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

Abstract

The invention discloses application of a TaRomo1 protein in regulation and control of male fertility of wheat. The amino acid sequence of the TaRomo1 protein is shown as SEQ ID NO:2 and/or SEQ ID NO:4, respectively. The silent TaRomo1 protein can cause the male sterility of wheat, and is specifically shown in the aspects of opening of glumes of ears, reduction of pollen fertility, reduction of fructification rate and the like. Therefore, the TaRomo1 protein can regulate the male fertility of wheat. The invention has important application value.

Description

Application of TaRomo1 protein in regulation of male fertility of wheat

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of TaRomo1 protein in regulation of male fertility of wheat.

Background

The hybrid has the characteristics of high yield, excellent agronomic characters and the like, and is widely applied to crop production. Wheat is one of the most important grain crops in the world, and the improvement of the yield per unit of wheat has great significance for guaranteeing national grain safety. Wheat is a strict male and female homofloral self-pollinated crop, and the production of hybrid seeds completely depends on the use of a wheat male sterile line. Compared with rice, the available wheat male sterile line material and male sterile gene resource are very deficient, so that the production level of hybrid seeds is far behind that of rice. Therefore, the cloning of the new male sterile gene enriches the existing male sterile gene resources, is an important way for creating wheat male sterile line materials, and has important practical production significance.

Through map-based cloning, the wheat has been cloned into 3 male nuclear sterile genes, which are Ms1, ms2 and Ms5 respectively. Both Ms1 and Ms5 encode glycosyl phosphatidylinositol anchored lipid transfer protein, the specific expression of the gene ensures the integrity of the pollen outer wall, and the gene deletion causes the accumulation of anther long-chain fatty acid and the structural damage of the pollen outer wall, resulting in male sterility. The Ms2 gene codes an orphan protein with unknown function, but the sterility is dominant due to Ms2, so that the Ms2 gene is difficult to be used for large-scale wheat hybrid seed production. Besides the genes, other recessive male nuclear sterile genes with clear functions are not identified at present, and related sterile gene resources are very deficient.

Disclosure of Invention

The invention aims to obtain protein related to wheat male sterility or male partial sterility.

The invention firstly protects the TaRomo1 protein from wheat. The TaRomo1 protein may be a 1) or a 2) or a 3) or a 4) as follows:

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

a2 In SEQ ID NO:2 and/or SEQ ID NO:4, the N end or/and the C end of the protein shown in the figure is connected with a label to obtain fusion protein;

a3 A protein which is derived from wheat and is related to the male fertility of the wheat and is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in a 1) or a 2);

a4 And SEQ ID NO:2 and/or SEQ ID NO:4 has 80 percent or more than 80 percent of homology, is derived from wheat and is related to the male fertility of the 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.

To facilitate purification of the protein of a 1), the protein of SEQ ID NO 2 and/or SEQ ID NO:4 to which the tags shown in table 1 were attached at the amino terminus or the carboxyl terminus of the protein shown in table 4.

TABLE 1 sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
FLAG	8	DYKDDDDK
			Strep-tag II	8	WSHPQFEK
c-myc	10	EQKLISEEDL

The protein according to a 3) above, wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The protein in a 3) can be artificially synthesized, or can be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding the protein of a 3) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in SEQ ID NO.1, SEQ ID NO. 3 or SEQ ID NO. 5, and/or by performing missense mutation of one or several base pairs, and/or by connecting the coding sequence of the tag shown in Table 1 above to the 5 'end and/or 3' end thereof.

The invention also protects a nucleic acid molecule for coding any TaRomo1 protein.

The nucleic acid molecule for coding any TaRomo1 protein can be a DNA molecule shown in the following b 1) or b 2) or b 3) or b 4):

b1 The coding region is SEQ ID NO: 1. the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:5, a DNA molecule shown in the figure;

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

b3 A DNA molecule which has 75 percent or more than 75 percent of homology with the nucleotide sequence limited by b 1) or b 2), is derived from wheat and encodes any of the TaRomo1 proteins;

b4 A DNA molecule hybridizing under stringent conditions with a nucleotide sequence defined in b 1) or b 2) and encoding any of the above-mentioned TaRomo1 proteins.

