lys Met Glu Ile Pro Arg Ala Phe Val Cys Ala Glu Ser Lys Ile
435 440 445
Phe Asp Val Ser Glu Trp Ala Thr Cys Gln Gly Met Glu Cys Lys Pro
450 455 460
Asn Thr His Gly Pro Thr Phe Met Val Asn Met Val Gly Ala Asp Arg
465 470 475 480
Met Pro Gln Arg Ser Tyr Ser Ser Arg Tyr Pro Phe Ser Leu Asp Ala
485 490 495
Glu Met Ile Pro Glu Asp Glu Phe Glu Leu Trp Leu Gln Gln Ala Leu
500 505 510
Ala Ser Gly Val Phe Ala Asp Ser Pro Lys Arg Arg Lys Ser Trp Ser
515 520 525
Pro Phe Lys Leu Pro Gln Lys Gly Ile Lys Ser Trp Arg Arg Ser Ser
530 535 540
<210> 60
<211> 545
<212> PRT
<213> Zea_mays
<400> 60
Met Glu Asp Leu Gly Leu Trp Asn Gln Ala Trp Met Trp Val Leu Ser
1 5 10 15
Gln Lys His Ile Leu Ala Trp Ala His Thr Ala Ala Cys Gly Ser Arg
20 25 30
Glu Arg Leu Ala Phe Leu Val Asp Arg His Trp Pro Ala Val Ser Arg
35 40 45
Gly Cys Ala Thr Ser Ser Arg Leu Thr Leu Ala Ala Leu Arg Gln Trp
50 55 60
Arg Gly Cys Met Ala Arg Gly Val Leu Ala Val Ala Ser Leu Gly Pro
65 70 75 80
Ala Ala Val Phe Val Ile Leu Trp Ser Phe Phe Val Cys Met Thr Ser
85 90 95
Pro Ala Cys Ala Leu Tyr Ala Leu Leu Ser Leu Gly Ala Ala Ala Ala
100 105 110
Val Val His Tyr Met Gly Tyr Thr Pro Gly Leu Leu Ile Val Gly Leu
115 120 125
Phe Gly Ile Leu Ile Met Trp Met Tyr Gly Tyr Phe Trp Ile Thr Gly
130 135 140
Met Leu Leu Val Ala Gly Gly Cys Met Cys Ser Leu Lys His Ala Arg
145 150 155 160
Tyr Val Thr Pro Val Leu Thr Ser Tyr Ala Ile Tyr Cys Val Ala Val
165 170 175
Arg Val Gly Trp Leu Gly Val Phe Leu Thr Phe Asn Leu Ser Phe Leu
180 185 190
Thr Asn Asp Leu Leu Asn Lys Leu Ala Gln Gly Tyr Glu Gly Ser Thr
195 200 205
Glu Glu Ser Gln Phe Glu Asp Met Lys Asp Ser Asp Pro Val Met Asp
210 215 220
Glu Phe Tyr Arg Ser Cys Glu Phe Pro Ser Val Pro Asp Ser Glu Pro
225 230 235 240
Glu Thr Val Ser Ser Ala Lys Pro Tyr Cys Ser Ala Pro Ile Gln Asp
245 250 255
Val Leu His Val Gln Lys Glu Glu Pro Pro Ser Lys Ile Val Lys Ser
260 265 270
Asp Ser Ser Ser Ser Asp Glu Ile Lys Arg Ile Met Asp Gly Ser Asn
275 280 285
His Tyr Glu Val Leu Gly Val Pro Arg Asn Arg Ser Ile Asp Gln Lys
290 295 300
Ala Leu Lys Lys Glu Tyr His Arg Met Val Leu Leu Val His Pro Asp
305 310 315 320
Lys Asn Met Gly Asn Pro Leu Ala Cys Glu Ser Phe Lys Lys Leu Gln
325 330 335
Ser Ala Tyr Glu Val Leu Ser Asp Phe Thr Lys Lys Asn Ser Tyr Asp
340 345 350
Gln Gln Leu Arg Lys Glu Glu Ser Gln Lys Met Thr Pro Arg Ser Arg
355 360 365
Ala Val Ser Gln Gln Ser Gly Val Glu Phe Leu Ser Glu Glu Ser Arg
370 375 380
Arg Ile Gln Cys Thr Lys Cys Gly Asn Phe His Ile Trp Ile Cys Thr
385 390 395 400
Lys Arg Ser Lys Thr Lys Ala Arg Phe Cys Gln Gly Cys Asp Gln Phe
405 410 415
His Gln Ala Lys Asp Gly Asp Gly Trp Val Glu Thr Arg Phe Ser Ser
420 425 430
Ser Ile Lys Met Glu Ile Pro Arg Ala Phe Val Cys Ala Glu Ser Lys
435 440 445
Ile Phe Asp Val Ser Glu Trp Ala Thr Cys Gln Gly Met Glu Cys Lys
450 455 460
Pro Asn Thr His Gly Pro Thr Phe Met Val Asn Met Val Gly Ala Asp
465 470 475 480
Arg Met Pro Gln Arg Ser Tyr Ser Ser Arg Tyr Pro Phe Ser Leu Asp
485 490 495
Ala Glu Met Ile Pro Asp Asp Glu Phe Glu Met Trp Leu Gln Gln Ala
500 505 510
Leu Ala Ser Gly Val Phe Ala Asp Ser Pro Lys Arg Arg Lys Ser Trp
515 520 525
Ser Pro Phe Lys Leu Pro Gln Lys Gly Ile Lys Ser Trp Arg Arg Ser
530 535 540
Ser
545
<210> 61
<211> 545
<212> PRT
<213> Sorghum_bicolor
<400> 61
Met Ala Asp Leu Gly Leu Trp Lys Gln Ala Trp Met Trp Val Leu Ser
1 5 10 15
Gln Lys His Ile Leu Ala Trp Ala His Thr Ala Ala Cys Gly Ser Arg
20 25 30
