CN115448982A - Application of TaRomo1 protein in regulation of male fertility of wheat - Google Patents

Abstract

The invention discloses the application of TaRomo1 protein in regulating wheat male fertility. The amino acid sequence of TaRomo1 protein is shown in SEQ ID NO:2 and/or SEQ ID NO:4. Silencing TaRomo1 protein can lead to wheat male semi-sterility, which is manifested in the opening of glumes at the panicle, decreased pollen fertility, and decreased seed setting rate. It can be seen that TaRomo1 protein can regulate the male fertility of wheat. The invention has important application value.

Description

Application of TaRomo1 Protein in Regulating Wheat Male Fertility

technical field

The invention belongs to the field of biotechnology, and in particular relates to the application of TaRomo1 protein in regulating wheat male fertility.

Background technique

Hybrids have the characteristics of high yield and excellent agronomic traits, and are widely used in crop production. Wheat is one of the most important food crops in the world, and increasing the yield per unit area of wheat is of great significance to ensuring national food security. Wheat is a strictly monoecious, self-pollinated crop, and the production of hybrids is entirely dependent on the use of wheat male sterile lines. Compared with rice, available wheat male sterile line materials and male sterile gene resources are very scarce, resulting in the production level of its hybrids lagging far behind that of rice. Therefore, cloning new male sterile genes and enriching existing male sterile gene resources is an important way to create wheat male sterile line materials, and has important practical significance in production.

Through map-based cloning, three male sterility genes have been cloned in wheat, namely Ms1, Ms2 and Ms5. Both Ms1 and Ms5 encode glycosylphosphatidylinositol-anchored lipid transfer protein. The specific expression of the gene ensures the integrity of the pollen exine, while the deletion of the gene causes the accumulation of long-chain fatty acids in anthers and the destruction of the structure of the pollen exine, resulting in male sterility. The Ms2 gene encodes an orphan protein with unknown function, but because ms2 causes sterility to be dominant, it is difficult to be used in large-scale wheat hybrid seed production. In addition to the above genes, no other recessive male sterility genes with definite functions have been identified so far, and the resources of related sterility genes are very scarce.

Contents of the invention

The purpose of the present invention is to obtain the protein related to wheat male sterility or male partial sterility.

The present invention firstly protects the TaRomo1 protein derived from wheat. The TaRomo1 protein may be a1) or a2) or a3) or a4) as follows:

a1) the amino acid sequence is the protein shown in SEQ ID NO: 2 and/or SEQ ID NO: 4;

a2) a fusion protein obtained by connecting a tag to the N-terminal or/and C-terminal of the protein shown in SEQ ID NO: 2 and/or SEQ ID NO: 4;

a3) A protein derived from wheat and related to wheat male fertility obtained by substituting and/or deleting and/or adding one or several amino acid residues to the protein shown in a1) or a2);

a4) A protein having 80% or more homology with the amino acid sequence defined by SEQ ID NO: 2 and/or SEQ ID NO: 4, derived from wheat and related to male fertility of wheat.

Wherein, SEQ ID NO: 2 consists of 75 amino acid residues. SEQ ID NO: 4 consists of 77 amino acid residues. SEQ ID NO: 6 consists of 77 amino acid residues.

In order to make the protein in a1) easy to purify, the amino terminus or carboxyl terminus of the protein shown in SEQ ID NO: 2 and/or SEQ ID NO: 4 can be linked with the tags shown in Table 1.

Table 1. Sequence of tags

Label Residues sequence Poly-Arg 5-6 (usually 5) RRRRR FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

For the protein in a3) above, the substitution and/or deletion and/or addition of one or several amino acid residues is a substitution and/or deletion and/or addition of no more than 10 amino acid residues.

The protein in a3) above can be synthesized artificially, or its coding gene can be synthesized first, and then obtained by biological expression.

The coding gene of the protein in the above a3) can be deleted by the codon of one or several amino acid residues in the DNA sequence shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, and/or carry out A missense mutation of one or several base pairs, and/or the coding sequence of the tag shown in Table 1 is attached to its 5' end and/or 3' end.

