Application of TaRomo1 protein in regulating male fertility in wheat

Abstract

This invention discloses the application of TaRomo1 protein in regulating male fertility in wheat. 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 male semi-sterility in wheat, specifically manifested as opening of the glume in the ear, reduced pollen fertility, and decreased seed setting rate. Therefore, TaRomo1 protein can regulate male fertility in wheat. This invention has significant application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of TaRomo1 protein in regulating male fertility in wheat. Background Technology

[0002] Hybrids are characterized by high yield and superior agronomic traits, and are widely used in crop production. Wheat is one of the world's most important food crops, and increasing wheat yield is of great significance to ensuring national food security. Wheat is a strictly hermaphroditic, self-pollinating crop, and the production of hybrids depends entirely on the use of wheat male-sterile lines. Compared with rice, the production of usable wheat male-sterile lines and male-sterile gene resources is extremely scarce, resulting in a much lower level of hybrid production compared to rice. Therefore, cloning new male-sterile genes and enriching existing male-sterile gene resources is an important approach to creating wheat male-sterile lines and has significant practical production implications.

[0003] Three male sterility genes for wheat have been cloned using map-based cloning: Ms1, Ms2, and Ms5. Ms1 and Ms5 both encode glycosylphosphatidylinositol-anchored lipid transfer proteins. Specific expression of these genes ensures the integrity of the pollen exine, while gene deletion leads to the accumulation of long-chain fatty acids in the anthers and disruption of the pollen exine structure, resulting in male sterility. The Ms2 gene encodes an orphan protein with an unknown function; however, because Ms2 causes dominant sterility, it is difficult to use it for large-scale wheat hybrid seed production. Besides these genes, no other recessive male sterility genes with clearly defined functions have been identified, indicating a severe shortage of related sterility gene resources. Summary of the Invention

[0004] The purpose of this invention is to obtain proteins related to male sterility or partial male sterility in wheat.

[0005] This invention first protects the TaRomo1 protein derived from wheat. The TaRomo1 protein may be a1), a2), a3), or a4):

[0006] a1) The amino acid sequence is that of the protein shown in SEQ ID NO: 2 and / or SEQ ID NO: 4;

[0007] a2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO: 2 and / or SEQ ID NO: 4;

[0008] a3) Proteins derived from wheat and associated with wheat male fertility obtained by substituting and / or deleting and / or adding one or more amino acid residues of the proteins shown in a1) or a2).

[0009] a4) A protein derived from wheat and associated with wheat male fertility that has 80% or more homology with the amino acid sequence defined by SEQ ID NO: 2 and / or SEQ ID NO: 4.

[0010] Of these, 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.

[0011] To facilitate the purification of the protein in a1), a tag as shown in Table 1 may be attached to the amino or carboxyl terminus of the protein shown in SEQ ID NO:2 and / or SEQ ID NO:4.

[0012] Table 1. Sequence of Labels

[0013] Label residues sequence Poly-Arg 5-6 (usually 5) RRRRR FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

[0014] The protein in a3) 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 no more than 10 amino acid residues.

[0015] The proteins mentioned in a3) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0016] The gene encoding the protein in a3) above can be obtained by deleting one or more 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 a missense mutation on one or more base pairs, and / or by attaching the coding sequence of the tag shown in Table 1 to its 5′ end and / or 3′ end.

[0017] The present invention also protects nucleic acid molecules encoding any of the TaRomo1 proteins described above.

[0018] The nucleic acid molecule encoding any of the TaRomo1 proteins described above may be a DNA molecule as shown in b1), b2), b3), or b4):

[0019] b1) The coding region is a DNA molecule shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5;

[0020] b2) The nucleotide sequence is the DNA molecule shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5;

[0021] b3) Having 75% or more homology with the nucleotide sequence defined by b1) or b2), and being a DNA molecule derived from wheat that encodes any of the TaRomo1 proteins described above;

[0022] b4) Hybridizes under stringent conditions to the nucleotide sequence defined by b1) or b2) and to a DNA molecule encoding any of the TaRomo1 proteins described above.