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

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.

The nucleotide sequence encoding the TaRomo1 protein of the present invention can be easily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 75% or more identity to the nucleotide sequence of the TaRomo1 protein isolated in the present invention, as long as they encode the TaRomo1 protein, are derived from and identical to the nucleotide sequence of the present invention.

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

Expression cassettes, recombinant vectors or recombinant microorganisms containing any of the above-described nucleic acid molecules are also within the scope of the present invention.

The invention also provides application of any one of the above TaRomo1 proteins, or any one of the above nucleic acid molecules, or an expression cassette, a recombinant vector or a recombinant microorganism containing any one of the above nucleic acid molecules in regulation of male fertility of wheat.

The invention also provides application of any one of the above-mentioned TaRomo1 proteins, or any one of the above-mentioned nucleic acid molecules, or an expression cassette, a recombinant vector or a recombinant microorganism containing any one of the above-mentioned nucleic acid molecules in cultivation of male sterile or male partially sterile transgenic wheat.

The invention also provides a method for cultivating transgenic wheat, which comprises the step of inhibiting the expression quantity and/or activity of any TaRomo1 protein in receptor wheat to obtain transgenic wheat; the transgenic wheat is male-sterile or male-partially sterile compared to the recipient wheat.

In the above method, the "inhibiting the expression level and/or activity of any of the above-mentioned TaRomo1 proteins in the recipient wheat" may be achieved by a method known in the art, such as gene site-directed editing, RNA interference, homologous recombination, gene knockout, and the like, to inhibit the expression level and/or activity of the TaRomo1 protein.

In the above method, the step of "inhibiting the expression level and/or activity of any of the above-mentioned TaRomo1 proteins in recipient wheat" may be a step of introducing a substance that inhibits the expression level and/or activity of any of the above-mentioned TaRomo1 proteins into recipient wheat.

In the above method, the substance inhibiting the expression amount and/or activity of any of the above TaRomo1 proteins may be specifically the vector Pro-stem-loop mentioned in the examples. The transgenic wheat may be the Pro: : romo1-RNAi # 2-Pro: : romo1-RNAi #5.

The invention also provides a wheat breeding method, which comprises the following steps: reducing the content and/or activity of any of the above-described TaRomo1 proteins in wheat, whereby the male sterility or male partial sterility of wheat is achieved.

Any of the above wheat may be wheat Fielder.

The male sterility or the male partial sterility of any one of the above-mentioned wheat may be expressed as a glume opening of the ear.

The male sterility or partial male sterility of any one of the above-described wheat plants may be expressed as reduced pollen fertility (e.g., reduced to less than 50% of normal).

The male sterility or male partial sterility of any of the above wheat may be manifested as a reduction in maturing rate.

Experiments prove that silencing of the TaRomo1 gene can cause wheat male half-sterility, namely that the TaRomo1-1AL protein, the TaRomo1-1BL protein and the TaRomo1-1DL protein can regulate and control the male fertility of wheat. The TaRomo1 gene is a wheat male sterile gene with potential utilization value. The TaRomo1 protein can regulate the male fertility of wheat. The invention has important application value.

Drawings

FIG. 1 is a schematic diagram of a partial structure of vector pANDA (NPT II is Kanamycin resistance gene, HPT is Hygromycin resistance gene, ubqpro. Is Maize ubiquitin1 promoter +1st intron and hybridizing receptor site, attR is LR cloning cassette (Invitrogen, cat. No.11828-019, rfA), attR1& attR2 is LR cloning cassettes, cmR is Chramphenicol resistance gene, ccdB is ccd B gene, nt is terminator, bar is Bialaphos resistance gene, and vector backbone is pBI 101).

FIG. 2 is Pro: : romo1-RNAi # 2-Pro: : bar gene identification result of Romo1-RNAi #5.

FIG. 3 is Pro: : romo1-RNAi # 2-Pro: : romo1-RNAi #5 spike glume status.

FIG. 4 is Pro: : romo1-RNAi # 2-Pro: : pollen staining results for Romo1-RNAi #5.