The present invention also protects the nucleic acid molecule encoding any one of the aforementioned TaRomo1 proteins.

The nucleic acid molecule encoding any of the TaRomo1 proteins described above can be a DNA molecule as shown in b1) or b2) or b3) or b4) as follows:

b1) the coding region is the DNA molecule shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5;

b2) the nucleotide sequence is a DNA molecule shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5;

b3) has 75% or more homology with the nucleotide sequence defined in b1) or b2), and is derived from wheat and encodes any one of the above-mentioned TaRomo1 protein DNA molecules;

b4) a DNA molecule that hybridizes to the nucleotide sequence defined in b1) or b2) under stringent conditions and encodes any one of the above-mentioned TaRomo1 proteins.

Wherein, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

Wherein, SEQ ID NO:1 consists of 228 nucleotides, SEQ ID NO:3 consists of 234 nucleotides, and SEQ ID NO:5 consists of 234 nucleotides. The nucleotide sequence shown in SEQ ID NO:1 encodes the amino acid sequence shown in SEQ ID NO:2. The nucleotide sequence shown in SEQ ID NO:3 encodes the amino acid sequence shown in SEQ ID NO:4. The nucleotide sequence shown in SEQ ID NO:5 encodes the amino acid sequence shown in SEQ ID NO:6.

Those skilled in the art can easily use known methods, such as directed evolution and point mutation methods, to mutate the nucleotide sequence encoding the TaRomo1 protein of the present invention. Those artificially modified nucleotides having 75% or higher identity with the nucleotide sequence of the TaRomo1 protein isolated in the present invention, as long as they encode the TaRomo1 protein, are all derived from the nucleosides of the present invention acid sequence and is equivalent to the sequence of the present invention.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "Identity" includes 75% or higher, or 80% or higher, with the nucleotide sequence of the TaRomo1 protein encoded by the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 of the present invention , or nucleotide sequences of 85% or greater, or 90% or greater, or 95% or greater identity. Identity can be assessed visually or with computer software. Using computer software, identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

Expression cassettes, recombinant vectors or recombinant microorganisms containing any of the nucleic acid molecules described above also belong to the protection scope of the present invention.

The present invention also protects any of the above-mentioned TaRomo1 protein, or, any of the above-mentioned nucleic acid molecules, or, the expression cassette, recombinant vector or recombinant microorganism containing any of the above-mentioned nucleic acid molecules, in regulating male fertility in wheat application.

The present invention also protects any of the above-mentioned TaRomo1 proteins, or, any of the above-mentioned nucleic acid molecules, or, an expression cassette, a recombinant vector or a recombinant microorganism containing any of the above-mentioned nucleic acid molecules, when cultivating male sterility or male parts Application of sterile transgenic wheat.

The present invention also protects a method for cultivating transgenic wheat, including the step of inhibiting the expression and/or activity of any one of the above-mentioned TaRomo1 proteins in the recipient wheat to obtain the transgenic wheat; compared with the recipient wheat, the Male sterility or partial male sterility in genetically modified wheat.

In the above method, the "inhibiting the expression level and/or activity of any one of the above-mentioned TaRomo1 proteins in the recipient wheat" can be carried out by methods well known in the art, such as gene site-directed editing, RNA interference, homologous recombination, gene knockout, etc. The purpose of inhibiting the expression and/or activity of TaRomo1 protein is achieved.

In the above method, the "inhibiting the expression and/or activity of any of the above-mentioned TaRomo1 proteins in the recipient wheat" can be introduced into the recipient wheat to inhibit the expression and/or activity of any of the above-mentioned TaRomo1 proteins. substance.

In the above method, the substance that inhibits the expression and/or activity of any one of the above-mentioned TaRomo1 proteins can specifically be the carrier Pro-stem-loop mentioned in the examples. The transgenic wheat can be any one of Pro::Romo1-RNAi#2-Pro::Romo1-RNAi#5 mentioned in the examples.

The present invention also protects a wheat breeding method, which may include the following steps: reducing the content and/or activity of any one of the above-mentioned TaRomo1 proteins in wheat, so that the wheat is male sterile or partially male sterile.