[0023] 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.

[0024] Of these, 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 nucleotides shown in SEQ ID NO:1 encode the amino acid sequence shown in SEQ ID NO:2. The nucleotides shown in SEQ ID NO:3 encode the amino acid sequence shown in SEQ ID NO:4. The nucleotides shown in SEQ ID NO:5 encode the amino acid sequence shown in SEQ ID NO:6.

[0025] Those skilled in the art can readily mutate the nucleotide sequence encoding the TaRomo1 protein of this invention using known methods, such as directed evolution and point mutation. Any artificially modified nucleotides having 75% or higher identity with the nucleotide sequence of the TaRomo1 protein isolated according to this invention, as long as they encode the TaRomo1 protein, are derived from and equivalent to the nucleotide sequence of this invention.

[0026] As used herein, the term "identity" refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences having 75% or higher, or 80% or higher, or 85% or higher, or 90% or higher, or 95% or higher identity with the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:4 of the present invention. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0027] Expression cassettes, recombinant vectors, or recombinant microorganisms containing any of the aforementioned nucleic acid molecules are also within the scope of protection of this invention.

[0028] This invention also protects the use of any of the above-described TaRomo1 proteins, or any of the above-described nucleic acid molecules, or expression cassettes, recombinant vectors, or recombinant microorganisms containing any of the above-described nucleic acid molecules in regulating male fertility in wheat.

[0029] The present invention also protects the use of any of the above-described TaRomo1 proteins, or any of the above-described nucleic acid molecules, or expression cassettes, recombinant vectors, or recombinant microorganisms containing any of the above-described nucleic acid molecules in the cultivation of transgenic wheat that is male-sterile or partially male-sterile.

[0030] The present invention also protects a method for breeding transgenic wheat, comprising the step of inhibiting the expression level and / or activity of any of the TaRomo1 proteins described above in recipient wheat to obtain transgenic wheat; wherein, compared with the recipient wheat, the transgenic wheat is male-sterile or partially male-sterile.

[0031] In the above method, the "inhibition of the expression level and / or activity of any of the TaRomo1 proteins in the recipient wheat" can be achieved by methods well known in the art, such as gene editing, RNA interference, homologous recombination, and gene knockout, to inhibit the expression level and / or activity of TaRomo1 proteins.

[0032] In the above method, "inhibiting the expression level and / or activity of any of the TaRomo1 proteins in the recipient wheat" can be achieved by introducing a substance into the recipient wheat that inhibits the expression level and / or activity of any of the TaRomo1 proteins.

[0033] In the above method, the substance that inhibits the expression level and / or activity of any of the TaRomo1 proteins mentioned above can specifically be the vector 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.

[0034] The present invention also protects a wheat breeding method, which may include the following steps: reducing the content and / or activity of any of the TaRomo1 proteins described above in wheat, thereby causing male sterility or partial male sterility in wheat.

[0035] The wheat mentioned above can be wheat Fielder.

[0036] The male sterility or partial male sterility of any of the above-mentioned wheat can specifically manifest as the opening of the glume of the ear.

[0037] The male sterility or partial male sterility of wheat described above can specifically manifest as a reduction in pollen fertility (e.g., reduced to less than 50% of normal).

[0038] The male sterility or partial male sterility of wheat described above can specifically manifest as a reduced seed setting rate.

