CN108315336B - Application of gene PIS1 for controlling development of rice spikelets - Google Patents

Abstract

The invention belongs to the technical field of plant genetic engineering, and discloses a gene for controlling development of rice spikeletsPIS1The use of (1). In particular to the positioning, cloning, functional verification and application of a gene for controlling the development of rice spikelets. SaidPIS1The nucleotide sequence of the gene is shown as a sequence table SEQ ID number 1, and the coded amino acid sequence is shown as a sequence table SEQ ID number 2.PIS1Determine the development of floral organs of the rice spikelet, particularly the development of pistils and stamens. The expression of the gene in rice is purposefully regulated by utilizing the genetic engineering technology, so that the fertility of the rice can be regulated, and a new germplasm for rice breeding is created. Therefore, the invention has good application prospect for genetically modified rice.

Description

Application of gene PIS1 for controlling development of rice spikelets

Technical Field

The invention belongs to the field of plant genetic engineering. In particular to a gene for cloning rice floral organ development by using a map-based cloning technologyPIS1(Panile In Spikelet) and the function of the gene was verified by using a transgene complementation experiment. At the same time, it also relates to the use of said gene to regulate the development of rice sexual organ, i.e. to regulate by using genetic engineering methodPIS1The expression of the gene can control the fertility of rice and create new rice breed. Therefore, the invention has good application prospect for genetically modified rice.

Background

Rice is the first crop cultivated in China and plays a significant role in national economy. The rice spike is one of the most important agronomic traits of rice and consists of a plurality of small spikes. The rice spikelet is a reproductive organ and also a basis for forming grains, and any important gene related to the rice spikelet has functional mutation, so that the abnormal development of the floral organ of the rice can be caused, and the rice cannot bear fruit. Therefore, spikelets are always the focus of attention of rice genetic improvement workers, and deep understanding of the molecular mechanism of spikelet formation has great significance in guiding rice production, particularly the variety breeding of hybrid rice.

Flowers are the most prominent feature of plants, and flower formation is a major turning point in the life history of plants. The formation of plant floral organs is a complex biological process in which multiple genes are involved in regulation. More than 20 years ago, the research is carried out by utilizing the mutant of dicotyledonous plants such as arabidopsis thaliana and the like, the molecular genetic research of the development of the floral organs is greatly progressed, a plurality of floral organ characteristic genes which mainly comprise MADS genes are cloned in sequence, and a genetic model which determines the formation of the floral organs is provided. The structure of the rice floret is specific and is obviously different from that of dicotyledonous plants, but at present, the MADS gene is still generally considered to be relatively conservative in function of characteristic genes of the main floral organs, and a floral organ development model based on the dicotyledonous plants is also basically suitable for the monocotyledonous plants such as rice and the like.

Although the role of floral organ signature genes is important, they are not the only determinants of floral organ formation in plants. Recently, it has been discovered that upstream regulatory genes (mainly transcription factors) as flower organ characteristic genes have been confirmed to be involved in determining the formation of plant flower organs by activating or inhibiting the expression of the flower organ characteristic genes at different times, at different sites or in different regulatory pathways. In recent years, a small number of plants, but widely distributed in plants, have been identifiedRBR(RB-related) gene. The genes are not only involved in regulating the process of the cell cycle and maintaining the balance between cell proliferation and differentiation, but also in controlling the size of cells and the death of cells and maintaining the division, proliferation and differentiation capacity of stem cells, thereby playing a vital role in controlling the tissue differentiation and organ formation of roots, stems, leaves, flowers, seeds and the like of plants.

2 of the rice plantsRBRGenes, but their specific functions are not clear. The invention clones a factor for controlling the development of rice spikelets by using a map-based cloning methodPIS1A gene encoding an RBR protein (OsRBR 1).PIS1The functional deficiency of (A) causes abnormal development of the young panicle of rice and complete degeneration of sexual organs, thereby reducingPIS1The expression of (a) then causes male sterility. Therefore, the expression of the gene in rice is purposefully regulated by utilizing the genetic engineering technology, the fertility of the rice can be regulated, and a new rice breeding germplasm is created.

Disclosure of Invention

The invention aims to provide a gene for controlling development of rice spikelets, wherein the inactivation of the gene causes abnormal development of rice floral organs, and the reduction of the expression level of the gene causes male sterility. The invention also provides application of the transgenic plant of the gene in improving rice fertility.

