阅读说明：本技术 抑制OaGS3基因表达的物质在调控四倍体野生稻籽粒长度中的应用 (Application of substance for inhibiting OaGS3 gene expression in regulating and controlling length of tetraploid wild rice grains ) 是由 李家洋 余泓 孟祥兵 张静昆 刘贵富 荆彦辉 陈明江 于 2021-02-02 设计创作，主要内容包括：本发明提供了调控OaGS3基因的物质在调控四倍体野生稻产量/籽粒长度中的应用。本发明的具体实施例证明通过编辑基因OaGS3,可实现籽粒长度的增加,从而选育产量较高的四倍体野生稻,推进四倍体野生稻的驯化与利用。(The invention provides application of a substance for regulating an OaGS3 gene in regulating and controlling the yield/seed length of tetraploid wild rice. The specific embodiment of the invention proves that the increase of the grain length can be realized by editing the gene OaGS3, so that the tetraploid wild rice with higher yield is bred, and the domestication and utilization of the tetraploid wild rice are promoted.)

1. The application of a substance inhibiting the expression of an OaGS3 gene in increasing the yield or the grain length of tetraploid wild rice is characterized in that the OaGS3 gene encodes an OaGS3 protein, and the OaGS3 protein is a protein of A1, A2 or A3 as follows:

a1, protein of which the amino acid sequence is shown in any one of sequence 5 and sequence 6 in a sequence table;

a2, a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in any one of the sequence 5 and the sequence 6 in the sequence table, has more than 80% of identity with the protein shown in A1), and has the same function;

a3, a fusion protein obtained by attaching a protein tag to the N-terminus or/and the C-terminus of A1) or A2).

2. The use according to claim 1, wherein the OaGS3 gene is specifically the gene as shown in E1 or E2 below:

e1, the coding sequence of the coding chain is a DNA molecule shown in a sequence 3 or a sequence 4 in a sequence table;

e2, the nucleotide sequence is a DNA molecule shown in sequence 1 or sequence 2 in the sequence table.

3. The use of claim 2, wherein said inhibition of OaGS3 gene expression is achieved by genome-directed editing of the OaGS3 gene in tetraploid wild rice by the CRISPR/Cas9 system, said CRISPR/Cas9 system targeting the XXX sequence located on any of the nucleotide sequences of the OaGS3 gene, including xxxgg; wherein XXX is any nucleic acid sequence of 19-20bp in the DNA molecule sequence, and N is any nucleotide of A, T, G, C.

4. The use of claim 3, wherein the target sequence is a sequence from 392 th to 411 th of sequence 1 in the sequence listing.

5. The use according to any one of claims 1 to 4 wherein the agent is the CRISPR/Cas9 system; the CRISPR/Cas9 system includes the following 1) or 2):

1) the sgRNA targets are sequences shown as 392 th to 411 th positions of a sequence 1 or 579 th to 598 th positions of a sequence 2;

2) a CRISPR/Cas9 vector expressing the sgRNA.

6. The method for breeding the tetraploid wild rice with higher yield is characterized by comprising the following steps: inhibiting the expression of the OaGS3 gene in the receptor tetraploid wild rice to obtain the target tetraploid wild rice with higher yield than the receptor tetraploid wild rice.

7. The method for breeding the tetraploid wild rice with longer kernel length is characterized by comprising the following steps: inhibiting the expression of the OaGS3 gene in the receptor tetraploid wild rice to obtain the target tetraploid wild rice with the kernel length longer than that of the receptor tetraploid wild rice.

8. An agent, wherein the active ingredient of the agent is a substance that inhibits the expression of a gene encoding the OaGS3 protein, reduces the abundance of the OaGS3 protein, and/or knockouts the gene encoding the OaGS3 protein.

A CRISPR/Cas9 system, characterized in that the CRISPR/Cas9 system comprises 1) or 2) as follows:

1) the sgRNA targets are sequences shown as 392 th to 411 th positions of a sequence 1 or 579 th to 598 th positions of a sequence 2;

2) a CRISPR/Cas9 vector expressing the sgRNA.

10. Any one of the following Y1-Y4:

y1, the use according to any one of claims 1 to 5 in domesticated breeding of tetraploid wild rice;

use of Y2 or the method of any one of claims 6 to 7 in domesticated breeding of tetraploid wild rice;

use of Y3, the agent of claim 8, in domestication breeding of tetraploid wild rice;

use of Y4, the CRISPR/Cas9 system of claim 9 in tetraploid wild rice acclimation breeding;

y5, the use of the substance for regulating the OaGS3 gene as claimed in any one of claims 1 to 5 in rice breeding.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of a substance for inhibiting OaGS3 gene expression in regulating and controlling the length of tetraploid wild rice grains.

Background

Rice is one of the most important food crops in the world. The high and stable yield of the rice is of great significance for guaranteeing the world grain safety. However, the urbanization process aggravates to cause the acute reduction of the cultivated land area, the frequent increase of the risk of stable yield of grains in extreme weather and the slow increase of the crop yield brought by the traditional breeding technology. The grain safety problem is increasingly prominent and becomes a new challenge to people.