Any of the above-mentioned wheats can be wheat Fielder.

The male sterility or partial male sterility of any one of the above-mentioned wheats can be manifested specifically as the opening of glumes at the panicle.

The male sterility or partial male sterility of any one of the above-mentioned wheats can specifically be manifested as a decrease in pollen fertility (eg, decrease to below 50% of normal).

The male sterility or partial male sterility of any one of the above-mentioned wheats can be specifically manifested as a decrease in seed setting rate.

Experiments have proved that silencing TaRomo1 gene can lead to wheat male semi-sterility, that is, TaRomo1-1AL protein, TaRomo1-1BL protein and TaRomo1-1DL protein can regulate the male fertility of wheat. TaRomo1 gene is a wheat male sterility gene with potential utilization value. TaRomo1 protein can regulate wheat male fertility. The invention has important application value.

Description of drawings

Figure 1 is a schematic diagram of the partial structure of the vector pANDA (NPT II is Kanamycin resistance gene, HPT is Hygromycin resistance gene, Ubqpro. is Maize ubiquitin1 promoter+1st intron&splicing acceptor site, attR is LR clonase recombination cassette (Invitrogen, Cat.No.11828- 019, rfA), attR1&attR2 are LR clonase recombination sites, CmR is Chloramphenicol resistance gene, ccdB is ccd B gene, Nt is NOS terminator, bar is Bialaphos resistance gene, and the vector backbone is pBI101).

Figure 2 is the bar gene identification results of Pro::Romo1-RNAi#2-Pro::Romo1-RNAi#5.

Fig. 3 is the glume state of Pro::Romo1-RNAi#2-Pro::Romo1-RNAi#5 ear.

Figure 4 shows the pollen staining results of Pro::Romo1-RNAi#2-Pro::Romo1-RNAi#5.

Figure 5 is the pollen viability statistical results of Pro::Romo1-RNAi#2-Pro::Romo1-RNAi#5.

Fig. 6 is the statistical result of seed setting rate of Pro::Romo1-RNAi#2-Pro::Romo1-RNAi#5.

detailed description

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention. The examples provided below can be used as a guideline for those skilled in the art to make further improvements, and are not intended to limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Embodiment 1, the acquisition of TaRomo1 gene

TaRomo1 genes include TaRomo1-1AL gene from wheat A genome, TaRomo1-1BL gene from wheat B genome and TaRomo1-1DL gene from wheat D genome.

The TaRomo1-1AL gene, TaRomo1-1BL gene and TaRomo1-1DL gene were artificially synthesized.

The nucleotide sequence of the TaRomo1-1AL gene is shown in SEQ ID NO:1, which encodes the TaRomo1-1AL protein shown in SEQ ID NO:2.

The nucleotide sequence of the TaRomo1-1BL gene is shown in SEQ ID NO:3, which encodes the TaRomo1-1BL protein shown in SEQ ID NO:4.

The nucleotide sequence of the TaRomo1-1DL gene is shown in SEQ ID NO:5, which encodes the TaRomo1-1DL protein shown in SEQ ID NO:6.

TaRomo1 proteins include TaRomo1-1AL protein, TaRomo1-1BL protein and TaRomo1-1DL protein.

Embodiment 2, the application of TaRomo1 protein in regulating wheat male fertility

1. Construction of vector pANDA-antiRomo1

1. Using the nucleotide sequence of the TaRomo1-1AL gene shown in SEQ ID NO: 1 as a template, use TaRomo1RNAi-F: 5'- GCAGGCTCAGGGGATATC TTCAGCAGCCCAGGGACCCT-3' (the underline is the same sequence as the pQBV3 vector for homologous Recombination) and TaRomo1 RNAi-R: 5'- CAGGGCGATATCGATATC CGAGGAGGGATAGCTGCTT-3' (the underline is the same sequence as the pQBV3 vector, used for homologous recombination) for PCR amplification, and a PCR amplification product of about 163bp was recovered.

The reaction program is: 94°C for 2min; 94°C for 30sec, 60°C for 30sec, 68°C for 1min, 34cycles; 68°C for 10min.

