CN112239762A

CN112239762A - Plant pollen tube growth gene and application

Info

Publication number: CN112239762A
Application number: CN202010879353.9A
Authority: CN
Inventors: 张毅; 陈云; 杜双林
Original assignee: Yunnan University YNU
Current assignee: Yunnan University YNU
Priority date: 2020-08-27
Filing date: 2020-08-27
Publication date: 2021-01-19
Anticipated expiration: 2040-08-27
Also published as: WO2022042619A1; CN112239762B

Abstract

The invention discloses a rice Pollen Tube growth related gene OsPTD1(Pollen Tube Development) and application thereof. The protein coded by the gene is an amino acid sequence shown in SEQ ID NO.1, or a protein which is substituted, deleted or added with at least one amino acid and has the same function with the OsPTD1 protein; the nucleotide sequence of the protein consisting of the amino acid sequence shown in SEQ ID NO.1 is shown in SEQ ID NO. 2. The OsPTD1 gene disclosed by the invention codes ascorbic acid oxidase, is an essential gene for normal growth of a pollen tube, and the abnormal function of the gene can lead to premature rupture of the pollen tube, can not finish insemination, can generate gametophyte male sterility, can be used for preparing gametophyte male sterility, and has an important role in preventing transgene from drifting along with pollen, breeding sporophyte male sterility and the like.

Description

Plant pollen tube growth gene and application

Technical Field

The invention belongs to the field of genetic engineering, and relates to a rice pollen tube growth related gene OsPTD1 and application thereof, wherein gametophyte male sterility can be generated due to abnormal function of the gene, so that the gene can be used for changing pollen fertility, controlling transgene drift, preparing male gametophyte sterility, reproducing sporophyte male nuclear sterility and the like.

Background

The male nuclear sterility of plants plays an important role in the research of pollen development and the utilization of heterosis. Male sterility includes sporozoite sterility and gametophyte sterility.

The sterility of the sporophyte is controlled by the genotype of the plant, all pollen in the anther is sterile, and the anther is usually abnormal in shape, color and size and is not fruity in the field, so that the anther is easy to discover and identify; the sporophyte sterile gene can be transmitted between generations through a female gamete and can also be transmitted between generations through a male gamete. The gametophyte sterility is controlled by the pollen genotype, half of the pollen in the anther can be bred, half of the pollen is sterile, the field has no obvious difference with the wild type, the seed setting is normal, and the direct discovery and identification are difficult; male gametophytic sterility genes can only be transmitted through female gametes. Due to the difference, the preparation, discovery and identification of the gametophyte sterility are very difficult, and the preparation, discovery and identification need to be carried out by combining indoor microscopic observation, PCR, electrophoresis and other means, so that the gametophyte sterility materials are rare objectively, and the related basis and application research lags behind the sporophyte sterility. The environment-insensitive type in sporophyte nuclear sterility has great potential in heterosis utilization, but the population with thorough sterility and sterile plant rate of 100 percent is difficult to reproduce by itself, so that the application of the population is limited; and gametophyte male sterility can be used to reproduce sporophyte male sterility. Therefore, it is necessary to discover and verify some gametophyte male sterility related genes and create some gametophyte male sterility materials.

Plant copper-rich oxidase including laccase and ascorbate oxidase participates in regulating plant growth, reproduction, aging and environmental stress response, and can regulate flowering phase (Shen et al 2009). There are more than 10 genes coding for multicopper oxidase in rice, but their biological functions are not clear, and it is important to find gametophyte male sterility related gene and create gametophyte male sterility, and use it for propagation of sporophyte sterility and reduction of transgene drift.

Disclosure of Invention

In view of the above, an object of the present invention is to provide a gene OsPTD1 for controlling rice pollen tube development, a second object of the present invention is to provide an application of the gene OsPTD1 related to rice pollen tube growth in restoring mutant pollen fertility, and a third object of the present invention is to provide an application of suppressing the expression of the gene OsPTD1 related to rice pollen tube growth in preparing gametophyte male sterility; the fourth purpose of the invention is to provide a method for preparing gametophyte male sterility by inhibiting the expression of the rice pollen tube growth related gene OsPTD 1; the fifth purpose of the invention is to provide a method for expressing and propagating sporophyte nuclear male sterility by using a rice pollen tube growth related gene OsPTD1 in a down-regulation manner and application thereof (the coded amino acid sequence of the gene is shown as SEQ ID NO.1, the CDS nucleotide sequence of the gene is shown as SEQ ID NO.2, and the genome nucleotide sequence of the gene is shown as SEQ ID NO. 3).