In order to realize the purpose, the following technical scheme is adopted:

PIS1the gene has a DNA sequence shown in SEQ ID No.1, and also comprises a gene sequence which has at least 90 percent of homology with the DNA sequence shown in SEQ ID No. 1. The protein encoded by SEQ ID No.2 of the present invention is a plant RBR protein, which includes functional analogs obtained by performing one or more amino acid substitutions, insertions or deletions. In addition, mutants, alleles or derivatives produced by adding, substituting, inserting or deleting one or more nucleotides in SEQ ID No.1 are also included, and sequences having the same function can also achieve the object of the present invention.

Rice cloned according to the inventionPIS1Single base mutants of the gene showed marked abnormalities in spikelet development, but were completely normal for other traits (see example 1). Will function normallyPIS1After genetic transformation of the mutant, the plants recovered to their normal phenotype (see example 2).

The invention also provides a usePIS1The invention provides a method for carrying out efficient plant transformation on a gene, and particularly provides a carrier of a sequence gene shown in SEQ ID No.1 or a part of similar functional fragments of the gene, as shown in figure 4 (see example 2). The vector also has a host cell containing the above expression vector. Such host cells include E.coli, Agrobacterium and plant cells.

The invention utilizes antisense RNA technology to inhibit endogenous sources of ricePIS1The expression of the gene causes male sterility of rice but other characters are completely normal, and can be used for cultivating rice sterile line materials (see example 3). Specifically, the method comprises the following steps ofPIS1The gene is fused with other regulatory elements such as a constitutive promoter or an organ-specific promoter to construct a gene suppression expression vector, and the fertility of rice is artificially controlled by a transgenic technology (such as antisense RNA or RNAi) to create a new rice male sterile germplasm, which can be used for breeding new rice varieties.

The specific technical steps for realizing the invention are as follows:

ricepis1Isolation and genetic analysis of mutants:

the rice variety Minghui 86 is mutagenized by radiation, and the mutant with obviously abnormal development of spikelet but completely normal other characters as shown in figure 1 is obtained by the inventionpis1. Except for the normal development of the palea, other floral organs of the spikelet of the mutant are obviously abnormal, the determinative loss of the floral meristem of the mutant is realized, and a spike-shaped structure grows in the center of most spikelets. Through mutation heterozygote plant selfing and positive and negative crossing experiments with wild plants, the method proves thatpis1Is a genetic rule conforming to the control of a single gene.

Second, map location cloningPIS1Gene:

1．PIS1positioning:

to separatePIS1The gene, the method of map-based cloning adopted by the invention, firstly creates an F₂Locate a group consisting ofPIS1F obtained by hybridizing mutant heterozygote serving as female parent and DZ60 serving as male parent₂In (1)psd1And (4) mutant composition. Using rice microsatellite marker and InDel marker pairsPIS1The gene is located, andPIS1genes were finely localized to a range of about 32kb between InDel4 and InDel5 on BAC clone AP004592 as shown in FIG. 2, and candidate genes were identified by analyzing genomic sequence differences of mutants from wild type in this section. The positioning results are shown in fig. 2.

2．PIS1Identification and functional analysis of genes:

a complementary experimental vector as shown in FIG. 3 was constructed. The invention transfers complementary vector into the gene transfer technologypis1The transgenic rice with the normal phenotype as shown in figure 4 is obtained after the mutant, and the correct cloning of the invention is provedPIS1A gene; the analysis of the amino acid sequence shows that,PIS1encodes an RBR protein.

Thirdly, inhibiting the endogenous source of the ricePIS1To create a novel male sterile line material of rice

Inhibition of endogenous sources in rice by antisense RNA technologyPIS1The expression of the gene causes male sterility of the small ears of rice but can not bear fruit, and can be used for cultivating a novel sterile line material of the rice (see example 3).

The invention has the advantages that:at present, the heterosis of hybrid rice is mainly used for cultivating high-yield rice varieties. The mutant of rice with obviously abnormal development but completely normal other characterspis1The mutation is a single-gene recessive mutation and accords with the Mendelian inheritance rule. The invention obtains through a map location cloning technologyPIS1The gene and the function of the gene is identified by a function complementation experiment. Amino acid sequence analysis shows that the gene codes an RBR protein. Thereby reducing the endogenous source of the ricePIS1The expression of the gene causes the complete male sterility of the rice spikelets. Therefore, the expression of the hybrid rice can be purposefully regulated in rice by utilizing the genetic engineering technology, and further, a new rice sterile line germplasm can be cultured so as to improve the yield of rice, improve the quality of hybrid seed production and reduce the production cost. Therefore, the gene has very important application value and wide application prospect.