FIG. 5 shows Pro: : romo1-RNAi # 2-Pro: : pollen viability statistics of Romo1-RNAi #5.

FIG. 6 is Pro: : romo1-RNAi # 2-Pro: : statistical results of seed set rate of Romo1-RNAi #5.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 obtaining of the TaRomo1 Gene

The TaRomo1 gene includes a TaRomo1-1AL gene from the wheat A genome, a TaRomo1-1BL gene from the wheat B genome, and a TaRomo1-1DL gene from the wheat D genome.

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

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

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

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

The TaRomo1 protein comprises a TaRomo1-1AL protein, a TaRomo1-1BL protein and a TaRomo1-1DL protein.

Example 2 application of TaRomo1 protein in regulation of male fertility of wheat

1. Construction of vector pANDA-anti Romo1

1. Taking the nucleotide sequence of the TaRomo1-1AL gene shown in SEQ ID NO.1 as a template, adopting TaRomo1RNAi-F:5' -GCAGGCTCAGGGGATATCTtcagcagcagcccagggacctc-3' (underlined is the same sequence as the pQBV3 vector for homologous recombination) and TaRomo1 RNAi-R:5' -CAGGGCGATATCGATATCThe primer pair consisting of CGAGGAGGATAGCTGCTT-3' (underlined is the same sequence as the pQBV3 vector, used for homologous recombination) was subjected to PCR amplification, and about 163bp of PCR amplification product was recovered.

The reaction procedure is as follows: 2min at 94 ℃; 30sec at 94 ℃, 30sec at 60 ℃, 1min at 68 ℃,34cycles; 10min at 68 ℃.

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

Sequencing results show that the PCR amplification product contains a DNA fragment with a nucleotide sequence shown as SEQ ID NO. 7.

3. The PCR amplification product and the vector pQBV3 (described in Liu, J., cheng, X., liu, P., and Sun, J. (2017); miR156-Targeted SBP-Box Transcription Factors Interact with DWARF53 to regulated TEOSINTE BRANCHED1 and BARREN STALK1 Expression in Bread white plant Physiology 174, 1931-1948.) were subjected to homologous recombination to give the intermediate vector pQBV3-anti Romo1.

4. Prepare Gateway reaction system. The Gateway reaction system was 5. Mu.l, and included the intermediate vector pQBV3-anti Romo1 (ca. 80 ng), vector pANDA (ca. 80 ng), 0.5. Mu.l LR enzyme and TE buffer.

The vector pANDA is described in the following documents: chukwurah, P.N., poku, S.A., yokoyama, A., takeda, A., shishido, M., nakamura, I. (2019). Mitigating root mapping on transgenic nanobaco via in Plant hairpin RNA expression of Meloidogyne incognita-specific PolA1 sequence, american Journal of Plant Science,10, 866-884.

A schematic diagram of a portion of the vector pANDA is shown in FIG. 1.

5. And (5) treating the Gateway reaction system prepared in the step (4) at 25 ℃ for 1h to obtain a vector pANDA-anti Romo1.

2. Construction of the vector Pro-stem-Loop

1. Taking a vector pANDA-anti Romo1 as a template, adopting SL-F:5 'aggtagggttcagcagcagcagcagcagccagcccagggacct-3' and SL-R:5 'GAACGATCTGTTCCTAGGTTAACTTCAGCAGCCCAGGACCCT-3' and recovering a PCR amplification product (i.e. stem-loop sequence) of about 1214 bp.

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

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

3. Using a vector Blunt-KX943032.1 as a template, adopting Pro-F:5 'GAAGGAGCCACTCAGCAAGCTTCATCACCTCCCTGTCTCA-3' and Pro-R:5' GGGCTGCTGAActccctctccggcgcggccttc-.

The vector Blunt-KX943032.1 is a recombinant plasmid obtained by inserting a target gene (KX 943032.1) into a vector pEASY-Blunt (TransGen Biotech, CB 101); specifically, the following documents are mentioned: xia, C, zhang, L., zou, C., gu, Y., duan, J., zhao, G., wu, J., liu, Y., fang, X., gao, L., jiano, Y., sun, J., pan, Y., liu, X., jia, J., and Kong, X. (2017). A TRIM insertion in the promoter of Ms2 consumers master in the world communications8,15407.