In order to achieve the purpose, the invention provides the following technical scheme:

1. a rice pollen tube growth related gene OsPTD1, wherein the amino acid sequence coded by the rice pollen tube growth related gene is shown in SEQ ID NO. 1; or the amino acid sequence shown in SEQ ID NO.1 is substituted, deleted or added with at least one amino acid and has the amino acid sequence with the same function as the OsPTD1 protein; the CDS nucleotide sequence of the rice pollen tube growth related gene OsPTD1 is shown as SEQ ID NO. 2; or a nucleotide sequence which is shown in SEQ ID NO.2, is substituted, deleted or added with at least one nucleotide and has the same function with the rice pollen tube growth related gene OsPTD 1; the genome nucleotide sequence of the rice pollen tube growth related gene OsPTD1 is shown in SEQ ID NO. 3; or a nucleotide sequence which is shown in SEQ ID NO.3, is substituted, deleted or added with at least one nucleotide and has the same function with the rice pollen tube growth related gene OsPTD 1.

2. The application of the rice pollen tube growth related gene OsPTD1 in restoring mutant pollen fertility is disclosed, wherein the mutant is a pollen sterile mutant generated by OsPTD1 gene or homologous gene variation.

3. The rice pollen tube growth related gene OsPTD1 expression is down-regulated or mutated to be applied to the preparation of gametophyte male sterility or propagation sporophyte nuclear sterility.

Preferably, the method for down-regulating the expression of the rice pollen tube growth related gene OsPTD1 is interference with the expression of the rice pollen tube growth related gene OsPTD 1.

Preferably, the method for interfering the expression of the rice pollen tube growth related gene OsPTD1 comprises the steps of firstly constructing a vector for interfering the expression of the rice pollen tube growth related gene OsPTD1, transfecting plants through agrobacterium mediation, and screening transgenic positive plants.

Preferably, the expression vector comprises an interference stem-loop, an interference left arm and an interference right arm, wherein the sequence of the interference stem-loop is the interference stem-loop shown in SEQ ID NO. 8; the interference left arm takes sequences shown in SEQ ID NO.27 and SEQ ID NO.28 as primers and is obtained by amplification from the Zhonghua 11; the interference right arm is obtained by taking sequences shown in SEQ ID NO.29 and SEQ ID NO.30 as primers and amplifying from the Zhonghua 11.

4. The method for preparing gametophyte male sterility by down-regulating the expression of a rice pollen tube growth related gene OsPTD1 comprises the following steps:

the method comprises the steps of transfecting a plant by a down-regulation element Xi of a gene OsPTD1 related to the growth of a pollen tube regulated and controlled by a pollen specific promoter through agrobacterium mediation, and screening a transgenic positive strain, wherein the transgenic positive strain is gametophyte male sterility.

5. The method for preparing sporophyte male sterility by down regulating the expression of a rice pollen tube growth related gene OsPTD1 comprises the following steps: linking sporophyte male fertile gene Y with OsPTD1 gene interference fragment regulated and controlled by promoter POsPTD1, transferring into sterile mutant of sporophyte male fertile gene Y, screening transgenic positive plant, pollinating the sterile mutant of sporophyte male fertile gene Y by using the positive plant as male parent, and reproducing sporophyte nuclear male sterility.

In the present invention, the sporophyte male fertile gene Y can be any sporophyte male fertile gene, such as sporophyte male fertile gene OsABCG15, and the sterile mutant of the sporophyte male fertile gene Y is Zhongjiu B-OsABCG 15: a pollen-free male sterile mutant of OsABCG15 gene is used as a non-recurrent female parent, and sterile medium nine B-OsABCG15 is obtained through backcross. Other sporozoite male fertile genes may also be selected.

As a general technical scheme, the promoter, the interference fragment, the terminator and the species and variety to be transformed can be flexibly selected; the sporophyte fertility gene can be flexibly selected; the way of inhibiting the expression of the gene can be interference, antisense, miRNA and the like; the loading sequence and relative position of various fragments can be adjusted according to specific conditions. In addition, the carrier names used in the method statement are used only for convenience of statement and do not limit the use of other symbols to represent the relevant carrier in practical application. Thus, the practical use of the alternatives described above does not indicate a substantial difference from the invention, and thus does not affect the protection of the invention.

The invention has the beneficial effects that: OsPTD1 is a rice endogenous gene, and gametophytic sterility can be generated by carrying out down-regulation expression on the gene without the help of exogenous genes or fragments, so that a plant variety with transgenic components not drifting along with pollen is cultivated (if the method is used in other species, gametophytic sterility related genes in target species can be selected); the method can also be used for breeding sporophyte genic sterility, solves the breeding problem of sporophyte sterility, is favorable for freely selecting male parents and fully utilizes heterosis; the method for preparing and identifying the gametophyte male sterility has the advantages of intuition, simplicity, accuracy, time saving, economy and the like.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 shows the analysis of OsPTD1 gene expression profile (A is RT-qPCR analysis of OsPTD1 gene in various wild organs and flower development stages and expression in late stage of interference homozygous progeny flower development; B is GUS staining detection of promoter POsPTD1 driving GUS gene to express specifically in pollen, showing GUS staining of transgenic heterozygote pollen; C is GUS staining detection of promoter POsPTD1 driving GUS gene to express specifically in pollen, showing GUS staining of transgenic homozygote pollen);

FIG. 2 shows RACE analysis and full-length amplification of cDNA of OsPTD1 gene (A and B are 3 rounds of amplification of 5 and 3 ends, respectively, and C is full-length amplification of cDNA);

FIG. 3 is a vector engineering map;

FIG. 4 shows pollen germination (A is wild type pollen and B is interference T)₀Pollen substitute (about half pollen breaks early));

FIG. 5 shows transgenic T₀Generation positive strain hybrid F₁The transmission of the purple line.