The high stalk tetraploid wild rice O.alta is a perennial alloploid rice and is mainly distributed in south America. Compared with diploid cultivated rice, the tetraploid wild rice with tetraploid stalks has the advantages of large biomass, perennial period, strong regeneration capacity, strong stress adaptability and the like. But the seeds are small without artificial domestication, and the thousand kernel weight is only 8.79 g. The grain length of the high-stalk tetraploid wild rice is increased through a genome editing technology, so that the grain weight is increased, and meanwhile, the purpose of increasing the yield is achieved by combining various advantages of the high-stalk tetraploid wild rice.

Disclosure of Invention

The technical problem to be solved by the invention is how to increase the yield of tetraploid wild rice.

In order to solve the technical problems, the invention provides an application of a substance inhibiting the expression of an OaGS3 gene in increasing the yield or the seed length of tetraploid wild rice, wherein the OaGS3 gene encodes an OaGS3 protein, and the OaGS3 protein is a protein of A1, A2 or A3 as follows:

a1, protein of which the amino acid sequence is shown in any one of sequence 5 and sequence 6 in a sequence table;

a2, a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in any one of the sequence 5 and the sequence 6 in the sequence table, has more than 80% of identity with the protein shown in A1), and has the same function;

a3, a fusion protein obtained by attaching a protein tag to the N-terminus or/and the C-terminus of A1) or A2).

In the application, the sequence 5 in the sequence table is composed of 221 amino acid residues, and the sequence 6 in the sequence table is composed of 211 amino acid residues.

In the above applications, identity refers to the identity of amino acid sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Perresilute Gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of identity can be obtained.

In the above applications, the 80% or greater identity may be at least 81%, 85%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

In the above application, the OaGS3 gene may be specifically a gene shown as E1 or E2 as follows:

e1, wherein the coding sequence (ORF) of the coding strand is a DNA molecule shown as a sequence 3 or a sequence 4 in the sequence table;

e2, the nucleotide sequence is a DNA molecule shown in sequence 1 or sequence 2 in the sequence table.

In the application, the inhibition of the expression of the OaGS3 gene can be realized by carrying out chemical mutagenesis, physical mutagenesis, RNAi, genome site-directed editing or homologous recombination on the OaGS3 gene in tetraploid wild rice.

In the above application, the genome site-directed editing may be implemented by using Zinc Finger Nuclease (ZFN), Transcription activator-like effector nuclease (TALEN), clustered regularly spaced short palindromic repeats (clustered regularly interspaced short palindromic repeats/CRISPR associated, CRISPR/Cas 9), and other technologies capable of implementing genome site-directed editing.

In the application, the genome site-specific editing can be realized by virtue of a CRISPR/Cas9 system. The CRISPR/Cas9 system, wherein the target sequence is XXX sequence located on any nucleotide sequence of OaGS3 gene including XXXGG; wherein XXX is any nucleic acid sequence of 19-20bp in the DNA molecule sequence, and N is any nucleotide of A, T, G, C.

The specific target sequence can be a sequence shown from 392 rd position to 411 th position of a sequence 1 in a sequence table or a sequence shown from 579 th position to 598 th position in a sequence 2.

In the above application, the substance may be a CRISPR/Cas9 system;

the CRISPR/Cas9 system includes the following 1) or 2):

1) the sgRNA targets are sequences shown as 392 th to 411 th positions of a sequence 1 or 579 th to 598 th positions of a sequence 2;

2) a CRISPR/Cas9 vector expressing the sgRNA.

In a specific embodiment of the invention, the recombinant vector of the CRISPR/Cas9 system comprises recombinant vector VK005-OaGS3 gRNA; the recombinant vector VK005-OaGS3gRNA contains a sgRNA expression cassette and a Cas9 encoding gene, and can express the sgRNA and Cas 9.

The invention also provides a method for breeding tetraploid wild rice (O.alta) with higher yield, which comprises the following steps: inhibiting the expression of the OaGS3 gene in the receptor tetraploid wild rice to obtain the target tetraploid wild rice with higher yield than the receptor tetraploid wild rice.

The invention also provides a method for breeding tetraploid wild rice (O.alta) with longer grain length, which comprises the following steps: inhibiting the expression of the OaGS3 gene in the receptor tetraploid wild rice to obtain the target tetraploid wild rice with the kernel length longer than that of the receptor tetraploid wild rice.

In the above method, the target tetraploid wild rice is tetraploid wild rice satisfying the following conditions: both the CC subgenome and the DD subgenome are mutated in the target region of the sgRNA.

In the above method, the tetraploid wild rice of interest is understood to include not only the first-generation to second-generation transgenic tetraploid wild rice but also its progeny. For transgenic tetraploid wild rice, the gene can be inherited in that species, or can be transferred into other varieties of the same species using conventional breeding techniques. The transgenic tetraploid wild rice includes seed, callus, complete plant and cell.

In order to solve the above technical problems, the present invention also provides an agent for increasing the yield or grain length of tetraploid wild rice, the agent having as an active ingredient a substance that inhibits the expression of a gene encoding the OaGS3 protein, reduces the abundance of the OaGS3 protein, and/or knockouts the gene encoding the OaGS3 protein.