2. Sequencing the PCR amplification product recovered in step 1.

Sequencing results showed that the PCR amplification product contained a DNA fragment with a nucleotide sequence as shown in SEQ ID NO:7.

3. The PCR amplification product and vector pQBV3 (recorded in the following documents: Liu, J., Cheng, X., Liu, P., and Sun, J. (2017). miR156-Targeted SBP-Box Transcription Factors Interact withDWARF53 to Regulate TEOSINTE BRANCHED1 and BARREN STALK1 Expression in BreadWheat. Plant Physiology 174, 1931-1948.) for homologous recombination to obtain the intermediate vector pQBV3-antiRomo1.

4. Prepare the Gateway reaction system. The Gateway reaction system is 5 μl, including intermediate vector pQBV3-antiRomo1 (about 80 ng), vector pANDA (about 80 ng), 0.5 μl LR enzyme and TE buffer.

The vector pANDA is described in: Chukwurah, P.N., Poku, S.A., Yokoyama, A., Takeda, A., Shishido, M., Nakamura, I. (2019). Mitigating root knot nematodepropagation on transgenic tobacco via in Planta hairpin RNA expression of Meloidogyne incognita-specific PolA1 sequence. American Journal of Plant Science, 10, 866-884.

The schematic diagram of the partial structure of the vector pANDA is shown in Fig. 1 .

5. Take the Gateway reaction system prepared in step 4 and treat it at 25°C for 1 hour to obtain the vector pANDA-antiRomo1.

2. Construction of carrier Pro-stem-loop

1. Using the vector pANDA-antiRomo1 as a template, use the primer pair consisting of SL-F: 5'-aggtagggagTTCAGCAGCCCAGGGACCCT-3' and SL-R: 5'-GAACGATCTGTTCCTAGGGTTAACTTCAGCAGCCCAGGGACCCT-3' for PCR amplification, and recover about 1214bp of PCR amplification Product (ie stem-loop sequence).

2. Sequencing the PCR amplification product recovered in step 1.

Sequencing results show that the PCR amplification product recovered in step 1 contains a DNA fragment whose nucleotide sequence is shown in SEQ ID NO:8.

3. Using the vector Blunt-KX943032.1 as a template, use the primer pair consisting of Pro-F: 5'-GAAGGAGCCACTCAGCAAGCTTCATCACCTCCTCCGTCCTCAC-3' and Pro-R: 5'-GGGCTGCTGAActccctacctccggccggccttc-3' to perform PCR amplification, and recover about 3894bp PCR Amplified product (ie Pro sequence).

The vector Blunt-KX943032.1 is the recombinant plasmid obtained by inserting the target gene (KX943032.1) into the vector pEASY-Blunt (TransGen Biotech, CB101); details are described in the following documents: Xia, C, Zhang, L., Zou, C ., Gu, Y., Duan, J., Zhao, G., Wu, J., Liu, Y., Fang, X., Gao, L., Jiao, Y., Sun, J., Pan, Y. .,Liu,X.,Jia,J.and Kong,X.(2017).A TRIM insertion in the promoter of Ms2causes male sterility in wheat.Nature Communications8,15407.

4. Sequencing the PCR amplification product recovered in step 3.

Sequencing results show that the PCR amplification product recovered in step 3 contains a DNA fragment with a nucleotide sequence as shown in SEQ ID NO:9.

5. Digest the vector pUbi-pAHC25 with restriction enzymes HindIII and HpaI (recorded in the following documents: He, X., Qu, B., Li, W., Zhao, X., Teng, W., Ma, W., Ren, Y., Li, B., Li, Z., and Tong, Y. (2015). The Nitrate-Inducible NAC Transcription Factor TaNAC2-5A Controls Nitrate Response and Increases Wheat Yield. Plant physiology 169, 1991 -2005.), Recovery of vector backbones.

6. Prepare the reaction system. The reaction system is 10 μl, including the vector skeleton recovered in step 5 (about 30 ng), stem-loop sequence (about 50 ng), Pro sequence (about 50 ng), 5 μl Ligase-free cloning Mix and H ₂ O.