Detailed Description

The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.

Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The experimental procedures, for which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions, for example as described in the molecular cloning protocols (fourth edition, Cold spring harbor laboratory Press) or in the compendium for molecular biology protocols (fifth edition, scientific Press), or according to the conditions recommended by the manufacturer.

In the examples, various vectors known in the art, such as commercially available vectors including plasmids, cosmids, and the like, may be used.

The full-length sequence of related nucleotides or fragments thereof can be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed based on the nucleotide sequences disclosed in this example, and the sequences can be amplified using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art, or genomic DNA as a template. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order. Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

Example 1 selection of male gametophyte function-related gene OsPTD 1:

in order to find some gametophyte development related genes, a BAR rice gene expression database (http:// BAR. utoronto. ca/efprice/cgi-bin/efpWeb. cgi) is analyzed, a plurality of genes expressed in flowers at the middle and later stages are found, and a batch of genes possibly related to the development of male gametophytes are selected through RT-qPCR verification. The rice Loc _ Os05g40740 gene is specifically and highly expressed in late flowers, the arabidopsis thaliana homologous gene is specifically expressed in pollen, the Loc _ Os05g40740 gene is preliminarily listed as a male gametophyte development related gene and named as OsPTD1, the amino acid sequence is shown as SEQ ID No.1, the nucleotide sequence is shown as SEQ ID No.2, and the genome sequence is shown as SEQ ID No. 3.

1. RT-qPCR analysis

The spatiotemporal expression pattern of the gene is analyzed by RT-qPCR, and the qPCR primers of the OsPTD1 gene are as follows: upstream primer MQF 2: 5'-ggcgtcttcaggtacaaccag-3' (SEQ ID NO.9), and downstream primer MQR25'-ggtacacctggaccgagtgc-3' (SEQ ID NO. 10). The used internal reference primers are as follows: the upstream primer OsActin 2F: 5'-ctctgtatgccagtggtcgt-3' (SEQ ID NO.11), downstream primer OsActin 2R: 5'-ccgttgtggtgaatgagtaac-3' (SEQ ID NO. 12). The qPCR results show that: the OsPTD1 gene is not expressed in roots, stems, leaves, leaf sheaths and seeds, and is highly expressed in flowers in the middle and later stages of development (FIG. 1). Further analyzing the expression of the gene at the later development stage of the interference homozygous plant flowers, and finding that the interference causes the serious reduction of the expression of the gene.

2. GUS staining analysis

To determine whether the gene is specifically expressed in pollen, the promoter of the gene was cloned, placed in front of the GUS gene of pCAMBIA1301, and examined for transgene and GUS staining. The specific operation is as follows:

with promoter upstream primer P5F 1: 5'-gcgtcgacgtcccatgtcaccgacagtact-3' (SEQ ID NO.13 with cleavage site SalI) and the reverse primer P5R 1: 5'-catgccatggcgtggaaatgtgatcgctaggct-3' (SEQ ID NO.14, with restriction enzyme site NcoI) the promoter of Loc _ Os05g40740 (POsPTD 1 for short) was amplified from Zhonghua 11, and its nucleotide sequence was shown in SEQ ID NO. 6. The vector pCAMBIA1301 was preceded by GUS gene using SalI and NcoI (incomplete digestion) to obtain p POsPTD 1-GUS.

pPOsPTD1-GUS was transferred into Agrobacterium LBA4404, and positive Agrobacterium was transfected into middle flower 11. GUS staining observation is carried out on roots, leaves and flowers of transgenic positive offspring in the flowering period, and the result shows that: the roots, leaves, glumes, filaments, pistils, etc. are not dyed, while the anthers are dyed blue. The anthers were further mashed and observed under a microscope to see: the transgenic heterozygous plants had only about 50% of GUS-stained pollen, and the homozygous plants had almost all pollen stained by GUS (FIG. 1). The above results indicate that the promoter POsPTD1 is indeed a pollen-specific promoter.

Example 2 cloning and structural analysis of OsPTD1 Gene

To obtain the gene structure of OsPTD1, analysis was performed using the RACE kit of the precious organism: taking total RNA reverse transcription products of rice anthers as templates, carrying out PCR three-wheel amplification by using universal primers provided by a kit and 5 'RACE primers and 3' RACE primers designed by the kit (figure 2), recovering and connecting products of the last round to a pMD18-T vector, and finally determining 3 'UTR, 5' UTR and CDS of OsPTD1 gene cDNA through bacterial liquid PCR detection and sample sending sequencing: the gene has a 5 'UTR of 201bp, a 3' UTR of 261bp, a CDS total length of 1671bp and a cDNA total length of 2338 bp. The 5 'RACE specific primers are respectively shown in SEQ ID NO.15-17, the 3' RACE specific primers are respectively shown in SEQ ID NO.18-20, and the full-length amplification primers are respectively shown in SEQ ID NO. 21-22.