[0039] Experiments have shown that silencing the TaRomo1 gene can lead to male semi-sterility in wheat, meaning that the TaRomo1-1AL, TaRomo1-1BL, and TaRomo1-1DL proteins can regulate male fertility in wheat. The TaRomo1 gene is a wheat male sterility gene with potential application value. The TaRomo1 protein can regulate male fertility in wheat. This invention has significant application value. Attached Figure Description

[0040] Figure 1 This is a partial structural diagram of the vector pANDA (NPT II is the Kanamycin resistance gene, HPT is the Hygromycin resistance gene, Ubqpro. is the Maize ubiquitin1 promoter + 1st intron & splitting acceptor site, attR is the LR clonase recombination cassette (Invitrogen, Cat. No. 11828-019, rfA), attR1 & attR2 are LR clonase recombination sites, CmR is the Chloramphenicol resistance gene, ccdB is the ccd B gene, Nt is the NOS terminator, bar is the Bialaphos resistance gene, and the vector backbone is pBI101).

[0041] Figure 2 The results of bar gene identification for Pro::Romo1-RNAi#2—Pro::Romo1-RNAi#5.

[0042] Figure 3 The glumes of the spike are in the state of Pro::Romo1-RNAi#2—Pro::Romo1-RNAi#5.

[0043] Figure 4 Pollen staining results for Pro::Romo1-RNAi#2—Pro::Romo1-RNAi#5.

[0044] Figure 5 The pollen viability statistics for Pro::Romo1-RNAi#2—Pro::Romo1-RNAi#5.

[0045] Figure 6 The statistical results of the fruit set rate for Pro::Romo1-RNAi#2—Pro::Romo1-RNAi#5. Detailed Implementation

[0046] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0047] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0048] Example 1: Obtaining the TaRomo1 gene

[0049] The TaRomo1 gene includes the TaRomo1-1AL gene from the wheat A genome, the TaRomo1-1BL gene from the wheat B genome, and the TaRomo1-1DL gene from the wheat D genome.

[0050] The TaRomo1-1AL, TaRomo1-1BL, and TaRomo1-1DL genes were artificially synthesized.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] TaRomo1 proteins include TaRomo1-1AL, TaRomo1-1BL, and TaRomo1-1DL proteins.

[0055] Example 2: Application of TaRomo1 protein in regulating male fertility in wheat

[0056] I. Construction of the vector pANDA-antiRomo1

[0057] 1. Using the nucleotide sequence of the TaRomo1-1AL gene shown in SEQ ID NO:1 as a template, TaRomo1RNAi-F: 5'- GCAGGCTCAGGGGATATC TTCAGCAGCCCAGGGACCCT-3' (underlined sequence is identical to the pQBV3 vector, used for homologous recombination) and TaRomo1 RNAi-R: 5'- CAGGGCGATATCGATATC PCR amplification was performed using a primer pair consisting of CGAGGAGGGATAGCTGCTT-3' (the underlined sequence is the same as that in the pQBV3 vector and is used for homologous recombination), and approximately 163 bp of PCR amplification product was recovered.

[0058] The reaction program was as follows: 94℃ for 2 min; 94℃ for 30 sec, 60℃ for 30 sec, 68℃ for 1 min, 34 cycles; 68℃ for 10 min.

[0059] 2. Sequencing the PCR amplification products recovered in step 1.

[0060] Sequencing results showed that the PCR amplification product contained a DNA fragment with a nucleotide sequence as shown in SEQ ID NO:7.

[0061] 3. Homologous recombination was performed between the PCR amplification product and the vector pQBV3 (described in the following literature: Liu, J., Cheng, X., Liu, P., and Sun, J. (2017). miR156-Targeted SBP-Box Transcription Factors Interact with DWARF53 to Regulate TEOSINTE BRANCHED1 and BARREN STALK1 Expression in BreadWheat. Plant Physiology 174, 1931-1948.) to obtain the intermediate vector pQBV3-antiRomo1.

[0062] 4. Prepare the Gateway reaction system. The Gateway reaction system consists of 5 μl of intermediate vector pQBV3-antiRomo1 (approximately 80 ng), vector pANDA (approximately 80 ng), 0.5 μl of LR enzyme, and TE buffer.

[0063] The vector pANDA is described in the following literature: Chukwurah, PN, Poku, SA, 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.

[0064] A partial structural diagram of the pANDA carrier is shown below. Figure 1 .