7. Take the reaction system prepared in step 6 and treat it at 50°C for 40 minutes to obtain the carrier Pro-stem-loop.

3. Acquisition of recombinant Agrobacterium

Using the heat shock transformation method, the vector Pro-stem-loop was introduced into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium, which was named EHA105/Pro-stem-loop.

4. Obtaining genetically modified wheat

Using the Agrobacterium-mediated genetic transformation method, EHA105/Pro-stem-loop was transformed into wheat Fielder, and then selfed for ₂ generations to obtain 4 transgenic wheat T2 lines, which were named Pro::Romo1-RNAi#2—Pro :: Romo1-RNAi#5.

5. Identification of transgenic wheat

The wheat to be tested is Wheat Fielder, Pro::Romo1-RNAi#2, Pro::Romo1-RNAi#3, Pro::Romo1-RNAi#4 or Pro::Romo1-RNAi#5.

1. Take about 0.5 g of wheat leaves to be tested, crush them into powder in liquid nitrogen, then add 400 μl of EB2 extraction buffer (a component in the QuickStix kit (EnviroLogix, product number AS013LS)), mix well, and obtain a sample solution.

2. Insert the end of the test strip (a component in the QuickStix kit (EnviroLogix, product number AS013LS)) into the sample solution, observe the test strip, and make the following judgment: if two bands are displayed, the wheat bar gene to be tested is inserted; If one band is displayed, the tested wheat does not have the bar gene inserted.

The identification results are shown in Figure 2 (WT is wheat Fielder). The results showed that Pro::Romo1-RNAi#2—Pro::Romo1-RNAi#5 were all bar gene insertions, that is, they were all transgenic lines.

6. Identification of male fertility

The wheat to be tested is Wheat Fielder, Pro::Romo1-RNAi#2, Pro::Romo1-RNAi#3, Pro::Romo1-RNAi#4 or Pro::Romo1-RNAi#5.

1. The wheat to be tested grows to the flowering stage, and observes the state of glumes at the ear.

The results are shown in Figure 3 (WT is wheat Fielder). The results showed that the glumes of Pro::Romo1-RNAi#2-Pro::Romo1-RNAi#5 were open and sterile; the glumes of wheat Fielder were closed.

2. Take the anthers of the wheat to be tested, apply the pollen in the anthers on the glass slide, drop an appropriate amount of starch potassium iodide solution for staining, cover the glass with a cover glass and observe under the microscope, the pollen with vitality is black.

The staining results are shown in Figure 4 (WT is wheat Fielder). The statistical results are shown in Figure 5 (WT is wheat Fielder). The results showed that the pollen fertility of wheat Fielder was normal; compared with wheat Fielder, the pollen fertility of Pro::Romo1-RNAi#2—Pro::Romo1-RNAi#5 was reduced to less than 50%.

3. The wheat to be tested is mature, and the seed setting rate is counted.

The results are shown in Figure 6 (WT is wheat Fielder). The results showed that compared with wheat Fielder, the seed setting rate of Pro::Romo1-RNAi#2—Pro::Romo1-RNAi#5 was significantly lower.

The above results indicated that silencing TaRomo1 gene can lead to wheat male semi-sterility, that is, TaRomo1-1AL protein, TaRomo1-1BL protein and TaRomo1-1DL protein can regulate the male fertility of wheat. TaRomo1 gene is a wheat male sterility gene with potential utilization value.

The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experiments, the present invention can be practiced in a wider range under equivalent parameters, concentrations and conditions. While specific embodiments of the invention have been shown, it should be understood that the invention can be further modified. In a word, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by using conventional techniques known in the art and departing from the disclosed scope of this application. Applications of some of the essential features are possible within the scope of the appended claims below.