Example 3 suppression of OsPTD1 Gene expression preparation of Male gametophyte sterility

In order to determine whether the OsPTD1 gene regulates pollen fertility and can be used for preparing and identifying male gametophyte sterile materials, the interference fragment of the OsPTD1 gene driven by the POsPTD1 promoter is used for implementing male gametophyte sterility creation and identification:

1. modifying a carrier: in order to facilitate loading of related target genes or DNA fragments and accurately and directly identify transgenic positive strains and male gametophyte sterility, a modified gene OsMYB76R of a rice anthocyanin synthesis related gene is used for replacing a GUS gene, and a multiple cloning site of a vector pCAMBIA1301 is modified, and the specific operations are as follows:

(1) OsMYB76 gene was modified and replaced with GUS: the OsMYB76 gene is an essential gene for anthocyanin synthesis in rice and can be used as an endogenous reporter gene. In order to avoid the influence of enzyme cutting sites carried by the OsMYB76 gene on loading of a target fragment, common enzyme cutting sites in the OsMYB76 gene are removed through base substitution, a CDS sequence of the OsMYB76R gene after the OsMYB76 gene is modified is obtained and artificially synthesized, and the sequence is shown as SEQ ID No. 4. Taking the synthesized OsMYB76R gene as a template, and carrying out reaction by using a primer OsMYB76 RF: 5'-ggactcttgaccatgatgggacgcagggcttgct-3' (SEQ ID NO.23, BglII-near homologous fragment with pCAMBIA 1301) and OsMYB76 RR: 5'-attcgagctggtcactcacgcacacaggttccaag-3' (SEQ ID NO.24, BstEII near homologous fragment with pCAMBIA 1301) to obtain the target fragment, and ligating the target fragment into pCAMBIA1301 by homologous recombination to obtain vector pOsMYB 76R.

(2) And (3) multi-cloning site modification: the kind and sequence of the enzyme cutting sites of the multiple cloning sites of the vector pOsMYB76R are modified. Firstly, artificially synthesizing a new multiple cloning site sequence, wherein the nucleotide sequence of the new multiple cloning site sequence is shown as SEQ ID NO. 5. Then the synthetic sequence is replaced by the original sequence of the multiple cloning site through homologous recombination, the new polyclonal enzyme cutting site is shown in figure 3, and the vector after the multiple cloning site is modified is named as pOsMYB76 RM.

2. Loading of the inhibitory element Xi of OsPTD1 Gene

(1) Using stem-loop structure upstream primer LF 2: 5'-gaagatctacgagctggtgagctagcta-3' (SEQ ID NO.25, with restriction site BglII) and the downstream primer LR 2: 5'-ctggtcacccctcaaacctgaaaattcag-3' (SEQ ID NO.26, with restriction site BstEII) the first intron of the OsMYB76 gene was amplified from R25 as an interference stem-loop (SEQ ID NO.8) and loaded into pOsMYB76RM to obtain vector pOsMYB76 RL.

(2) With the interference left arm forward primer I5F 1: 5'-cggaattcgcagaaggtgatcctgatcaac-3' (SEQ ID NO.27, EcoRI with the cleavage site) and the downstream primer I5R 1: 5'-gaagatctgcaggcggctgacgacgctgat-3' (SEQ ID NO.28, with restriction site BglII) expanded the interfering left arm of OsPTD1 from Zhonghua 11; with the interfering right arm forward primer I5F 2: 5'-gacctgcagggcagaaggtgatcctgatcaac-3' (SEQ ID NO.29 with an enzyme cleavage site SbfI) and the reverse primer I5R 2: 5'-ctggtcaccgcaggcggctgacgacgctgat-3' (SEQ ID NO.30, with restriction site BstEII) the interfering right arm of Loc _ Os05g40740 was expanded from middle flower 11. Loading the left arm and the right arm into two sides of a stem loop in the vector pOsMYB76RL respectively, and loading a Tnos terminator behind the interference right arm by using SbfI and AscI to obtain a vector pOsMYB76 RLi; the nucleotide sequence of the interference fragment is shown as SEQ ID NO. 7.

(3) With promoter upstream primer P5F 1: 5'-gcgtcgacgtcccatgtcaccgacagtact-3' (SEQ ID NO.8 with a cleavage site SalI) and the reverse primer P5R 2: 5'-cggaattccgtggaaatgtgatcgctaggct-3' (SEQ ID NO.20, EcoRI with the restriction site) the promoter of Loc _ Os05g40740 was amplified from Zhonghua 11, the nucleotide sequence of which is shown in SEQ ID NO. 6. Loading it into vector pOsMYB76RLi to obtain male gametophyte sterile vector pXi-OsMYB76 RM.

3. pXi-OsMYB76RM was transferred to Agrobacterium LBA4404, and positive Agrobacterium was transfected into a rice variety with colorless organs.