Glu Arg Leu Ala Phe Leu Val Asp Arg His Trp Pro Ala Val Ser Arg
35 40 45
Ala Cys Ala Thr Ser Ser Arg Leu Ala Leu Ala Ala Leu Arg Gln Trp
50 55 60
Arg Gly Cys Thr Ala Arg Gly Val Leu Ala Val Ala Ser Leu Gly Pro
65 70 75 80
Ala Ala Val Phe Val Ile Leu Trp Ser Phe Phe Val Cys Met Thr Ser
85 90 95
Pro Ala Cys Ala Leu Tyr Ala Leu Leu Ser Leu Gly Ala Ala Ala Ala
100 105 110
Val Val His Tyr Met Gly Tyr Thr Pro Gly Leu Phe Ile Val Gly Leu
115 120 125
Phe Gly Ile Leu Ile Met Trp Met Tyr Gly Tyr Phe Trp Ile Thr Gly
130 135 140
Met Leu Leu Val Ala Gly Gly Cys Met Cys Ser Leu Lys His Ala Arg
145 150 155 160
Tyr Val Ile Pro Val Leu Thr Ser Tyr Ala Ile Tyr Ser Val Ala Val
165 170 175
Arg Val Gly Trp Leu Gly Val Phe Leu Thr Leu Asn Leu Ser Phe Leu
180 185 190
Thr Asn Asp Leu Leu Asn Lys Leu Ala Gln Gly Tyr Glu Gly Ser Thr
195 200 205
Glu Glu Ser Gln Phe Glu Asp Ile Lys Gly Ser Asp Pro Val Met Asp
210 215 220
Glu Phe Tyr Arg Ser Cys Glu Phe Pro Pro Val Pro Asp Ser Glu Pro
225 230 235 240
Glu Thr Val Ser Ser Ala Lys Pro Tyr Cys Thr Ala Pro Val Gln Asp
245 250 255
Val Leu His Val Gln Lys Glu Glu Pro Pro Ser Lys Val Val Lys Ser
260 265 270
Asp Ser Ser Ser Leu Asp Glu Ile Lys Arg Ile Met Asp Gly Ser Asn
275 280 285
His Tyr Glu Val Leu Gly Val Pro Arg Asn Arg Ser Ile Asp Gln Lys
290 295 300
Thr Leu Lys Lys Glu Tyr His Arg Met Val Leu Leu Val His Pro Asp
305 310 315 320
Lys Asn Met Gly Asn Pro Leu Ala Cys Glu Ser Phe Lys Lys Leu Gln
325 330 335
Ser Ala Tyr Glu Val Leu Ser Asp Phe Thr Lys Lys Asn Ser Tyr Asp
340 345 350
Glu Gln Leu Arg Lys Glu Glu Ser Leu Lys Met Thr Pro Arg Ser Arg
355 360 365
Val Val Ser Gln Gln Ser Gly Val Glu Phe Leu Ser Glu Glu Ser Arg
370 375 380
Arg Ile Gln Cys Thr Lys Cys Gly Asn Phe His Ile Trp Ile Cys Thr
385 390 395 400
Lys Arg Ser Lys Thr Arg Ala Arg Phe Cys Gln Gly Cys Asp Gln Phe
405 410 415
His Gln Ala Lys Asp Gly Asp Gly Trp Val Glu Thr Arg Phe Ser Ser
420 425 430
Ser Ile Lys Met Glu Ile Pro Arg Ala Phe Val Cys Ala Glu Ser Lys
435 440 445
Ile Phe Asp Val Ser Glu Trp Ala Thr Cys Gln Gly Met Glu Cys Lys
450 455 460
Pro Asn Thr His Gly Pro Thr Phe Met Val Asn Met Val Gly Thr Asp
465 470 475 480
Arg Met Pro Gln Arg Ser Tyr Ser Ser Arg Tyr Pro Phe Ser Leu Asp
485 490 495
Ala Glu Met Ile Pro Glu Asp Glu Phe Glu Leu Trp Leu Gln Gln Ala
500 505 510
Leu Ala Ser Gly Val Phe Ala Asp Ser Pro Lys Arg Arg Lys Ser Trp
515 520 525
Ser Pro Phe Lys Leu Pro Gln Lys Gly Ile Lys Ser Trp Arg Arg Ser
530 535 540
Ser
545

Claims (11)

1. a kind of method of regulation size of plant seed, passes through the expression regulation plant for influencing the gene of regulation size of plant seed Seed size, it is characterised in that it is described regulation size of plant seed gene nucleotide sequence be selected from following group sequence it One:
(a) nucleotide sequence as shown in the gene LOC_Os12g41820 of regulation size of plant seed；
(b) nucleotide sequence as shown in SEQ ID NO:1,2,20 or 21；
(c) its encoding amino acid sequence nucleotide sequence as shown in SEQ ID NO:3,55,56,57,58,59,60 or 61；
It (d) can be with the DNA sequence dna of the DNA hybridization of sequence described in (a)-(c) under high stringency conditions；Or
(e) there is at least 80%(to be preferably at least 85%) sequence similarity with (a)-(d) sequence, and there is regulation vegetable seeds The DNA sequence dna of magnitude function；Or
(f) DNA sequence dna complementary with any sequence of (a)-(e).