In the reagent, the substance is a CRISPR/Cas9 system;

the CRISPR/Cas9 system includes the following 1) or 2):

1) the sgRNA targets are sequences shown as 392 th to 411 th positions of a sequence 1 or 579 th to 598 th positions of a sequence 2;

2) a CRISPR/Cas9 vector expressing the sgRNA.

The active ingredient of the agent may further contain other biological ingredients or/and non-biological ingredients, and the other active ingredients of the agent can be determined by those skilled in the art according to the yield-increasing effect or the grain length-increasing effect of tetraploid wild rice.

The invention also protects a CRISPR/Cas9 system comprising 1) or 2) as follows:

1) the sgRNA targets are sequences shown as 392 th to 411 th positions of a sequence 1 or 579 th to 598 th positions of a sequence 2;

2) a CRISPR/Cas9 vector expressing the sgRNA.

The invention also provides any one of the following applications of Y1-Y5:

y1, the use of the above in domestication and breeding of tetraploid wild rice;

y2, the application of the method in the domestication and breeding of tetraploid wild rice;

y3, use of the above reagent in domestication breeding of tetraploid wild rice;

y4 and the application of the CRISPR/Cas9 system in the domestication and breeding of tetraploid wild rice;

y5, and the application of the substance for regulating the OaGS3 gene in rice breeding.

The invention provides application of a substance for regulating an OaGS3 gene in regulating and controlling the yield/seed length of tetraploid wild rice. The specific embodiment of the invention proves that the increase of the grain length can be realized by editing the gene OaGS3, so that the tetraploid wild rice with higher yield is bred, and the domestication and utilization of the tetraploid wild rice are promoted.

Drawings

FIG. 1 is a dot diagram of the OaGS3 gene target site in O.acta in example 1 of the present invention.

FIG. 2 is a diagram showing the construction of VK005-OaGS3gRNA in example 1 of the present invention.

FIG. 3 shows the bands resulting from partial T0 amplification of OaGS3-CC and OaGS3-DD in example 2 of the present invention.

FIG. 4 is a graph showing the sequencing result of a partial genome editing T0 material mutation in example 2 of the present invention. Wherein, O.alta is wild type, and T0-1 and T0-2 are plants of T0 material with genome editing.

FIG. 5 is a photograph of a portion of genome editing material and a wild-type grain length phenotype in example 2 of the present invention. Wherein, O.alta is wild type, and T0-1 and T0-2 are genome editing material strains.

FIG. 6 is a graph showing the statistical results of the data of the partial genome editing materials and the length of wild-type kernels in example 2 of the present invention. Wherein, O.alta is wild type, and T0-1 and T0-2 are genome editing material strains. Data shown in the figure are mean ± sd, repeat number 20, representing significance analysis result P < 0.01.

Detailed Description

The following examples are intended to illustrate the invention without limiting its scope. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The binary Vector VK005-01 is Cas9/gRNA Vector in a plant Cas9/gRNA plasmid construction kit (cargo number: VK005-01) of Beijing Weishanglide Biotech Co.

The tetraploid wild rice in the following examples is tetraploid wild rice Oryza alta 2007-24, which is a high stalk tetraploid wild rice seed material (o. alta Swallen, chromosome set CCDD, No. YD-7900) in left column 1.1 of page 906 of document 1, and publicly available from institute of genetics and developmental biology or national germplasm naning tetraploid wild rice. Document 1: douglas et al, research on callus-induced differentiation of high-stalk tetraploid wild rice, journal of agriculture in the southwest, 2014,27(3), 905 and 909.

The transcript used in the examples below is T01, as an example only, and does not limit the editing sites in the application. The examples were carried out according to the usual experimental conditions or the product specifications, unless otherwise specified.

In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.

Example 1 selection of tetraploid OaGS3 target site and construction of CRISPR/Cas9 Gene editing vector

Selection of target sequences

Tetraploid high culm tetraploid wild rice Oryza alta is an allotetraploid, and most genes are copied on CC and DD subgenomes. Through alignment analysis, two homologous genes of OaGS3 exist in Oryza alta, the gene positioned on the chromosome 3 of the CC subgenomic group is named as OaGS3-CC, and the other gene positioned on the chromosome 3 of the DD subgenomic group is named as OaGS3-DD (the nucleotide sequence is shown as sequence 2).

OaGS3-CC comprises 5926 nucleotides, and the nucleotide sequence is shown as a sequence 1. The CDS sequence is shown in sequence 3, and the coded protein sequence is shown in sequence 5.

OaGS3-DD consists of 6193 nucleotides, and the nucleotide sequence is shown as a sequence 2. The CDS sequence is shown in sequence 4, and the coded protein sequence is shown in sequence 6.

As shown in FIG. 1, target sequences were selected on conserved regions of the first exons of OaGS3-CC and OaGS3-DD as follows:

target sequence: 5'-CCGGCAGCGCCCGACCCCTG-3' (as shown in sequence 1 from 392 rd to 411 th or as shown in sequence 2 from 579 th to 598 th);

the sgRNA was designed according to the target sequence and designated as OaGS 3-sgRNA.