4. At T₀Positive plants are selected according to the existence of purple at the stem base, and the pollen germination and purple transmission conditions are investigated to find that:

(1) about half of pollen tubes of the pollen of the positive strains can not grow normally on the culture medium, and show premature rupture (figure 4), which indicates that the pollen tube growth is abnormal due to the interference of the OsPTD1 gene, and the OsPTD1 gene is involved in regulating the pollen fertility.

(2) Selfing the positive strain, and pollinating the middle nine B which is not transgenic by using the positive strain as a male parent. The purple line investigation is carried out on the inbred and hybrid seeds, and the inbred F is found₂The separation ratio of the seed purple line is 1:1, and is not 3:1, and the cross F of 96.4 percent₁The seeds showed no purple line (FIG. 5), indicating that the transgene components are difficult to transfer with pollen between generations, but with female gametes, which is characteristic of male gametophyte sterility.

(3) Therefore, the OsPTD1 gene is related to the development of male gametophyte pollen, and by interfering with the development of male gametophyte pollen, male gametophyte sterile materials are obtained and genetic identification is successfully carried out by means of genetic investigation of linked purple line characters.

Example 4 Down-Regulation of expression of Nonine B-osabcg15 in reproductive sporophyte Nuclear Male sterility Using the OsPTD1 Gene

In order to determine whether sporophyte nuclear male sterility can be propagated by using the OsPTD1 gene, nine B-OsABCG15 in sterile materials generated by the OsABCG15 gene (Wu et al, 2014) is taken as an object, and an interference equal fragment of the OsPTD1 gene is linked with the OsABCG15 gene, so that the propagation practice of the nine B-OsABCG15 is carried out, and the specific steps are as follows:

1. and (3) breeding of Zhongjiu B-osabcg 15: the medium nine B (male fertile, without purple) with abnormal OsMYB76 gene is taken as a recurrent male parent, a pollen-free male sterile mutant (Wu et al, 2014) with OsABCG15 gene is taken as a non-recurrent female parent, and the medium nine B-OsABCG15 without purple and sterile is obtained through backcross.

2. The normal OsABCG15 gene is ligated into pXi-OsMYB76RM to obtain vector p OsABCG15-Xi-OsMYB76 RM.

3. The pOsABCG15-Xi-OsMYB76RM is transferred into Zhongjiu B-osabcg15, 11 positive plants are screened out according to the existence of purple, and all purple plants show fertile pollen.

4. Pollinating nine B-osabcg15 in the mutant by taking the positive strain as a male parent to obtain F₁429 seeds are adopted, hybrid seeds are sown and transplanted to obtain 386 flowering plants, wherein 376 plants are colorless sterile, 10 plants are purple fertile, colorless and sterile are separated together, purple and fertile are separated together, and the sterile plant rate is 97.4%.

Under natural conditions, the method with the highest sterile plant rate of the nine B-osabcg15 in propagation is brother-sister crossing, the sterile plant rate is 50% at most, and the sterile plant rate is more than 95% by the method. It is demonstrated that the method of the present invention can be used to propagate sporozoite nuclear male sterility.

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

Sequence listing

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aacgtcatca ccgccgagtt ccgcaccttc atcgagatca tcttcgagaa ccccgagaag 1320

agcatggact ccgtccacct cgacggctac gccttcttcg ccgtcgggat ggggccgggg 1380

aagtggtcgg cggaggagag gaagacgtac aacctgctgg acggggtgag ccggcactcg 1440

gtccaggtgt acccgaggtc gtggacggcg atcatgctga cgttcgacaa cgccgggatg 1500

tggaacgtga ggtccaacat ctgggagagg cactacctcg gcgagcagct ctacatcagc 1560

gtcgtctcgc cggcgaggtc gctccgggac gagtacaaca tgccggagaa cgccctccgc 1620

tgcggcaagg tcgtcggcct gccgctgccg ccgtcctacc tcccggccta a 1671

<210> 3

<211> 2235

<212> DNA

<213> Rice (oryza. sativa L.)