<110> Institute of Crop Science, Chinese Academy of Agricultural Sciences

<120> Application of TaRomo1 Protein in Regulation of Wheat Male Fertility

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gaaatggatc ggcccggaca caaaacggac cagataggtt caggccgtcg cgcgctgggc 1140

gtggggatttg ttcttttgtc ccaaatggac ggggccggac gggatggggt cgcgcgcaag 1200

ggcgagagca gaaccatcga ccgcaagcgg tgattttctg cgcaccttct gccataggtg 1260

tagctcgtcg accgccaagt atatcactgt ctcgcgattt gagcatagag tcaatcgatt 1320

ttcctggcca atggcgtcaa ggggagagat ttggtcaaat gggcggaagt cgcagaccca 1380

tgtatatgtg cacgggtggg tgggtgcctt agggcattta caacgcaagg cgctaaggcg 1440

ggcgccaggg tcaggatcct agtcgtttgg cttagttccc gtccaaattt gagaattgag 1500

ctggcatcga tgccatataa gtcgtcgggc gtcgggcgct aactcagttt tctgtctatt 1560

ttatgtgtgt agcgctcata cgtggctctc agcgttggaa gagggactct tagcccaggc 1620

gctaggaaga aaatactatt ttatttccag tcaagtgcct gattaggcgc cctccattgg 1680

agatgccctt atgtgcctct ctacgccgca gcagccggaa actacggcgc accagtactg 1740

gacggctcgt ttcttattct aaacacagat actagtgttg ttgccgccag tccctcgccg 1800

ccggagctct ctctctctct ctccctcgca caaacataga agaaagaagg aagaggagcg 1860

atgcagtgga cacaacaagc tttacgcggt gcacgtacgc tgccggccgc acgaacagcc 1920

gatcgttttc attcctgagc tcgaactcag ccaccggaca acaacgagta cacagagggc 1980

cttctatacc caagctacac acatcaggct agctaccaca cgcaagcacg catgcatcca 2040

ctgcagcgaa agctaactac atgcacgcat gcagcccacg acccggctgc atgacgcccg 2100

cgcctgccga gtccacgatc cgcacggcgt gaccaactaa ctgcatgcaa ctagacggag 2160

cgcccgca acgcccgccc cgcgctcctc agctcccgcg cccgccgcgc acgcacgcca 2220

acgggatacg actggttcca gcgcctggcg cggtcacacc tcgcgcgtcc gtctaaccaa 2280

cacacacaca catgaccccg ccgcgcaccc gccgcgcccg acacgcccgg cgcaatcgcg 2340

gtggcttatg cccaacactc accccccctta gccacgaatt acagcaggtg agttcatcat 2400

cgtcgatgtc gccatggccg tcgcatcgca ccgctgcggc ctccgccatg ccgtcgacgt 2460

cgttgtagcc gccgccgtcc tgacgtcgct gccaacacctg ccaccgtgcc gccgtgccct 2520

tcgcgtgcac tccccgcgct cccggcccgc gctcccgcgc gcacgtacgc tatctgcgca 2580

actaggtcca gtgtctcgac gcggtccact cccacggtcc cgacgcgtct ggtgcgcacc 2640

cataacacgc accggtcgcg cccggctcgc caccgcgtct tattgccctg cactgccgtg 2700

ccgtcaaccg tagcgcagcg cctccacggt cgtcgcgccg agccgccgcg gcctctgcga 2760

caccacgcag gtcctccgcg acctcctcgt ctccgcgacc gccactgctc gccgcgcgca 2820

cggcatcacg ccacaccgcc gtggactcgc cgcgcgttgc cgacgccacg cgctcgccgc 2880

acgcccggca tcacgccaca ccgccgtgga ctcgccgcgc gttgccgaga tcttcatgtc 2940

cgccgcgcgc cacggccgcc ccccgaacct gtggctctga taccaaatgt tgttgccgcc 3000

agtccctcgc cgccggagct ctctctctct ctctccctcg cacaaacata gaagaaagaa 3060

ggaagaggag cgatgcagtg gacacaacaa gctttacgcg gtgcacgtac gctgccggcc 3120

gcacgaacag ccgatcgttt tcattcctga gctcgaactc agccaccgga caacaacgag 3180

tacacagagg gccttctata cccaagctac acacatcagg ctagctacca cacgcaagca 3240

cgcatgcatc cactgcagcg aaagctaact acatgcacgc atgcagccca cgacccggct 3300

gcatgacgcc cgcgcctgcc gagtccacga tccgcacggc gtgaccaact aactgcatgc 3360

aactagacgg agcgcccacg caacgcccgc cccgcgctcc tcagctcccg cgcccgccgc 3420

gcacgcacgc caacgggata cgactggttc cagcgcctgg cgcggtcaca cctcgcgcgt 3480

ccgtctaacc aacacacaca cacatgaccc cgccgcgcac ccgccgcgcc cgacacgccc 3540

ggcgcaatcg cggtggctta tgcccaacaa ctagtgtgca cctcgttgag agtgcggcac 3600

ccgactgcac agtgcacatg catgcagctg gctctttctc ttgacttgac acgctctcgc 3660