Drawings

FIG. 1: rice wild type and corresponding mutantpis1(iii) a phenotype of (a); a: wild type and corresponding mutantspis1Spikelet phenotype of (a); b: phenotype of wild spikelets (opening the inner and outer palea pieces); c:pis1phenotype of mutant spikelets.

FIG. 2:PIS1fine localization of genes and candidate gene mapping.

FIG. 3: map of the complementary experimental vector.

FIG. 4: wild type, wild type,pis1Mutant and complementation experiment T₀Representative diagram of generation spikelets.

FIG. 5: RNAi experiment vector pTCK303-OsRBR1A map of (a).

FIG. 6: wild type and RNAi-T₀A fertility phenotype diagram of the representative spikelets; a: male fertile and RNAi-T of wild type₀A phenotypic map of male sterility of the generations (inner and outer palea pieces removed); b: high fruit set percentage and RNAi-T of wild type₀Representing a representative diagram without firmness.

Detailed Description

Example 1: rice (Oryza sativa L.) with improved resistance to stressPIS1Map-based cloning of genes

1. Rice material and location populations:

rice protrusions as shown in FIG. 1Variantspis1The original wild-type parent is indica-type rice variety Minghui 86, which is obtained by the method of radiation mutagenesis. Crossing the mutant heterozygote with the wild type variety DZ60, F₁Selfing for generation to obtain F2 population, which is based on the heading date of ricepis11148 plants were selected as the mapping population for the mutant phenotype.

2. Positioning by using rice microsatellite marker (RM or SSR) and InDel markerPIS1Gene:

the total DNA of the rice is rapidly extracted by adopting a rice micro-method. Approximately 0.3 g of rice leaf was taken, put into a 1.5ml centrifuge tube, frozen with liquid nitrogen, and then DNA was rapidly extracted, and the obtained DNA was dissolved in 100 ul of ultrapure water. Preliminary positioning by using a DNA pool positioning methodPIS1A gene. Uniformly selecting microsatellite primers distributed on each chromosome to carry out PCR amplification according to a published rice microsatellite genetic map, separating on 6% non-denaturing polyacrylamide gel, dyeing with silver nitrate, and detecting polymorphism of a PCR product. 208 mutants in the F2 population were then selected for linkage analysis, andPIS1the gene is initially located between two markers RM256 and RM447 on the long arm of chromosome 8 of rice.

In the fine positioningPIS1In gene, linkage analysis of microsatellite markers and InDel markers was performed on 1148 mutant individuals in the F2 population. According to BAC sequence analysis between molecular markers RM256 and RM447, 28 pairs of SSR primers and 15 pairs of InDel primers are designed for fine positioning by using published rice genome sequencePIS1Genes, of which 4 pairs of SSR primers and 4 pairs of InDel primers (primer sequences are shown in Table 1), have polymorphisms between the two parents. The 1148 mutant individuals in the F2 population were subjected to linkage analysis using these 8 pairs of polymorphic markers. The result of the fine positioning is shown in fig. 2.

TABLE 1 for Fine positioningPIS1Primer sequences for genes

3. Gene prediction and comparative analysis

According to the result of the fine positioning,PIS1the gene was located in the range of about 32kb between ID4 and ID5 on BAC clone AP 004592. According to the gene annotation information provided on the TIGR (http:// rice. plant. msu. edu /) website,PIS1the 32kb interval of the gene is provided with 5 genes which respectively code mtN9 protein, RBR protein, PPR protein, zinc finger gene protein and an unknown protein. The genome sequence analysis result shows that one of the regionsOsRBR1The gene is mutated by a single base (TGG → TAG) resulting in premature termination of transcription. While the genomic sequences of the remaining 4 genes were completely identical in the mutant and in the wild type. Therefore, willOsRBR1As a genePIS1The candidate gene of (1). The results of candidate gene analysis and determination are shown in FIG. 2.