Second, construction of recombinant plasmid

The following single-stranded primers with linker sequences (underlined) were synthesized:

VK005-OaGS3-F：(the sequence indicated by the wavy line is the target sequence and the sequence indicated by the underlining is the linker sequence);

VK005-OaGS3-R：(the sequence indicated by the wavy line is complementary to Target site 1 in reverse, and the sequence indicated by underlining is the linker sequence).

VK005-OaGS3-F and VK005-OaGS3-R were primer annealed to form Oligo dimers. BspQI single enzyme digestion is carried out on the binary vector VK005-01, vector fragments of about 17kb are recovered by gel purification, and enzyme connection is carried out on the vector fragments and the Oligo dimer under the action of T4 ligase, so as to obtain a recombinant vector, and sequencing is carried out by the following sequencing primers:

VK005 primer:5’-GCCATGAATAGGTCTATGACC-3’。

the structure of the positive plasmid after sequencing verification is shown in fig. 2, namely a sgRNA expression cassette is correctly inserted into the enzyme cutting site of BspQI, and the positive plasmid is named as VK005-OaGS3 gRNA. VK005-OaGS3gRNA contains a sgRNA expression cassette and a Cas9 encoding gene, and can express the sgRNA and Cas 9.

Example 2 transformed tetraploid Rice healing and phenotypic identification

1. Cultivating tetraploid wild rice O.alta plant with OaGS3 gene knockout

The plasmid VK005-OaGS3gRNA obtained in example 1 was used to transform Agrobacterium EHA105 by an electric stimulation method, and the callus of tetraploid wild rice O.alta was infected with Agrobacterium to culture the above callus to obtain transformed seedlings. Extracting the genome DNA of the transformed seedling, and amplifying OaGS3-CC and OaGS3-DD by using specific primers.

The primer pair for amplifying OaGS3-CC consists of OaGS3-CC-F and OaGS 3-CC-R:

OaGS3-CC-F:5’-CCTCCGCCATTTATAATCCA-3’；

OaGS3-CC-R:5’-TATGCATTCGTGGTTTCAGC-3’。

the primer pair for amplifying the OaGS3-DD consists of OaGS3-DD-F and OaGS 3-DD-R:

OaGS3-DD-F:5’-GCTGCCTTTCCATCATCATT-3’；

OaGS3-DD-R:5’-ATGTTGGGCCATGCATATTT-3’。

the electrophoresis pattern of the amplified product is shown in FIG. 3, and the genome editing material is selected and sequenced to determine the mutation type. Partial results are shown in FIG. 4, and OaGS3-CC and OaGS3-DD have frame shift mutation, which results in protein sequence change and protein function disruption. The transformed seedling containing the gene editing mutation is T0 generation transgenic tetraploid wild rice (numbered T0-1 and T0-2).

2. OaGS3 knock-out tetraploid wild rice phenotype O.alta

Respectively sowing T0 generations of T0-1 strain and T0-2 strain in the field of Hainan experimental base of institute of genetics and developmental biology of Chinese academy of sciences, harvesting seeds in the mature period of normal agronomic management, removing top awns, fully dehydrating, and performing seed length statistics to ensure at least 3 times of biological repetition.

The length photos of the seeds of the strains are shown in figure 6, and the lengths of the seeds of the gene editing strain T0-1 strain and the gene editing strain T0-2 strain are obviously longer than that of the wild type.

Therefore, in the tetraploid wild rice Oryza alta, rapid domestication of grains can be realized by editing the gene OaGS3, so that the grains of the tetraploid wild rice Oryza alta are lengthened, and the yield increasing potential of the high-stalk tetraploid wild rice is improved.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> institute of genetics and developmental biology of Chinese academy of sciences

<120> application of substance inhibiting OaGS3 gene expression in regulating length of tetraploid wild rice seeds

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ctactattac aaatctgaaa aaaaaacctg ttcaaattcg taatactagg atatcatatt 1980

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ccgcttgtct gcatcgacgc agctactggc catgggaagc ctccccatgc aaccaagctt 3720

cactgagagg ccgcacccct accgaccgag gagaggtggc cgccgcggag aggagggccg 3780

tcgccatcgc gtcgccagtt agaggcaagc cgtcaccttg gcgtggccgc gtagaggagg 3840

gccgctgctg tcgtgtcgcg atcgtcatcg tcggcttggc ttggagaggt ggagagatgg 3900

aggaggagat aaggtgagag agatgagaga aaatattctg acggcttgtt tggcaacttg 3960

agggaagagg attgggagtt tagacgggaa aattgatggt gagatctgtc gatgggttat 4020

acctgcaaac cgtattcgga agatatcggg gtacgctggt actagggcct acacaatatc 4080

gtatcaagca ctcagagaca aatgtttata cgagttcaag ccctactcca gtttatatgg 4140

attatggatg gaacaccaca aggattacaa taggaaaaca taacaaccta tctaatttgc 4200

cgactaggat ccaccaagaa tggcccaacg agatctaatc gacgaatacg actgcggctt 4260

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ttgatcgctg cctttctctt caagtagaac tcaacaagac atatttggat acgagcctga 4380