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

The sequencing result shows that the PCR amplification product recovered in the step 3 contains a DNA fragment with a nucleotide sequence shown as SEQ ID NO. 9.

5. The vector backbone was recovered by double-digesting The vector pUbi-pAHC25 (described in He, X., qu, B., li, W., zhao, X., teng, W., ma, W., ren, Y., li, B., li, Z., and Tong, Y. (2015), the Nitrate-index NAC transformation vector NAC2-5A Controls Nitrate Response and Increases turbine yield 169, 1991-2005.) with restriction endonucleases HindIII and HpaI.

6. Preparing a reaction system. The reaction system was 10. Mu.l, and included the vector backbone recovered in step 5 (about 30 ng), the stem-loop sequence (about 50 ng), the Pro sequence (about 50 ng), 5. Mu.l of Ligase-free cloning Mix and H ₂ O。

7. And (3) treating the reaction system prepared in the step (6) at 50 ℃ for 40min to obtain the Pro-stem-loop carrier.

3. Obtaining of recombinant Agrobacterium

And introducing the vector Pro-stem-loop into the agrobacterium tumefaciens EHA105 by adopting a heat shock transformation method to obtain the recombinant agrobacterium tumefaciens named EHA105/Pro-stem-loop.

4. Obtaining transgenic wheat

Adopting agrobacterium-mediated genetic transformation method, transforming EHA105/Pro-stem-loop into wheat Fielder, and then selfing for 2 generations to obtain 4 transgenic wheat T ₂ Family, named Pro: : romo1-RNAi # 2-Pro: : romo1-RNAi #5.

5. Identification of transgenic wheat

The wheat to be detected is wheat Fielder, pro: : romo1-RNAi #2, pro: : romo1-RNAi #3, pro: : romo1-RNAi #4 or Pro: : romo1-RNAi #5.

1. About 0.5g of wheat leaves to be tested are taken and smashed into powder in liquid nitrogen, and then 400 mul of EB2 extraction buffer solution (component in QuickStix kit (Envirologix, cat. AS013 LS)) is added and mixed evenly to obtain a sample solution.

2. The test strips (components of the QuickStix kit (EnviroLogix, cat No. AS013 LS)) were inserted at their ends into the sample solution, observed, and judged AS follows: if two strips are displayed, inserting the bar gene of the wheat to be detected; if a band is displayed, the wheat to be tested has no bar gene insertion.

The identification result is shown in FIG. 2 (WT is wheat Fielder). The results indicate that Pro: : romo1-RNAi # 2-Pro: : romo1-RNAi #5 are all bar gene insertions, i.e. are all transgenic lines.

6. Male fertility identification

The wheat to be detected is wheat Fielder, pro: : romo1-RNAi #2, pro: : romo1-RNAi #3, pro: : romo1-RNAi #4 or Pro: : romo1-RNAi #5.

1. And observing the glume state of the ear part when the wheat to be detected grows to the flowering period.

The results are shown in FIG. 3 (WT is wheat Fielder). The results indicate that Pro: : romo1-RNAi # 2-Pro: : the glume of Romo1-RNAi #5 is open, and sterility exists; chaff of wheat Fielder is closed.

2. The method comprises the steps of taking anther of wheat to be detected, coating pollen in the anther on a glass slide, dropping a proper amount of starch potassium iodide solution for dyeing, covering a cover glass, and observing under a microscope, wherein the viable pollen is black.

The staining results are shown in FIG. 4 (WT is wheat Fielder). The statistical results are shown in FIG. 5 (WT is wheat Fielder). The result shows that the pollen fertility of the wheat Fielder is normal; compared to wheat Fielder, pro: : romo1-RNAi # 2-Pro: : the pollen fertility of Romo1-RNAi #5 is reduced to below 50%.

3. And (5) counting the maturing rate when the wheat to be detected is mature.

The results are shown in FIG. 6 (WT is wheat Fielder). The results show that Pro: : romo1-RNAi # 2-Pro: : the fructification rate of Romo1-RNAi #5 is significantly reduced.