Example 2 complementation experiment

1. Constructing a complementary experimental vector. According to the sequence of the indica rice Minghui 86 gene, a complementary experimental vector of the candidate gene shown in figure 3 is constructed by utilizing the pCAMBIA1300 vector with increased enzyme cutting sites. The specific process of vector construction is as follows: designing a pair of primers with enzyme cutting sites in a candidate OsRBR1 gene region and a candidate promoter region, carrying out high-fidelity PCR amplification and sequencing by using high-fidelity enzyme, selecting clone with completely correct sequence, carrying out enzyme cutting and connection to construct a vector shown in figure 3, and transferring the vector into agrobacterium. The DNA sequence of the complementary vector is 8547bp, including the fragments of 2000bp before the start code and 1000bp after the stop code. Primers used to amplify the DNA sequences for the complementary experiments were (underlined restriction sites): an upstream primer: CAGTG CTCTTCatagcagataaccccaatcaagtccat gag ctgtgatcc, respectively; a downstream primer: CAGTGCTCTTCagacatcaaaatccacata cacac tattaaaatgtacat。

2. Complementary experiment:

seeds cannot be harvested due to sterility of the mutant. Therefore, the mutant gene can only be preserved by the heterozygote seed, and the mutant can be separated from the selfed progeny. The seeds used in the complementation test are seeds harvested from mutant heterozygote plants, and single-seed induction of embryogenic callus is adopted to ensure that the calluses formed by each seed are not mixed. Due to the fact thatpis1The phenotype of (2) is caused by single base mutation, so that a sequencing identification method is adopted to selectThe genotype ispis12 for transformation experiments.

Transforming by callus infection methodpis1Callus of the mutant. Selecting embryogenic callus particles with rapid growth, bright yellow color, smooth surface, compact texture, and diameter of 2-3mm as transformation receptor. Impregnating the rice callus with agrobacterium EHA105 strain containing binary plasmid vector, and culturing in dark at 25 deg.C for 3 days; culturing with 30 mg/LHygromycin screening culture medium for 2 times, each for 15 days; culturing in 50 mg/L screening culture medium for 7 days; transferring the resistance callus obtained by 3 times of screening into a differentiation culture medium, and performing differentiation culture under the illumination condition; when the seedlings grow to 2-3cm, the seedlings are transferred to 1/2MS culture medium for rooting culture. The 17 regenerated plants obtained are identified and continuously observed, and the growth and development conditions of the transgenic rice plants are shown in figure 4, the flower organs of all the transgenic plants are normally developed and have no obvious difference compared with the wild type, which indicates that the candidate isOsRBR1The gene isPIS1A gene.

Example 3 inhibition of endogenous sources in RicePIS1（OsRBR1) Expressing and cultivating novel rice male sterile germplasm

According to the indica rice Minghui 86OsRBR1Gene sequence, construction of endogenous suppressor Using pTCK303 vectorOsRBR1A vector for gene expression. The specific process of vector construction is as follows: firstly, extracting total RNA of wild rice leaf, then making reverse transcription into cDNA, using cDNA as template and making PCR amplificationOsRBR1The cDNA sequence of (a); the PCR product is connected to a pGEMT-easy vector after recovery and purification; after the sequencing confirms complete correctness, the primer and the primer are used simultaneouslyBamH1 andSac1 double enzyme digestion pGEMT-easy vector and pTCK303 vector, respectively recovering enzyme digestion products, and then utilizing T4 DNA ligase to carry out ligationOsRBR1The cDNA of (a) was ligated in reverse direction to the downstream of Ubiquitin promoter of pTCK303 vector to construct the RNAi expression vector pTCK303-OsRBR1Then, it is transferred into agrobacterium. Amplification ofOsRBR1The primer sequences used for cDNA of (1) (the restriction sites are underlined, respectively)Sac1 andBamH1) an upstream primer: 5' -CGAGCTCccatacatcagaagatgagttgag-3', downstream primer：5'-CGGGATCCTGAAGGAAGGGG AGAGATTGAGCT -3。

The constructed RNAi vector is transformed into callus induced by flower seeds in rice varieties by using a callus infection method technology to obtain transgenic plants of antisense RNA (the transformation method is the same as the second part of the example 2). As a control, 17 transformation lines as shown in FIG. 6 were obtained in total from the plants differentiated from the empty vector of pTCK 303. The vegetative growth of these transgenic plants was normal, but the stamens of the floral organs were dysplastic and pollen-free, resulting in complete failure of fruit. Thus, genetic engineering methods can be used to control endogenous sources in ricePIS1Can breed the novel male sterile germplasm of rice.