ggagacctta tcttctttga ataggacttt aacaccttct gttccgagaa gataatttcc 4440

ttttatcctt gccgtatttt cttaattaaa ggtaccttcc cctatatttt taggtatatg 4500

gtattcgtat actccatgaa tctatgtatg gagatatgtt ttatccttta ccctgaaaag 4560

attaatctga gaatttgatt tttagtctca atctcttatt tgatagagat ggtgagaatt 4620

gatagagagt ttagttgaac attagatttt aaatgaaaat agaaggttga gatttgttta 4680

gacaaagtaa aggaggaatt ctctcttaat taccaccctc ttcccaggta ttagaaagaa 4740

gagagttcct cctgaatttc ctatccccat cccactacaa ctcccatatc tttcaaccaa 4800

acagaacatt taatagcctc atccctttaa acttttaatc cctttcaaaa actccctcca 4860

accaaacgga ccgcgaggag ataagagatg gatggtggag ataaggtcgc gctggtaagc 4920

ggggtccacg gtgatttttt tctccacccg tgttgctgct gcaattctat gtcggctaat 4980

aaaaaaatca gcatctatag tttctttatt tgtcccattg ttttaaaaaa ccgtacttat 5040

aataaatggt gtttttaata aaccagtacc tataaaatgt tataggtgtc ggtttttgat 5100

tttagattca tgtgaggagt cgagtaggcc agaaatagct tcatatttca tatatgcctg 5160

ttcaaggcat atatgaaggt gtccgttatt aggggttttg tagtaatgtt tgttggaatt 5220

aatcacttgt atattatgaa accgatggct ggcgaaactt tgtcaattgt gggcggtaag 5280

ctcacctaaa gcaaattgtt caaaacaagg ttaaagatga attttattaa tggattttgg 5340

tttggaaaaa ttttgtaggg ttgacgaatt catcggaaga actcccgaac cattcataac 5400

gatgtatgaa ttttcaggtc gagaatttgt ctttaacttg gcacaactgt tactttttct 5460

tattaattct ctgtttacaa gcagttcatc cgagaagcga agtcatgatc attctcacca 5520

cttttggaag aagtttcggt acttacttga ttcccggatc ttaatgtacg tatgcatctg 5580

cactgcgcta attattattt acaaaactga actatttaat aaacactaca aaatatttaa 5640

cttgcaaagt acatatttaa tcagggattt agtaaaccca ttccaatcta atgtcaccat 5700

atctgcatgc acggtccaca tggatgctgg tgtattaatt tctttttgtg ctagaatgga 5760

cgttttattt tcaattgtcg ttagtataca cctttacgca tcatctctct cctgcgcatt 5820

tgagaaaact tcttcctttg atgatagacg tggtcttatt cttcagtttg atttagtttc 5880

agatagaata aatattgttt attacttagt ttctctcctt cagtaa 5926

<210> 2

<211> 6193

<212> DNA

<213> wild rice (Oryza alta)