<400> 3

atgacgacga cgacgcgagt ggcggccgcc gccgccggcg tgctgctggt ggcggcggcg 60

ctggccggcg tggcgcgcgg cgaggacccg tacgtgttct tcgagtggaa ggtgacgtac 120

ggcaccaaga ccctcctgga cgcgccgcag aaggtgatcc tgatcaacgg cgagttcccg 180

ggcccgcgga tcaactgctc gtccaacaac aacatcgtgg tgaacgtgtt caaccagctg 240

gacgagccgc tgctcttcac ctggaacggg atgcagcacc gcaagaactc gtggcaggac 300

ggcctcgccg ggacgcagtg ccccatcgcg ccgggcacca actacacgta caagtggcag 360

cccaaggacc agatcggcag cttcttctac ttcccgtcgc tggggatgca ccgcgccgcc 420

ggcggctacg gcgggatcag cgtcgtcagc cgcctgctca tcccggtccc gttcgacccg 480

ccggccgacg accacatggt gctcatcggc gactggtaca ccaaggacca cgccgccatg 540

gccaagatgc tcgacgccgg caagagcttc ggccgcccgc acggggtggt catcaacggc 600

aagtccggca aggccgccgc cgacccgccc atgttcaccg tcgaggccgg caagacgtac 660

cggctccgcg tctgcaacgt cggcatcaag gcgtcgctca acttccgcat ccagggccac 720

gacatgaagc tggtggagat ggagggctcc cacacggtgc aggacatgta cgactccctc 780

gacgtccacg tcggccactg cctctccgtc ctcgtcgacg ccgaccagaa gcccggcgac 840

tactacgcgg tggcgtccac gcggttcatc cacgaggcca agtcggtgtc agccgtcatc 900

cgctacgccg gctcgagcac gccgccgtcg ccggccgtgc cggagccgcc ggcgggatgg 960

gcgtggtcga tcaaccagtg gaggtcgttc cggtggaacc tgacggcgag cgccgcccgc 1020

cccaacccgc aggggtccta ccactacggc cagatcaaca tcacgcgcac catcaggctc 1080

atggtctccc ggggccacat cgacggcaag ctcaagtacg gcttcaatgg cgtctcccac 1140

gtcgacgccg agacgccgct caagctcgcc gagtacttca acgtcaccga cggcgtcttc 1200

aggtacaacc agatgaccga cgtgccgccc gccgtcaatg gccccctcca cgtcgtcccc 1260

aacgtcatca ccgccgagtt ccgcaccttc atcgagatca tcttcgagaa ccccgagaag 1320

agcatggact ccgtccacct cgacggctac gccttcttcg ccgtcgggta cgtacataca 1380

tcgcccccca ttactactac ctccattaac cttttattaa agataggctt ggccattcat 1440

tttattttta aaaaattata taagtatcat ctattttatt gtaatttgat ttatcgtcaa 1500

gtgtgcttta aacataattt gatttttttt atatttgcat aaaaaattga atattatgaa 1560

tgatgtatat gatctgatac tactgttaac taacttactg atgcgcataa taataaaatt 1620

ttttaataag acgaatgata taaaaaaagt tttttaaatt acctccattt ataatatacg 1680

atgttttttt tacttttaac attcgtttat aatatatgtt ttttcacaaa cgtttgagtt 1740

ttgaccattt attttattta aaaataatta tgcagttatc atctatttta ttgtaatttt 1800

attgtaattt gattattgtc gagtgtactt taaatacgat ttgatttttc tatatttaca 1860

taaaattttt aaataagata aatgatggat acgatctgat gctaccgttg actgactgac 1920

tgatggcgca ggatggggcc ggggaagtgg tcggcggagg agaggaagac gtacaacctg 1980

ctggacgggg tgagccggca ctcggtccag gtgtacccga ggtcgtggac ggcgatcatg 2040

ctgacgttcg acaacgccgg gatgtggaac gtgaggtcca acatctggga gaggcactac 2100

ctcggcgagc agctctacat cagcgtcgtc tcgccggcga ggtcgctccg ggacgagtac 2160

aacatgccgg agaacgccct ccgctgcggc aaggtcgtcg gcctgccgct gccgccgtcc 2220

tacctcccgg cctaa 2235

<210> 4

<211> 819

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgggacgca gggcttgctg tgcaaaggaa ggaatgaagc ggggtgcctg gacgtccaag 60

gaggacgatg tcctggcgag ctacattaag tctcacggag agggtaagtg gcgggaggtt 120

ccgcagagag ccggacttcg gagatgcggc aagtcctgtc gccttaggtg gttgaactat 180

ctccgcccaa atatcaagag gggcaacatt gacgatgacg aggaagagct gatcgtgcgg 240

cttcatacgc tcctggggaa tagatggtcc ctgatcgcag gccgccttcc gggaaggacc 300

gataacgaga ttaagaacta ctggaatagt acactctcca ggaagatcgg aacggccgcg 360

accgccgccg ccggctcacg cggcggctcc acccctgata cagccagggc gactgacgcg 420

gcttccagct cttcagtggt cccaccagga cagcaacagc aaccagcaag ccgggcggat 480

actgacaccg caaccgccgc cgccgccgcc gccgccacta caacgactgt ttgggctcct 540

aaggcagtgc ggtgcacgag aggcttcttt ttccacgata gagaaactgc accattggct 600

gctgcggcgc ctgctcccgc aggagagctg ggagacggag atgacgtgga ttgcgactac 660

tattgttctg gttcgagttc cgctgcaacc acaacctcct cctcctccct cccggttgtg 720

gtcgagccat gctttagtgc cggcgatgac tggatggatg acgtccgcgc cttggcgtca 780

ttcctcgaca cagatgacgc ttggaacctg tgtgcgtga 819

<210> 5

<211> 227

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

acagctatga ccatgattac aagcttcata cctctagaca cccatggagt gtatgagtaa 60

ggtacacgtg cacgagctct ttttagcggg gtcgacacaa ccgaattcca cagatctatg 120

gaatgagtat gtgaggtgac ccaccctgca ggtttttgaa gggggcgcgc cacaaccact 180

agtcaccacg gtaccagcgg gggatccggc actggccgtc gttttac 227

<210> 6

<211> 1887

<212> DNA

<213> Rice (oryza. sativa l.)