The above description is only a few specific embodiments of the present invention, and it should be noted that all modifications that can be directly derived or suggested from the disclosure of the present invention by those skilled in the art are deemed to be within the scope of the present invention.

SEQUENCE LISTING

<110> Fujian agriculture and forestry university

<120> application of gene PIS1 for controlling development of rice spikelets

<130>22

<160>22

<170>PatentIn version 3.3

<210>1

<211>3036

<212>DNA

<213> Artificial sequence

<400>1

atggagggtg ccgcgccgcc agcgagctcc gggtcggagg tgacgggtgc gggctcgggg 60

aaggtggacg ctggcggcgg cgccgccatg gaggagcggt tcgccgatct gtgcaagagc 120

aagcttgggc tggacgagag cataacgagg caggcaatgc agctgttcaa ggagagcaag 180

agcatcctcc tatccagcat gtcatccttg ggcagtggat cgcctgagga gatcgagagg 240

ttctggtctg cttttgttct ttactgtgtg tcaaggcttg gcaaagcagg caaagggaag 300

gaagatggtg gcatctcact gtgccagata ttgagggcat ttagtctgaa catcgtcgat 360

ttcttcaagg agatgccaca gttctgcata aaggttgggt ctgttctggc tggtctatat 420

ggttcagatt gggagaagag gcttgagttg aaggaactgc aagcgaatgt cgtccattta 480

agcctgctaa gcaggtacta caagcgtgcc taccaggagc tgttcctatt aaatgatgcc 540

aagccaccag aaaattctgc agaaccaaat gcacaagcct ctgactatta tcgctttgga 600

tggttacttt tcttggtgtt aagaatccaa acattcagcc gatttaagga tcttgttaca 660

tccacaaatg gactagtttc tgtgttggcc gtacttattg ttcacattcc tgtgcggcta 720

aggaatttca atatcaaaga gtcttctagt tttgccaaga agtcagacaa gggagtaaat 780

cttattgctt ccctatgtga aaaataccat acatcagaag atgagttgag taaagcaatc 840

gagaagacaa atactctcat agtggatatt ctgaagaaga aaccatgtcc agctgcttca 900

gaatgtcagc aggataggtt gtcttttatt gatccagagg gcttgacata ttttaagaat 960

ttgctcgagg aagactcatt gaaattaagt ttgctaatgc tggaaaagga atatgagaat 1020

gcaattaaca ccaagggaga attagatgag cgcatgttcg caaacgatga ggacagcttg 1080

cttggcagtg gaagtctgtc aggaggtgcc atcaatttac caggcacaaa gagaaagtac 1140

gatgttatgg cctcacctgc aaaatcaata acaagtccaa gtccgatgtc tcctccacgg 1200

ttttgtgcat cccctactgg aaatggctac tgcagctcaa aaatggctcc tatcacccca 1260

gtgagcacag ccatgacaac agccaagtgg cttcggagca caatctctcc ccttccttca 1320

aaaccttccg gggagttact gcgcttcttc tcagcttgtg ataaggatgt gacagatgac 1380

attacacgca gagctggtat tatacttgga gccatattca caagcagttc tttcggcgaa 1440

cgtatatgta ccagcgtgcg aagcacaaac aggattgatg ctatctggac agagcaaaga 1500

aaaatggaag cacttaagct atattacagg gttctggaat caatgtgcag agcagagact 1560

cagatcttga gtgggaacaa tcttacatcg cttctgtcta atgagcgatt ccatcggtgt 1620

atgattgctt gttctgctga gttagtgctg gccactcaca agacagtcac aatgatgttc 1680

ccagctgtgt tagagaaaac tggcataaca gcttttgact tgagcaaagt catagagagt 1740

tttgttaggc atgaggatac acttccacga gagctgaaac gtcatttgaa ttcacttgag 1800

gaacggctcc tggaaagcat ggcatgggag aaaggctcat caatgtacaa ttctctgatt 1860

gttgccaggc caacactatc tgcagaaata aacaggctag gtttattggc tgaaccaatg 1920