<400> 2

acggctctga tccccgcggc gcagcggatc tagtccggat gggcagacga gacgagacga 60

gatgagatag atactagatc cgtccccgac aatctttcaa gccccgtggc cgtccctcct 120

ctgctcgatg tgccacgcct ctcagccttg ctgcctttcc atcatcatta ttcattcacg 180

cccaaagccc aaagcaattc acctctctct ctcgcacgaa tcgatagata cctgtaattt 240

cccccccgga aaagaaaaga gcggccattt ttctctcccc tccatcatta cttgcccaaa 300

aacggcaatc cctcctcccc catcgcctgc ttccatccat gtgcctccac cacctccgtc 360

ttccctccga ctgatcaaag cagctgctat cgctgctgca gcgccggcca tgcccgccct 420

ctagccccta cctaccctac cctaccctac tcggctactc ctactcctat atataccgtc 480

ctctctctct ctctctctct tgcgatatat ttcaagactc cagcgcgctc tccatcggag 540

tgatggcaat ggcggcggcg ccccggccca agtcgccgcc ggcagcgccc gacccctgcg 600

gccgccaccg cctccagctc gccgtcgacg cgctccaccg cgagatcgga ttcctcgagg 660

tacaacaata tccaatgtct tatcattccc tactccttct cttgcttcaa aaaaacatgt 720

cgtccatacc tcatattcat acacgtacgc tttttgctat cgctctctcc atgttttccc 780

aaccttaaaa accatcatgc atcatttgct tcttccgttc gacaaatcat ttgattagag 840

aagtttaaaa cattggaaca acaaatatgc atggcccaac attgctcctc tcgatctctt 900

ccctcagcca aattaacacg aacaaactta accaaacaag ttcaaccaac gatcggatag 960

gagtcaacca gcgctagcta gctgatgaac tagctagcaa gtccgttgac tgttcaacac 1020

taattaaggc accagttaat aagccgatta attaaacaat tatatgagct taacgggcgg 1080

ctagcttcgt cctctgggtc cgtgcggatc gtaccatcgg tttgcgcgtt cctccaccaa 1140

aactctctgc cttgttattc cctttgccta ggagcacata catttgcatg catcatcatc 1200

acatcaccag ttttaattgg attttaattt ggcggtctcc aaccatatca tattggtttt 1260

gttctctata ctagagtact ataatctcta gcggttttgt ggaactaaga aattataatt 1320

agccagggtt tgttaaccaa ttggtggttt agtttgttaa ttttagctag tactaggatt 1380

ttagacttag aggtcttcga gctactacta cgatatattg atgccatgca tcaatcgatg 1440

ctggtccatg ctagtttctg actagccctc ttaattgggc ttgacctact taattaatta 1500

accagtggct gcgtcactca ttgaccaaca ttgtcatgtt acccggactg atattttttt 1560

tataaaaaaa actaccaccg gtcgatatat tattagttag tgtatatatc tgctcaagca 1620

gcgcatgcac atggattttc gtcaaacaaa atatgtactg tatgctcaat gcatctgtgg 1680

gacgattgtc atattcgcct ttataattaa aatggtggtg cccagttgtt ttttttctcc 1740

aataatttat tgacttaaat ttcctgttct gttaggagta aaactattaa ttagcaccat 1800

tttaaaagtt tctaaaatct taataaagta tatgacactg caaccttact gtatatatat 1860

gttgtgtttg ttcattagca gtggcagtac ttgctgtcta actttaaatg ttttgggcgt 1920

taaaatatcc cccagacatc acctgaaaag ttgacaggct aaacacatgc ccatctccct 1980

cgtttactta aattaattcg aacaaacaac tatttcttgc agggtgaaat aaattcaatc 2040

gaagggatcc acgctgcctc cagatgctgc agagagtaag ttagccctgc tgtttctttt 2100

tgtacttcca tttcttctcg tctttaaact atccgcgacg ctacttactc taaattccta 2160

ctctaaagga atattccatg cctggtcagt gagattcctt actctatatc aaatcttccg 2220

cgctcgtgtt tactctatat cttcctctat aattttttaa ctatattttg aaaagtctat 2280

tttacctcct ccatcccccc aataaaaccc cgcactccta ctgcatcagc ggctcccccc 2340

cccgtcggtt gctccgccac cgccgatccc cctcccccgt gcccgaatcc ccccgccgca 2400

gccgtcgccg aatcccccct cccccagccg catcgcccct ccgccagccg catcggctcc 2460

tcccccgccg cccccgtcgc ccggcgtccc tctccctgcc gcggcttcgt cccgcatcag 2520

ctcctccccc ccgcctgtcg gccttcaccg cgccaacccg catcggctcg gaggcgcgga 2580

ggcggaggag ccgatggggc ggcgcgccgg ggcatcggcg gaggaagggc gtcgtggcgg 2640

atccgccgag ggagagcgct ctccgccgcg ccctccgccg agctctccgc ctcgctcgcc 2700

ctctccgcct cgcttgccct tccgctgtcg ctcgccgctg ctcctgcttc gttcccctcc 2760

tccccttctt ccgtccgtgc gcgggaggga cggacagttg gccgtcgccc gcgcgcgcgg 2820

cctatacagg ctgctacagt gctccgctac ggatagcgga gcactgtagc ccctggacga 2880

tacaggcgcc tttagcggat ggcgctgcag ccgcgccgtc cgctaccgcg gatagatcgc 2940

ggacggcgct gcggatagtc ttagtactct taccatgcat tcacaaaata tacttacctc 3000

ggttgatcat ggatagataa actttgaccg ttatcttttt aagcaacttc aataaaaata 3060

ggtttaaatg cagggtatta ggtatatatt ttcaattgca aatgtaacat acagcattga 3120

ttctaaaaca ttggcaaatc aaagtaaaaa actgaacatt tgatttaatt ctgaacgttg 3180

ggagtatttt ttttcatcat ttacaaatga gtacgttgtg gaattatatt gtgcaagctt 3240

ttgagactaa ttaggttata tgagatacta tctaccaagg ttgtcagtgt cttatgatac 3300

caggatccta ttaaagcgaa acgatattaa tcccatctag tatctaatta tcatagtatc 3360

tcatcttact atttatttcc atatgatcct acgaaatctt aggtagtatc ccgattctac 3420

gatccctctg atcctataaa atattattta aaataaggtg gattgaaaat aatatgaata 3480