<400> 6

gtcccatgtc accgacagta ctaaatgggt aaagattgga taaagtatat ggggtatttg 60

tgaggtatta ttagaaaact tcgtgtggtt ttgatggacc tgttttatgt gttgaaaata 120

tgaatggtta tagggtgtgt ttgcaagtgc aggatgggaa ctcatccctc ctgcacgcaa 180

aacggagcgg ctttttaaca catgattaat taaatattag ctaatttttt taaaaaaaat 240

ggattaattt gattttttta agcaactttc atatagaaat tttttgcaaa aaacacaccg 300

tttaatagtt taaaaacgtg cgcgcgaaaa acgagggaga ggggttggga acatgggttt 360

gcaaacacaa ccatagtatt ggcgattcct tttcgtttga gtaaatttta caaaactaca 420

ggtattttga ccaaattatc acaaaactac agatttaagg agttgtatca taaaactaca 480

catttagcat caaatttatc acaaaactgc agattttagg ttaagtatca caaaaataca 540

tatttaatat tgaacttatc acaaaactat aacttttgga gtttaaatcc ctagcaccat 600

tgttatggtg gagctataaa cattattact ttgtgattaa attggttcta aacctttagt 660

tttatgataa tttagtaact aaacgtgtag ttttgtaaca cttcatcttt aatatgtagt 720

tttgtgctaa atttggtgct aaatgtgtaa ttttgtgata taattcctta aatatgtagt 780

tttgtgatag tttggttata atatctgtag ttttatgaaa tttactcttt tcgttttcac 840

tgcaatttgg aatgatggaa ttgactagat ccggcattac cgatgggctg ccgaacgctg 900

tgatgcggtt gatcttgagc gatccgggac gccacaagca ccgatgggtt ctgggagttc 960

atacggctgg tgcagcagtg tgtcaatagc agccgggatg tgcgcccaac catggtcgcc 1020

gtcgagagga ggatcgaaga catcctgaac tcggttgtca ggtcatccac caccgggttc 1080

atgactgccg gaggcgacac acccagcaac gagccaaatc gtgaagataa cggaaacgag 1140

ccaaatccca gcaacgagat cgccagggac tagtagtacg tacagcagtg gtgatttgtc 1200

atataggtgt atatcggctg ttttcgcatc tcaaggcctc aagcagtgtg tgcaatctgg 1260

agtagtatat aaatatgtaa aatgttcatt tcgatatact gtcaaatgcg tgtaaattaa 1320

ccaatgctaa aacaacacac tgtgactaaa tttactgagt tggatgatga ggatgattat 1380

gttgcgtgca cacctgatca ggaggacata taatataggc catttgggcc gtcttggaca 1440

ccaccgtttg atttgtatga agttgggccg aactatgcaa gcccagaggc gctgcctctg 1500

tgccacggcc cacgggcatc gctggatggt caagcaggtg atcggtggag cgccaatggc 1560

ggcggcgaga cacacagcgc ggcgcgcgcg cgaacgtgcg gacgcgcgcg ccccggccac 1620

ggccgccgcg ctcgtctcct ggcctcccgc gcccgctaca aatggcggcc ccggcgtccc 1680

ctcctcactc cgaagcttcc cggttgacga cctctccggt ctcccccctc accccaccgc 1740

aacccgggac gtcttccatg gccgccgccg ccgccgcccc cgcctactaa accaccctac 1800

ccaccccctc caaactccca cacattacat ccttcaaaga gagcatcaca cacacacaca 1860

caccagccta gcgatcacat ttccacg 1887

<210> 7

<211> 311

<212> DNA

<213> Rice (oryza. sativa l.)

<400> 7

gcagaaggtg atcctgatca acggcgagtt cccgggcccg cggatcaact gctcgtccaa 60

caacaacatc gtggtgaacg tgttcaacca gctggacgag ccgctgctct tcacctggaa 120

cgggatgcag caccgcaaga actcgtggca ggacggcctc gccgggacgc agtgccccat 180

cgcgccgggc accaactaca cgtacaagtg gcagcccaag gaccagatcg gcagcttctt 240

ctacttcccg tcgctgggga tgcaccgcgc cgccggcggc tacggcggga tcagcgtcgt 300

cagccgcctg c 311

<210> 8

<211> 150

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

acgagctggt gagctagcta ttacctaatc gatcgatggt catcgatcat gagatgatga 60

tgatgagatt tgtacttaat tgtgatctgt atggatgctg ttgttgatca agttcttgcg 120

atcgatcgat ctgaattttc aggtttgagg 150

<210> 9

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ggcgtcttca ggtacaacca g 21

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ggtacacctg gaccgagtgc 20

<210> 11

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ctctgtatgc cagtggtcgt 20

<210> 12

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ccgttgtggt gaatgagtaa c 21

<210> 13

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gcgtcgacgt cccatgtcac cgacagtact 30