ccatcacttg atgccattgc ggcgcaccat aacatttcac tcgaggggct gccacctctt 1980

ccctttcaaa agcaagaaca ttcaccagac aaggatgaag ttagatctcc caaaagagca 2040

tgcactgaaa ggaggaatgt gctggtggac aacaattcat ttagatcacc agtcaaggat 2100

accctcaaat caaagttgcc tcctctccaa tcagcatttt taagtccaac aagacctaat 2160

cctgcagcag ggggagaatt atgcgcagag acaggaatag gtgtattttt gagcaagata 2220

gcaaagcttg cagctattag aatcagaggt ctttgtgaaa ggctgcaact ttctcagcaa 2280

gtcttggagc gagtgtactc ccttgtgcaa caaatcatta tccaacaaac tgctctattt 2340

ttcaatcggc acatcgatca gattatacta tgcagcatat atggagttgc taagatatct 2400

caattggcac tgacattcaa ggaaattatt ttcggctaca gaaaacaatc tcagtgcaag 2460

ccacaagttt tccgtagtgt ttatgttcat tgggcatcac gaagccgcaa tgggaaaaca 2520

ggggaggacc atgttgacat tatcactttc tacaacgaag tatttattcc tactgtgaaa 2580

cctttgctgg ttgagctagg gtctggtact agtccaaaca agaagaatga ggaaaaatgt 2640

gctgctgatg caggtccata tccagaatct cctcggctat ctcggtttcc aaaccttcct 2700

gacatgtctc ccaagaaagt ttctgctgct cataacgtat atgtttcacc cttgcgcaca 2760

tcaaagatgg atactttgct ttccccaagc tccaagagct attacgcctg tgtaggggag 2820

agtactcatg catttcagag cccctccaag gaccttaagg ttataaacaa ccgcctaaac 2880

agtgggaaga aagtcagtgg aagactgaac tttgatgttg tcagtgattt ggttgttgcc 2940

agaagtctca gcgatcaaaa tagcgcatca gcagcagcaa caacagcaga tatcgccaca 3000

aaaacaccag tgaaattgga gcagccagac tgctag 3036

<210>2

<211>1011

<212>PRT

<213> Artificial sequence

<400>2

Met Glu Gly Ala Ala Pro Pro Ala Ser Ser Gly Ser Glu Val Thr Gly

1 5 10 15

Ala Gly Ser Gly Lys Val Asp Ala Gly Gly Gly Ala Ala Met Glu Glu

20 25 30

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

35 40 45

Thr Arg Gln Ala Met Gln Leu Phe Lys Glu Ser Lys Ser Ile Leu Leu

50 55 60

Ser Ser Met Ser Ser Leu Gly Ser Gly Ser Pro Glu Glu Ile Glu Arg

65 70 75 80

Phe Trp Ser Ala Phe Val Leu Tyr Cys Val Ser Arg Leu Gly Lys Ala

85 90 95

Gly Lys Gly Lys Glu Asp Gly Gly Ile Ser Leu Cys Gln Ile Leu Arg

100 105 110

Ala Phe Ser Leu Asn Ile Val Asp Phe Phe Lys Glu Met Pro Gln Phe

115 120 125

Cys Ile Lys Val Gly Ser Val Leu Ala Gly Leu Tyr Gly Ser Asp Trp

130 135 140

Glu Lys Arg Leu Glu Leu Lys Glu Leu Gln Ala Asn Val Val His Leu

145 150 155 160

Ser Leu Leu Ser Arg Tyr Tyr Lys Arg Ala Tyr Gln Glu Leu Phe Leu

165 170 175

Leu Asn Asp Ala Lys Pro Pro Glu Asn Ser Ala Glu Pro Asn Ala Gln

180 185 190

Ala Ser Asp Tyr Tyr Arg Phe Gly Trp Leu Leu Phe Leu Val Leu Arg

195 200 205

Ile Gln Thr Phe Ser Arg Phe Lys Asp Leu Val Thr Ser Thr Asn Gly

210 215 220

Leu Val Ser Val Leu Ala Val Leu Ile Val His Ile Pro Val Arg Leu

225 230 235 240

Arg Asn Phe Asn Ile Lys Glu Ser Ser Ser Phe Ala Lys Lys Ser Asp

245 250 255

Lys Gly Val Asn Leu Ile Ala Ser Leu Cys Glu Lys Tyr His Thr Ser

260 265 270

Glu Asp Glu Leu Ser Lys Ala Ile Glu Lys Thr Asn Thr Leu Ile Val

275 280 285

Asp Ile Leu Lys Lys Lys Pro Cys Pro Ala Ala Ser Glu Cys Gln Gln