ttctcccgat tcctgcccgc gccggcgtct ccacccgact tgatcgacat cggcatcggc 3720

atcggcctcg gcatcggccc ctcgacgacg ctcagtatat aagcgatcgg gctggtggag 3780

ctgcttgcag tacccgcagt ggacacacgc ttagctttag ctacgtaggc gcagcagccg 3840

gaaactagct agcaggtcga gaaggccggc cggaggtagg gagttcagca gccc 3894

Claims (10)

1. TaRomo1 protein, as follows a1) or a2) or a3) or a4): a1) the amino acid sequence is the protein shown in SEQ ID NO: 2 and/or SEQ ID NO: 4; a2) a fusion protein obtained by connecting a tag to the N-terminal or/and C-terminal of the protein shown in SEQ ID NO: 2 and/or SEQ ID NO: 4; a3) A protein derived from wheat and related to wheat male fertility obtained by substituting and/or deleting and/or adding one or several amino acid residues to the protein shown in a1) or a2); a4) A protein having 80% or more homology with the amino acid sequence defined by SEQ ID NO: 2 and/or SEQ ID NO: 4, derived from wheat and related to male fertility of wheat. 2. A nucleic acid molecule encoding the TaRomol protein of claim 1. 3. nucleic acid molecule as claimed in claim 2, is characterized in that: described nucleic acid molecule is following b1) or b2) or b3) or the dna molecule shown in b4): b1) the coding region is the DNA molecule shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5; b2) the nucleotide sequence is a DNA molecule shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5; b3) has 75% or more homology with the nucleotide sequence defined in b1) or b2), and is derived from wheat and encodes the DNA molecule of TaRomo1 protein described in claim 1; b4) a DNA molecule that hybridizes to the nucleotide sequence defined in b1) or b2) under stringent conditions and encodes the TaRomo1 protein in claim 1. 4. An expression cassette, a recombinant vector or a recombinant microorganism containing the nucleic acid molecule of claim 2 or 3. 5. The TaRomo1 protein of claim 1, or, the nucleic acid molecule of claim 2 or 3, or, the expression cassette, recombinant vector or recombinant microorganism containing the nucleic acid molecule of claim 2 or 3, the application is D1) or D2): D1) regulating wheat male fertility; D2) Breeding male sterile or partially male sterile transgenic wheat. 6. The application according to claim 5, characterized in that: the wheat is wheat Fielder. 7. A method for cultivating transgenic wheat, comprising inhibiting the expression level and/or activity of the TaRomo1 protein described in claim 1 in the recipient wheat to obtain the step of transgenic wheat; compared with the recipient wheat, the transgenic Male sterility or male partial sterility in wheat. 8. The method according to claim 7, characterized in that: the expression level and/or activity of the TaRomo1 protein described in claim 1 in the suppressing recipient wheat is introduced into the recipient wheat to inhibit the expression of the TaRomo1 protein described in claim 1. The expression level and/or activity of the above-mentioned TaRomo1 protein. 9. A method for breeding wheat, comprising the steps of: reducing the content and/or activity of the TaRomo1 protein of claim 1 in wheat, so that the male sterility or partial male sterility of the wheat is achieved. 10. The method according to any one of claims 7-9, characterized in that: the wheat is wheat Fielder.

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