[0065] 5. Take the Gateway reaction system prepared in step 4 and treat it at 25℃ for 1 h to obtain the support pANDA-antiRomo1.

[0066] II. Construction of the Pro-stem-loop vector

[0067] 1. Using the vector pANDA-antiRomo1 as a template, PCR amplification was performed using primer pairs consisting of SL-F: 5'-aggtagggagTTCAGCAGCCCAGGGACCCT-3' and SL-R: 5'-GAACGATCTGTTCCTAGGGTTAACTTCAGCAGCCCAGGGACCCT-3', and the PCR amplification product (i.e. stem-loop sequence) of approximately 1214 bp was recovered.

[0068] 2. Sequencing the PCR amplification products recovered in step 1.

[0069] Sequencing results showed that the PCR amplification product recovered in step 1 contained a DNA fragment with a nucleotide sequence as shown in SEQ ID NO:8.

[0070] 3. Using the vector Blunt-KX943032.1 as a template, PCR amplification was performed using primer pairs consisting of Pro-F: 5'-GAAGGAGCCACTCAGCAAGCTTCATCACCTCCTCCGTCCTCAC-3' and Pro-R: 5'-GGGCTGCTGAActccctacctccggccggccttc-3', and the PCR amplification product (i.e., the Pro sequence) of approximately 3894 bp was recovered.

[0071] The target gene (KX943032.1) was inserted into the vector pEASY-Blunt (TransGen Biotech, CB101) to obtain the recombinant plasmid; the details are described in the following literature: 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 Communications 8, 15407.

[0072] 4. Sequencing the PCR amplification products recovered in step 3.

[0073] Sequencing results showed that the PCR amplification product recovered in step 3 contained a DNA fragment with a nucleotide sequence as shown in SEQ ID NO:9.

[0074] 5. The vector pUbi-pAHC25 was double-digested with restriction endonucleases HindIII and HpaI (described in the following literature: 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.), and the vector backbone was recovered.

[0075] 6. Preparation of the reaction system. The reaction system consists of 10 μl of the carrier backbone recovered in step 5 (approximately 30 ng), stem-loop sequence (approximately 50 ng), Pro sequence (approximately 50 ng), 5 μl of Ligase-free cloning Mix, and H2O.

[0076] 7. Take the reaction system prepared in step 6 and treat it at 50℃ for 40 min to obtain the support Pro-stem-loop.

[0077] III. Obtaining Recombinant Agrobacterium

[0078] The vector Pro-stem-loop was introduced into Agrobacterium tumefaciens EHA105 using the heat shock conversion method to obtain recombinant Agrobacterium, which was named EHA105 / Pro-stem-loop.

[0079] IV. Obtaining Genetically Modified Wheat

[0080] Using Agrobacterium-mediated genetic transformation, EHA105 / Pro-stem-loop was transformed into wheat Fielder, and then self-crossed for 2 generations to obtain 4 transgenic wheat T2 families, which were named Pro::Romo1-RNAi#2—Pro::Romo1-RNAi#5 in sequence.

[0081] V. Identification of Genetically Modified Wheat

[0082] The wheat samples to be tested were Wheat Fielder, Pro::Romo1-RNAi#2, Pro::Romo1-RNAi#3, Pro::Romo1-RNAi#4 or Pro::Romo1-RNAi#5.

[0083] 1. Take about 0.5g of wheat leaves to be tested, crush them into powder in liquid nitrogen, then add 400μl of EB2 extraction buffer (a component of the QuickStix kit (EnviroLogix, catalog number AS013LS)), mix well, and obtain the sample solution.

[0084] 2. Insert the end of the test strip (a component of the QuickStix kit (EnviroLogix, catalog number AS013LS)) into the sample solution and observe the test strip. Make the following judgments: If two bands are displayed, the bar gene is inserted in the wheat to be tested; if one band is displayed, the bar gene is not inserted in the wheat to be tested.