290 295 300

Asp Arg Leu Ser Phe Ile Asp Pro Glu Gly Leu Thr Tyr Phe Lys Asn

305 310 315 320

Leu Leu Glu Glu Asp Ser Leu Lys Leu Ser Leu Leu Met Leu Glu Lys

325 330 335

Glu Tyr Glu Asn Ala Ile Asn Thr Lys Gly Glu Leu Asp Glu Arg Met

340 345 350

Phe Ala Asn Asp Glu Asp Ser Leu Leu Gly Ser Gly Ser Leu Ser Gly

355 360 365

Gly Ala Ile Asn Leu Pro Gly Thr Lys Arg Lys Tyr Asp Val Met Ala

370 375 380

Ser Pro Ala Lys Ser Ile Thr Ser Pro Ser Pro Met Ser Pro Pro Arg

385 390 395 400

Phe Cys Ala Ser Pro Thr Gly Asn Gly Tyr Cys Ser Ser Lys Met Ala

405 410 415

Pro Ile Thr Pro Val Ser Thr Ala Met Thr Thr Ala Lys Trp Leu Arg

420 425 430

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

435 440 445

Phe Phe Ser Ala Cys Asp Lys Asp Val Thr Asp Asp Ile Thr Arg Arg

450 455 460

Ala Gly Ile Ile Leu Gly Ala Ile Phe Thr Ser Ser Ser Phe Gly Glu

465 470 475 480

Arg Ile Cys Thr Ser Val Arg Ser Thr Asn Arg Ile Asp Ala Ile Trp

485 490 495

Thr Glu Gln Arg Lys Met Glu Ala Leu Lys Leu Tyr Tyr Arg Val Leu

500 505 510

Glu Ser Met Cys Arg Ala Glu Thr Gln Ile Leu Ser Gly Asn Asn Leu

515 520 525

Thr Ser Leu Leu Ser Asn Glu Arg Phe His Arg Cys Met Ile Ala Cys

530 535 540

Ser Ala Glu Leu Val Leu Ala Thr His Lys Thr Val Thr Met Met Phe

545 550 555 560

Pro Ala Val Leu Glu Lys Thr Gly Ile Thr Ala Phe Asp Leu Ser Lys

565 570 575

Val Ile Glu Ser Phe Val Arg His Glu Asp Thr Leu Pro Arg Glu Leu

580 585 590

Lys Arg His Leu Asn Ser Leu Glu Glu Arg Leu Leu Glu Ser Met Ala

595 600 605

Trp Glu Lys Gly Ser Ser Met Tyr Asn Ser Leu Ile Val Ala Arg Pro

610 615 620

Thr Leu Ser Ala Glu Ile Asn Arg Leu Gly Leu Leu Ala Glu Pro Met

625 630 635 640

Pro Ser Leu Asp Ala Ile Ala Ala His His Asn Ile Ser Leu Glu Gly

645 650 655

Leu Pro Pro Leu Pro Phe Gln Lys Gln Glu His Ser Pro Asp Lys Asp

660 665 670

Glu Val Arg Ser Pro Lys Arg Ala Cys Thr Glu Arg Arg Asn Val Leu

675 680 685

Val Asp Asn Asn Ser Phe Arg Ser Pro Val Lys Asp Thr Leu Lys Ser

690 695 700

Lys Leu Pro Pro Leu Gln Ser Ala Phe Leu Ser Pro Thr Arg Pro Asn

705 710 715 720

Pro Ala Ala Gly Gly Glu Leu Cys Ala Glu Thr Gly Ile Gly Val Phe

725 730 735

Leu Ser Lys Ile Ala Lys Leu Ala Ala Ile Arg Ile Arg Gly Leu Cys

740 745 750

Glu Arg Leu Gln Leu Ser Gln Gln Val Leu Glu Arg Val Tyr Ser Leu

755 760 765

Val Gln Gln Ile Ile Ile Gln Gln Thr Ala Leu Phe Phe Asn Arg His

770 775 780

Ile Asp Gln Ile Ile Leu Cys Ser Ile Tyr Gly Val Ala Lys Ile Ser

785 790 795 800

Gln Leu Ala Leu Thr Phe Lys Glu Ile Ile Phe Gly Tyr Arg Lys Gln

805 810 815

Ser Gln Cys Lys Pro Gln Val Phe Arg Ser Val Tyr Val His Trp Ala

820 825 830

Ser Arg Ser Arg Asn Gly Lys Thr Gly Glu Asp His Val Asp Ile Ile

835 840 845

Thr Phe Tyr Asn Glu Val Phe Ile Pro Thr Val Lys Pro Leu Leu Val

850 855 860

Glu Leu Gly Ser Gly Thr Ser Pro Asn Lys Lys Asn Glu Glu Lys Cys