aatatactca agtttacata aaaccaatta aatttatcaa aatcaatatt atatgcttta 3540

atttgtacaa catattatat attttatata aaaatgtata ctttctaaaa tctgttagta 3600

ttccgatcct atgatactac caataaaaaa cgattctacc tggtatctcc atatgctatc 3660

tattagttgc ttgaggtaaa acaaaatgta gttaggttac atttactaaa ctgaacattt 3720

agtaatgttt tgttaaataa cggtaatttc aatgcatgca tgtcctcccg taaaaacagt 3780

gcagagactg ttcaaaaagt cattgcacaa taactattca catggaactg tgaaaagtat 3840

atattggaac ttactagatc cttttgggaa catgggaaaa gccaaagtca cgtgtggaat 3900

ccctattccc tgtgttcttt ttgaaaggat ccagttatgg cccgtttagt tcgcgaaatt 3960

tttttcaaaa acatcacatc aaacgtttga ccgaatgtcg ggaggggttt tcggacacga 4020

atgaaaaaac taatttcacg gttagcctgt aaaccacgag acgaattttt ttgagcctaa 4080

ttaagccgtc aacatgtagg ttactgtagc acttacggct aattatggcg taattatgac 4140

aaaattaggc tcaaaagatt cgtctcgtcg tttacaatcc aactgtgcaa ttagtttctt 4200

tttttatcta tatttaatgc ttcatgcatg agtccaaaag tttgatgtga tgtttttggg 4260

gttttcgttt tgggaactaa acaaggcctt agctagaaga gtggaaattg tactactact 4320

gagatgaaat tacagcagga gcagaaaggg ggacaaactg aaaattaaac aagcctcaaa 4380

attcaacatc agcaaccgac tcatctagtg ttcctgtgtt atgaaccagc cggcaactat 4440

gcctctatgt agcgcattga agcaccttaa ctaagggcta tcaaggctgt gttaaactgt 4500

taatggtaca tatgcctttc agtttctgtc tgtactttag gttgctatac atcctatgaa 4560

catgttttct tgtctttctt cttacctcgg ttgaaaaacc catacaaata tcatttttta 4620

actgactact cactaaataa aaagaatcaa atgtgccgat ttaagaactg atgcctttaa 4680

aattttaata ataagaagaa aaataccagt tcgatgcatc gtctcgaatc gaccgaatcg 4740

aaccgatgct tcaaccggcc cccatccctc gagaggacga ctcccattct agaaacatgg 4800

acatggtaac acagaaattt tcgaccagca catatgaaat aaccgaatca attagttcag 4860

aatccttcca ctttcgaatc ttgtagaact tctatccttt cggaaacaag agggggaagg 4920

aggaggcaac aacaaagtat gggaactgag aacaaaataa atatgccgat gtacacaagc 4980

atgaaaagtt aaagatctca aaggaaagga gatcataaat caagaaccaa gaaccagctc 5040

actcggtcca gttcatgctc gttagctagc ttccatgtag caagaaggca cgcagccatc 5100

gcacatagtg tttgtaattt cggtagccac cgaaatttcg gtggttaccg aaattttgaa 5160

aaaaatcatg aatttcggtg ttttctgact gatttttttt tcaattttaa ctgagtttga 5220

acaaatttgg atcaaattca caaatatttg aaaaaatcca aaaaatttgg ggagatttga 5280

tcgtgccgat agggtccgaa atttcaaacc atgatcgcac acttcaccgc ttgtctgcat 5340

cgacgctggc cgtgggaagc ttccccgtgc aaccaagctt caccgagcgg ccgcacccct 5400

gccgatcgaa ggagaggtgg ccgccgtgga gaggagggcc gctgccgtcg cgtcgccggg 5460

tagaggcaag ccttcacctt ggcgtggccg cagacaggag ggccgctgct gtcgcgtaac 5520

ggccgtcgtc gccggcttgg cttggagagg tggagagaga tggaggagga gatgaggtta 5580

gagagagaga ggagaaagag gtaagaaaat atccagagga gataagagat gcatggtgga 5640

gataacgtcg cgctgggcaa gcgtggtcca cactgatttt ttccccgccc gtgtcgctgc 5700

tgcaattctg tgctggctaa taaaaaaaat cagataccta taaaatgtta tagatgtcgg 5760

tttttaattt taaactcatg tgagtagtcg ggtggataaa aaatggtctc gtctgccagt 5820

tgaatatgca tatataaaga cgtccactat tagggtttta tagtagttag ttagaattaa 5880

ttagttgcat attaaccaga aggctggcga aactttgtca attgtgggcg ctaatctcac 5940

ctgaagcaaa ttgttcaaaa caaggttaaa gatgattttt attaatgaat tttggcttgg 6000

aaaaattttg tagggttgac gaattcatcg gaagaactcc cgatccattc ataacgatgt 6060

atggattttc aggtcgagaa tttgtctttt aacttggcac aaccgtactt tttcttatta 6120

attctctgtt tacaagcagt tcatcggaga agcgaagtca tgatcattct caccacttat 6180

tgaagaagtt tcg 6193

<210> 3

<211> 666

<212> DNA

<213> wild rice (Oryza alta)

<400> 3

atggcaatgg cggcggcggc ggcggcgccc cggcccaagt cgccgccggc agcgcccgac 60

ccctgcggcc gccaccgcct ccagctcgcc gtcgacgcgc tccaccgcga gatcggattc 120

ctcgagggtg aaataaattc aatcgaaggg atccacgctg cctccagatg ctgcagagag 180

gttgacgaat tcatcggaag aactcccgaa ccattcataa cgatttcatc cgagaagcga 240

agtcatgatc attctcacca cttttggaag aagtttcggt acttacttga ttcccggatc 300

ttaatgtacg tatgcatctg cactgcgcta attattattt acaaaactga actatttaat 360

aaacactaca aaatatttaa cttgcaaagt acatatttaa tcagggattt agtaaaccca 420

ttccaatcta atgtcaccat atctgcatgc acggtccaca tggatgctgg tgtattaatt 480

tctttttgtg ctagaatgga cgttttattt tcaattgtcg ttagtataca cctttacgca 540

tcatctctct cctgcgcatt tgagaaaact tcttcctttg atgatagacg tggtcttatt 600

cttcagtttg atttagtttc agatagaata aatattgttt attacttagt ttctctcctt 660

cagtaa 666

<210> 4

<211> 651

<212> DNA

<213> wild rice (Oryza alta)