[0085] The identification results are as follows Figure 2 (WT stands for wheat Fielder). The results showed that Pro::Romo1-RNAi#2—Pro::Romo1-RNAi#5 all had bar gene insertion, meaning they were all transgenic lines.

[0086] VI. Male fertility assessment

[0087] The wheat samples to be tested were Wheat Fielder, Pro::Romo1-RNAi#2, Pro::Romo1-RNAi#3, Pro::Romo1-RNAi#4 or Pro::Romo1-RNAi#5.

[0088] 1. Wait until the wheat to be tested grows to the flowering stage and observe the state of the glume on the ear.

[0089] See results Figure 3 (WT represents 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.

[0090] 2. Take the anthers of the wheat to be tested, spread the pollen from the anthers onto a glass slide, add an appropriate amount of starch-potassium iodide solution for staining, cover with a coverslip and observe under a microscope. Viable pollen will appear black.

[0091] Staining results are shown Figure 4 (WT stands for Wheat Fielder). Statistical results can be found... Figure 5 (WT stands for 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 below 50%.

[0092] 3. Once the wheat to be tested matures, the seed setting rate will be calculated.

[0093] See results Figure 6 (WT stands for 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 reduced.

[0094] The above results indicate that silencing the TaRomo1 gene can lead to male hyposterility in wheat, meaning that the TaRomo1-1AL, TaRomo1-1BL, and TaRomo1-1DL proteins can regulate male fertility in wheat. The TaRomo1 gene is a potentially valuable wheat male sterility gene.