865 870 875 880

Ala Ala Asp Ala Gly Pro Tyr Pro Glu Ser Pro Arg Leu Ser Arg Phe

885 890 895

Pro Asn Leu Pro Asp Met Ser Pro Lys Lys Val Ser Ala Ala His Asn

900 905 910

Val Tyr Val Ser Pro Leu Arg Thr Ser Lys Met Asp Thr Leu Leu Ser

915 920 925

Pro Ser Ser Lys Ser Tyr Tyr Ala Cys Val Gly Glu Ser Thr His Ala

930 935 940

Phe Gln Ser Pro Ser Lys Asp Leu Lys Val Ile Asn Asn Arg Leu Asn

945 950 955 960

Ser Gly Lys Lys Val Ser Gly Arg Leu Asn Phe Asp Val Val Ser Asp

965 970 975

Leu Val Val Ala Arg Ser Leu Ser Asp Gln Asn Ser Ala Ser Ala Ala

980 985 990

Ala Thr Thr Ala Asp Ile Ala Thr Lys Thr Pro Val Lys Leu Glu Gln

995 1000 1005

Pro Asp Cys

1010

<210>3

<211>20

<212>DNA

<213> Artificial sequence

<400>3

acttcatccg tttcacaatg 20

<210>4

<211>20

<212>DNA

<213> Artificial sequence

<400>4

atggaaacag tggttgacga 20

<210>5

<211>20

<212>DNA

<213> Artificial sequence

<400>5

tctgatgtga tgagttctgc 20

<210>6

<211>20

<212>DNA

<213> Artificial sequence

<400>6

ctaagctagt gttggaattg 20

<210>7

<211>19

<212>DNA

<213> Artificial sequence

<400>7

ctgcctgttg actgttgaa 19

<210>8

<211>18

<212>DNA

<213> Artificial sequence

<400>8

ttcatatatt tttagcct 18

<210>9

<211>19

<212>DNA

<213> Artificial sequence

<400>9

tcggcaccta cggcgccct 19

<210>10

<211>20

<212>DNA

<213> Artificial sequence

<400>10

catccctaac ccgagaagac 20

<210>11

<211>21

<212>DNA

<213> Artificial sequence

<400>11

attcttcttt ctgccgcttg c 21

<210>12

<211>18

<212>DNA

<213> Artificial sequence

<400>12

ccactttgcc ccattgaa 18

<210>13

<211>20

<212>DNA

<213> Artificial sequence

<400>13

acaagtcacc aacgaatctc 20

<210>14

<211>20

<212>DNA

<213> Artificial sequence

<400>14

actgaatgag caaaacgagt 20

<210>15

<211>20

<212>DNA

<213> Artificial sequence

<400>15

agtctcacac aacgacctct 20

<210>16

<211>20

<212>DNA

<213> Artificial sequence

<400>16

gtccgaacac agctaaccta 20

<210>17

<211>19

<212>DNA

<213> Artificial sequence

<400>17

aaatgggtcg tattgggct 19

<210>18

<211>20

<212>DNA

<213> Artificial sequence

<400>18

gggggtgact cagtttgatt 20

<210>19

<211>50

<212>DNA

<213> Artificial sequence

<400>19

cagtgctctt catagcagat aaccccaatc aagtccatga gctgtgatcc 50

<210>20

<211>50

<212>DNA

<213> Artificial sequence

<400>20

cagtgctctt cagacatcaa aatccacata cacactatta aaatgtacat 50

<210>21

<211>31

<212>DNA

<213> Artificial sequence

<400>21

cgagctccca tacatcagaa gatgagttga g 31

<210>22

<211>32

<212>DNA

<213> Artificial sequence

<400>22

cgggatcctg aaggaagggg agagattgag ct 32

Claims (1)

1.PIS1Application of gene in controlling development of stamens of rice, and application of genePIS1The nucleotide sequence of the gene is shown in a sequence table SEQID NO.1PIS1The amino acid sequence of the gene code is shown in the sequence table SEQ ID NO. 2.

Images