<210> 14

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

catgccatgg cgtggaaatg tgatcgctag gct 33

<210> 15

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gcaggcggct gacgacgctg at 22

<210> 16

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

gatctggtcc ttgggctgcc acttg 25

<210> 17

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

ggttgaacac gttcaccacg atgtt 25

<210> 18

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

ggcgtcttca ggtacaacca g 21

<210> 19

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

cgagttccgc accttcatcg agatc 25

<210> 20

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

cgatcatgct gacgttcgac aac 23

<210> 21

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

cctccaaact cccacacatt acatcct 27

<210> 22

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

gctcagcttc ttcataggac gtacgtt 27

<210> 23

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

ggactcttga ccatgatggg acgcagggct tgct 34

<210> 24

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

attcgagctg gtcactcacg cacacaggtt ccaag 35

<210> 25

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

gaagatctac gagctggtga gctagcta 28

<210> 26

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

ctggtcaccc ctcaaacctg aaaattcag 29

<210> 27

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

cggaattcgc agaaggtgat cctgatcaac 30

<210> 28

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

gaagatctgc aggcggctga cgacgctgat 30

<210> 29

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

gacctgcagg gcagaaggtg atcctgatca ac 32

<210> 30

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

ctggtcaccg caggcggctg acgacgctga t 31

Claims

1. A rice pollen tube growth related gene OsPTD1 is characterized in that: the amino acid sequence of the rice pollen tube growth related gene code is shown in SEQ ID NO. 1; or the amino acid sequence shown in SEQ ID NO.1 is substituted, deleted or added with at least one amino acid and has the amino acid sequence with the same function as the OsPTD1 protein.

2. The rice pollen tube growth related gene OsPTD1 as claimed in claim 1, wherein: the CDS nucleotide sequence of the rice pollen tube growth related gene OsPTD1 is shown as SEQ ID NO. 2; or a nucleotide sequence which is shown in SEQ ID NO.2, is substituted, deleted or added with at least one nucleotide and has the same function with the rice pollen tube growth related gene OsPTD 1.

3. The rice pollen tube growth related gene OsPTD1 as claimed in claim 1, wherein: the genome nucleotide sequence of the rice pollen tube growth related gene OsPTD1 is shown in SEQ ID NO. 3; or a nucleotide sequence which is shown in SEQ ID NO.3, is substituted, deleted or added with at least one nucleotide and has the same function with the rice pollen tube growth related gene OsPTD 1.

4. Use of the rice pollen tube growth related gene OsPTD1 as claimed in any one of claims 1-3 in restoring pollen fertility of mutant, wherein the mutant is a pollen sterile mutant produced by variation of OsPTD1 gene or homologous gene.

5. Use of the rice pollen tube growth related gene OsPTD1 expression of any one of claims 1-3 in the preparation of gametophyte male sterility or reproductive sporophyte nuclear sterility by down-regulation or mutation.

6. Use according to claim 5, characterized in that: the method for down-regulating the expression of the rice pollen tube growth related gene OsPTD1 is to interfere the expression of the rice pollen tube growth related gene OsPTD 1.

7. Use according to claim 6, characterized in that: the method for interfering the expression of the rice pollen tube growth related gene OsPTD1 is characterized by firstly constructing a vector for interfering the expression of the rice pollen tube growth related gene OsPTD1, transfecting plants through agrobacterium mediation, and screening transgenic positive plants.

8. Use according to claim 7, characterized in that: the expression vector contains an interference stem-loop, an interference left arm and an interference right arm, and the sequence of the interference stem-loop is the interference stem-loop shown in SEQ ID NO. 8; the interference left arm takes sequences shown in SEQ ID NO.27 and SEQ ID NO.28 as primers and is obtained by amplification from the Zhonghua 11; the interference right arm is obtained by taking sequences shown in SEQ ID NO.29 and SEQ ID NO.30 as primers and amplifying from the Zhonghua 11.

9. The method for preparing gametophyte male sterility by down-regulating the expression of a rice pollen tube growth related gene OsPTD1 is characterized by comprising the following steps:

transfecting a plant by the pollen specific promoter through agrobacterium-mediated transformation by a down-regulation element Xi of a gene OsPTD1 related to the growth of a rice pollen tube, and screening a transgenic positive strain, wherein the transgenic positive strain is gametophyte male sterility.

10. A method for breeding sporophyte male sterility by down-regulating the expression of a rice pollen tube growth related gene OsPTD1 is characterized in that:

linking a certain sporophyte male fertile gene Y with an OsPTD1 gene interference fragment regulated and controlled by a promoter POsPTD1, transferring into a sterile mutant of the sporophyte male fertile gene Y, screening a transgenic positive plant, pollinating the sterile mutant of the sporophyte male fertile gene Y by taking the positive plant as a male parent, and reproducing the sporophyte nuclear male sterility.