[0095] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims. <110> Institute of Crop Science, Chinese Academy of Agricultural Sciences <120> Application of TaRomo1 protein in regulating male fertility in wheat <160> 9 <170> PatentIn version 3.5 <210> 1 <211> 228 <212> DNA <213> Artificial sequence <400> 1 atggcgagga gggatagctg cttggcgcgc atcggcgccg gagtcgccat cggcggcgcg 60 gtcggcggag ccgtcggtgc tgtgtatggg acttatgccg ctatcagatt gagggtccct 120 gggctgctga agatcagaca catcggacag gccactgttg gcagcgctgc ggtattcggg 180 cttttcctgg gagctgggag cttgatacac tgtgggaaaa attactag 228 <210> 2 <211> 75 <212> PRT <213> Artificial sequence <400> 2 Met Ala Arg Arg Asp Ser Cys Leu Ala Arg Ile Gly Ala Gly Val Ala 1 5 10 15 Ile Gly Gly Ala Val Gly Gly Ala Val Gly Ala Val Tyr Gly Thr Tyr 20 25 30 Ala Ala Ile Arg Leu Arg Val Pro Gly Leu Leu Lys Ile Arg His Ile 35 40 45 Gly Gln Ala Thr Val Gly Ser Ala Ala Val Phe Gly Leu Phe Leu Gly 50 55 60 Ala Gly Ser Leu Ile His Cys Gly Lys Asn Tyr 65 70 75 <210> 3 <211> 234 <212> DNA <213> Artificial sequence <400> 3 atggctaggg gcgatagctg cttggcgcgc atcggcgccg gagtcgccat cggcggcgcg 60 gtcggcggag ccgtcggtgg tgctgtgtat gggacttatg ccgctatcag attgagggtg 120 gtccctgggc tgctgaagat cagacacatc ggacaggcca ccgttggcag cgctgcggta 180 ttcgggcttt tcctgggagc tgggagcttg atacactgtg ggaaaaatta ctag 234 <210> 4 <211> 77 <212> PRT <213> Artificial sequence <400> 4 Met Ala Arg Gly Asp Ser Cys Leu Ala Arg Ile Gly Ala Gly Val Ala 1 5 10 15 Ile Gly Gly Ala Val Gly Gly Ala Val Gly Gly Ala Val Tyr Gly Thr 20 25 30 Tyr Ala Ala Ile Arg Leu Arg Val Val Pro Gly Leu Leu Lys Ile Arg 35 40 45 His Ile Gly Gln Ala Thr Val Gly Ser Ala Ala Val Phe Gly Leu Phe 50 55 60 Leu Gly Ala Gly Ser Leu Ile His Cys Gly Lys Asn Tyr 65 70 75 <210> 5 <211> 234 <212> DNA <213> Artificial sequence <400> 5 atggcgagga gagatagctg cttggcgcgc atcggcgccg gagtcgccat cggcggcgcg 60 gtcggcggag ccgtcggtgg tgctgtgtat gggacttatg ccgctatcag attgagggtg 120 gtccctgggc tgctgaagat cagacacatc ggacaagcca ccgttggcag cgctgcggta 180 ttcgggcttt tcttgggagc tgggagtttg atacactgcg ggaaaaatta ctag 234 <210> 6 <211> 77 <212> PRT <213> Artificial sequence <400> 6 Met Ala Arg Arg Asp Ser Cys Leu Ala Arg Ile Gly Ala Gly Val Ala 1 5 10 15 Ile Gly Gly Ala Val Gly Gly Ala Val Gly Gly Ala Val Tyr Gly Thr 20 25 30 Tyr Ala Ala Ile Arg Leu Arg Val Val Pro Gly Leu Leu Lys Ile Arg 35 40 45 His Ile Gly Gln Ala Thr Val Gly Ser Ala Ala Val Phe Gly Leu Phe 50 55 60 Leu Gly Ala Gly Ser Leu Ile His Cys Gly Lys Asn Tyr 65 70 75 <210> 7 <211> 127 <212> DNA <213> Artificial sequence <400> 7 ttcagcagcc cagggaccct caatctgata gcggcataag tcccatacac agcaccgacg 60 gctccgccga ccgcgccgcc gatggcgact ccggcgccga tgcgcgccaa gcagctatcc 120 ctcctcg 127 <210> 8 <211> 1214 <212> DNA <213> Artificial sequence <400> 8 aggtagggag ttcagcagcc cagggaccct caatctgata gcggcataag tcccatacac 60 agcaccgacg gctccgccga ccgcgccgcc gatggcgact ccggcgccga tgcgcgccaa 120 gcagctatcc ctcctcgatc tacccgcttc gcgtcggcat ccggtcagtg gcagtgaagg 180 gcgaacagtt cctgattaac cacaaaccgt tctactttac tggctttggt cgtcatgaag 240 atgcggactt acgtggcaaa ggattcgata acgtgctgat ggtgcacgac cacgcattaa 300 tggactggat tggggccaac tcctaccgta cctcgcatta cccttacgct gaagagatgc 360 tcgactgggc agatgaacat ggcatcgtgg tgattgatga aactgctgct gtcggcttta 420 acctctcttt aggcattggt ttcgaagcgg gcaacaagcc gaaagaactg tacagcgaag 480 aggcagtcaa cggggaaact cagcaagcgc acttacaggc gattaaagag ctgatagcgc 540 gtgacaaaaa ccacccaagc gtggtgatgt ggagtattgc caacgaaccg gatacccgtc 600 cgcaagtgca cgggaatatt tcgccactgg cggaagcaac gcgtaaactc gacccgacgc 660 gtccgatcac