The results show that silencing of the TaRomo1 gene can cause wheat male half-sterility, namely that the TaRomo1-1AL protein, the TaRomo1-1BL protein and the TaRomo1-1DL protein can regulate and control the male fertility of wheat. The TaRomo1 gene is a wheat male sterile gene with potential utilization value.

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

<110> institute of crop science of Chinese academy of agricultural sciences

Application of <120> TaRomo1 protein in regulation and control of male fertility of wheat

<160> 9

<170> PatentIn version 3.5

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gacgagcgac gcgtccgttt gctgtccgcc gcgacccaaa tccggcacat gtttgcgctc 1080

gaaatggatc ggcccggaca caaaacggac cagataggtt caggccgtcg cgcgctgggc 1140

gtgggatttg 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

cgcccacgca acgcccgccc cgcgctcctc agctcccgcg cccgccgcgc acgcacgcca 2220

acgggatacg actggttcca gcgcctggcg cggtcacacc tcgcgcgtcc gtctaaccaa 2280

cacacacaca catgaccccg ccgcgcaccc gccgcgcccg acacgcccgg cgcaatcgcg 2340

gtggcttatg cccaacactc acccccctta gccacgaatt acagcaggtg agttcatcat 2400

cgtcgatgtc gccatggccg tcgcatcgca ccgctgcggc ctccgccatg ccgtcgacgt 2460

cgttgtagcc gccgccgtcc tgacgtcgct gccacacctg 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. A taromo1 protein which is a 1) or a 2) or a 3) or a 4) as follows:

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

   a2 In SEQ ID NO:2 and/or SEQ ID NO:4, the N end or/and the C end of the protein shown in the figure is connected with a label to obtain fusion protein;

   a3 A protein which is derived from wheat and is related to the male fertility of the wheat and is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in a 1) or a 2);

   a4 And SEQ ID NO:2 and/or SEQ ID NO:4 has 80 percent or more than 80 percent of homology, is derived from wheat and is related to the male fertility of the wheat.
2. 2. A nucleic acid molecule encoding a TaRomo1 protein of claim 1.
3. 3. The nucleic acid molecule of claim 2, wherein: the nucleic acid molecule is a DNA molecule shown as b 1) or b 2) or b 3) or b 4) as follows:

   b1 Code region is SEQ ID NO: 1. SEQ ID NO:3 or SEQ ID NO:5, a DNA molecule shown in the figure;

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

   b3 A DNA molecule having 75% or more homology with the nucleotide sequence defined in b 1) or b 2), derived from wheat and encoding the TaRomo1 protein of claim 1;

   b4 A DNA molecule hybridizing under stringent conditions with the nucleotide sequence defined in b 1) or b 2) and encoding a TaRomo1 protein according to claim 1.
4. 4. An expression cassette, recombinant vector or recombinant microorganism comprising the nucleic acid molecule of claim 2 or 3.
5. 5. Use of a TaRomo1 protein according to claim 1, or a nucleic acid molecule according to claim 2 or 3, or an expression cassette, a recombinant vector or a recombinant microorganism comprising a nucleic acid molecule according to claim 2 or 3, as D1) or D2):

   d1 Regulating male fertility of wheat;

   d2 Male sterile or male partially sterile transgenic wheat.
6. 6. The use of claim 5, wherein: the wheat is wheat Fielder.
7. 7. A method for producing transgenic wheat, comprising the step of inhibiting the expression level and/or activity of the TaRomo1 protein of claim 1 in recipient wheat to obtain transgenic wheat; the transgenic wheat is male sterile or male partially sterile as compared to the recipient wheat.
8. 8. The method of claim 7, wherein: the inhibition of the expression level and/or activity of the TaRomo1 protein of claim 1 in recipient wheat is the introduction of a substance that inhibits the expression level and/or activity of the TaRomo1 protein of claim 1 into recipient wheat.
9. 9. A wheat breeding method comprises the following steps: reducing the content and/or activity of a Taromo1 protein of claim 1 in wheat, whereby the wheat is male sterile or male partially sterile.
10. 10. The method of any of claims 7-9, wherein: the wheat is wheat Fielder.

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