2. method described in claim 1, which is characterized in that pass through the nucleotides sequence of the gene of mutation regulation size of plant seed The protein inactivation so that gene expression is arranged, so that the vegetable material with big kernel Characters phenotype is obtained, wherein the regulation The nucleotide sequence of the gene of size of plant seed is selected from following group of one of sequence:
(a) nucleotide sequence as shown in the gene LOC_Os12g41820 of regulation size of plant seed；
(b) nucleotide sequence as shown in SEQ ID NO:1,2,20 or 21；
(c) its encoding amino acid sequence nucleotide sequence as shown in SEQ ID NO:3,55,56,57,58,59,60 or 61；
It (d) can be with the DNA sequence dna of the DNA hybridization of sequence described in (a)-(c) under high stringency conditions；Or
(e) there is at least 80%(to be preferably at least 85%) sequence similarity with (a)-(d) sequence, and there is regulation vegetable seeds The DNA sequence dna of magnitude function；Or
(f) DNA sequence dna complementary with any sequence of (a)-(e).
3. method as claimed in claim 2, wherein the mutation includes the nucleotide in the gene of regulation size of plant seed It carries out replacing, missing or adding one or more nucleotide in sequence.
4. method as claimed in claim 3, wherein the mutation by physical mutagenesis, chemical mutagenesis or RNAi, TALEN, The site-directed mutagenesis techniques such as CRISPR/Cas9 obtain.
5. application of any method of claim 1-4 in regulation size of plant seed.
6. a kind of application of expression cassette, expression vector and engineering bacteria in regulation size of plant seed, it is characterised in that the table It include the gene of regulation size of plant seed up to box, expression vector and engineering bacteria, the gene of the regulation size of plant seed Nucleotide sequence is selected from following group of one of sequence:
(a) nucleotide sequence as shown in the gene LOC_Os12g41820 of regulation size of plant seed；
(b) nucleotide sequence as shown in SEQ ID NO:1,2,20 or 21；
(c) its encoding amino acid sequence nucleotide sequence as shown in SEQ ID NO:3,55,56,57,58,59,60 or 61；
It (d) can be with the DNA sequence dna of the DNA hybridization of sequence described in (a)-(c) under high stringency conditions；Or
(e) there is at least 80%(to be preferably at least 85%) sequence similarity with (a)-(d) sequence, and there is regulation vegetable seeds The DNA sequence dna of magnitude function；Or
(f) DNA sequence dna complementary with any sequence of (a)-(e).
7. a kind of preparation method of mutant material, it is characterised in that the mutant material is by regulation size of plant seed Caused by the mutation of gene, the plant containing the nucleotide sequence after the mutation has the phenotype of large seed, wherein the tune The nucleotide sequence for controlling the gene of size of plant seed is selected from following group of one of sequence:
(a) nucleotide sequence as shown in the gene LOC_Os12g41820 of regulation size of plant seed；
(b) nucleotide sequence as shown in SEQ ID NO:1,2,20 or 21；
(c) its encoding amino acid sequence nucleotide sequence as shown in SEQ ID NO:3,55,56,57,58,59,60 or 61；
It (d) can be with the DNA sequence dna of the DNA hybridization of sequence described in (a)-(c) under high stringency conditions；Or
(e) there is at least 80%(to be preferably at least 85%) sequence similarity with (a)-(d) sequence, and there is regulation vegetable seeds The DNA sequence dna of magnitude function；Or
(f) DNA sequence dna complementary with any sequence of (a)-(e).
8. method of claim 7, wherein the mutation is point mutation or DNA missing or insertion mutation, or logical Cross the generation of the gene silencings means such as RNAi, rite-directed mutagenesis.
9. method according to any one of claims 8, wherein the nucleotide sequence after the mutation as shown in SEQ ID NO:4, is compiled The amino acid sequence of code is as shown in SEQ ID NO:5.
10. the application of any method of claim 7-9 and its mutant material obtained in breeding.
11. application described in any one of claim 10 is controlled wherein the breeding refers to using the gene of regulation size of plant seed The size or grain weight of crop kernel, or the molecular labeling as identification crop big grain kind and granule kind.
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