<400> 4

atggcggcgg cgccccggcc caagtcgccg ccggcagcgc ccgacccctg cggccgccac 60

cgcctccagc tcgccgtcga cgcgctccac cgcgagatcg gattcctcga gggtgaaata 120

aattcaatcg aagggatcca cgctgcctcc agatgctgca gagaggttga cgaattcatc 180

ggaagaactc ccgatccatt cataacgatt tcatcggaga agcgaagtca tgatcattct 240

caccacttat tgaagaagtt tcggtactta cttgattccc ggatcttaat gtacatatgc 300

atctgcactg tgctaattgg tgtgcattat atggtcgtaa gtcctagtta cttattattt 360

acaaaactga actaataaac actacaaaat atttaacttg caaagtatcc aatctaatgt 420

cactatatct gcatgcatgg tccacatgga cgctggtgta ttattttttt tgtgctagta 480

tggacgtttt attttcaatt gtcgcttagt atacaccgtt acgcatcata tctctcctgc 540

gcatttgaga aaacttcttc ctttgatgat agacgtgggc ttattcttca gtttgattta 600

gtttcagata gaataaatat tgtttaatac ttagtttctc tccttcagta a 651

<210> 5

<211> 221

<212> PRT

<213> wild rice (Oryza alta)

<400> 5

Met Ala Met Ala Ala Ala Ala Ala Ala Pro Arg Pro Lys Ser Pro Pro

1 5 10 15

Ala Ala Pro Asp Pro Cys Gly Arg His Arg Leu Gln Leu Ala Val Asp

20 25 30

Ala Leu His Arg Glu Ile Gly Phe Leu Glu Gly Glu Ile Asn Ser Ile

35 40 45

Glu Gly Ile His Ala Ala Ser Arg Cys Cys Arg Glu Val Asp Glu Phe

50 55 60

Ile Gly Arg Thr Pro Glu Pro Phe Ile Thr Ile Ser Ser Glu Lys Arg

65 70 75 80

Ser His Asp His Ser His His Phe Trp Lys Lys Phe Arg Tyr Leu Leu

85 90 95

Asp Ser Arg Ile Leu Met Tyr Val Cys Ile Cys Thr Ala Leu Ile Ile

100 105 110

Ile Tyr Lys Thr Glu Leu Phe Asn Lys His Tyr Lys Ile Phe Asn Leu

115 120 125

Gln Ser Thr Tyr Leu Ile Arg Asp Leu Val Asn Pro Phe Gln Ser Asn

130 135 140

Val Thr Ile Ser Ala Cys Thr Val His Met Asp Ala Gly Val Leu Ile

145 150 155 160

Ser Phe Cys Ala Arg Met Asp Val Leu Phe Ser Ile Val Val Ser Ile

165 170 175

His Leu Tyr Ala Ser Ser Leu Ser Cys Ala Phe Glu Lys Thr Ser Ser

180 185 190

Phe Asp Asp Arg Arg Gly Leu Ile Leu Gln Phe Asp Leu Val Ser Asp

195 200 205

Arg Ile Asn Ile Val Tyr Tyr Leu Val Ser Leu Leu Gln

210 215 220

<210> 6

<211> 211

<212> PRT

<213> wild rice (Oryza alta)

<400> 6

Met Ala Ala Ala Pro Arg Pro Lys Ser Pro Pro Ala Ala Pro Asp Pro

1 5 10 15

Cys Gly Arg His Arg Leu Gln Leu Ala Val Asp Ala Leu His Arg Glu

20 25 30

Ile Gly Phe Leu Glu Gly Glu Ile Asn Ser Ile Glu Gly Ile His Ala

35 40 45

Ala Ser Arg Cys Cys Arg Glu Val Asp Glu Phe Ile Gly Arg Thr Pro

50 55 60

Asp Pro Phe Ile Thr Ile Ser Ser Glu Lys Arg Ser His Asp His Ser

65 70 75 80

His His Leu Leu Lys Lys Phe Arg Tyr Leu Leu Asp Ser Arg Ile Leu

85 90 95

Met Tyr Ile Cys Ile Cys Thr Val Leu Ile Gly Val His Tyr Met Val

100 105 110

Val Ser Pro Ser Tyr Leu Leu Phe Thr Lys Leu Asn Thr Leu Gln Asn

115 120 125

Ile Leu Ala Lys Tyr Pro Ile Cys His Tyr Ile Cys Met His Gly Pro

130 135 140

His Gly Arg Trp Cys Ile Ile Phe Phe Val Leu Val Trp Thr Phe Tyr

145 150 155 160

Phe Gln Leu Ser Leu Ser Ile His Arg Tyr Ala Ser Tyr Leu Ser Cys

165 170 175

Ala Phe Glu Lys Thr Ser Ser Phe Asp Asp Arg Arg Gly Leu Ile Leu

180 185 190

Gln Phe Asp Leu Val Ser Asp Arg Ile Asn Ile Val Tyr Leu Val Ser

195 200 205

Leu Leu Gln

210