ctgcgtcaat gtaatgttct gcgacgctca caccgatacc atcagcgatc 720 tctttgatgt gctgtgcctg aaccgttatt acggatggta tgtccaaagc ggcgatttgg 780 aaacggcaga gaaggtactg gaaaaagaac ttctggcctg gcaggagaaa ctgcatcagc 840 cgattatcat caccgaatac ggcgtggata cgttagccgg gctgcactca atgtacaccg 900 acatgtggag tgaagagtat cagtgtgcat ggctggatat gtatcaccgc gtctttgatc 960 gcgtcagcgc cgtcgtcggt gaacaggtat ggaatttcgc cgattttgcg acctcgcaag gcatattgcg cgttggcggt aacaagaaag ggatcttcac tcgcgaggag ggatagctgc 1080 ttggcgcgca tcggcgccgg agtcgccatc ggcggcgcgg tcggcggagc cgtcggtgct 1140 gtgtatggga cttatgccgc tatcagattg agggtccctg ggctgctgaa gttaacccta ggaacagatc gttc <210> 9 <211> 3894 <212> DNA <213> Artificial sequence <400> 9 gaggagcca ctcagcaagc ttcatcacct cctccgtcct caccatctcc agctcgggga tggcgacgtc gccggccgca gagagggcca tcatctcttc gaggccctcc cattggcgct catcgtgcgt cttcatggag tcctccatga cacgttgcat gagccgggcc tcctcctccg 180 ctgtcatgcg aggaggtggt ggtggcagcg gagatggtga aggcgacggc gtgggcgtga 240 ggccgcgcac ccgcgtacgc ctgcgcacct ggggagcagc cgctgcgcgc tctcgacgtg 300 gccgtcgcgg ccccgacaag gtgccggcga agaaaaaggc gcgacgcgtg tcgtgctcgt 360 cccgaaacca cgtgtcccac aggtcggagt cgggggcgta ccgagggtcg tagtacaggt 420 cgtccggcag gaggcggcgg cggcactgga tctcctcgcg gcgcgcacgg ccgctcgtcg 480 ggaccggcgg gatcggcacc cggttggcgg agagatgcca gttattgggg aggtgcacgt 540 cgctccatgg gagcggcgtc ctcgtctccc aataacgccg gcatacatcc gcgtggatgt 600 actgctggtc gcgcttgccg ccggccgtag ggccgatggt gaatggggca ggcgtggggg 660 ctcgatgcgg tggtgatgcg ggctcctctt tcatggagcc gcggcggcgg cccgaggaca 720 agccagcctc gtggtcgcgc ttccccttgc gaccggtgtt ccagaacccc atggctgcga 780 ggcggccggc cgacgagatc gaggacgggg agagggagag ctagggtttg gggtgtgtcg 840 ggtttcgagg aggcagcacg ggctggagtg gggagtgtgg acgacgaccg gtccacggtt 900 tcccatttaa gaaggacggc gactgtttgc tggacggatg acaggtgggg ccgaccgcgc 960 gtgtgcatta atgttggctg gtgggaggta ggtggccgcc tgctacgcgg cctcgaggcg 1020 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 caacaagc tttacgcggt gcacgtacgc tgccggccgc agaacagcc 1920 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 cacacaca catgaccccg ccgcgcaccc gccgcgcccg acacgccccgg 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

1. The application of TaRomo1 protein or nucleic acid molecule encoding TaRomo1 protein or expression cassette, recombinant vector or recombinant microorganism containing TaRomo1 protein in regulating male fertility in wheat, wherein TaRomo1 protein is the protein with the amino acid sequence shown in SEQ ID NO:

2.

2. The application of TaRomo1 protein or nucleic acid molecule encoding TaRomo1 protein or expression cassette, recombinant vector or recombinant microorganism containing TaRomo1 protein in the cultivation of male sterile wheat, wherein TaRomo1 protein is the protein with the amino acid sequence shown in SEQ ID NO:

2.

3. The application as described in claim 1 or 2, characterized in that: The nucleic acid is the DNA coding region shown in SEQ ID NO:

1.

4. The application as described in claim 1 or 2, characterized in that: The wheat in question is wheat fielder.

5. A method for breeding transgenic wheat, comprising the step of inhibiting the expression level and / or activity of the TaRomo1 protein as described in claim 1 in recipient wheat to obtain transgenic wheat; wherein the transgenic wheat is male-sterile compared to the recipient wheat; The expression level and / or activity of the TaRomo1 protein of claim 1 in the inhibitory receptor wheat are achieved by site-directed gene editing, RNA interference, homologous recombination, or gene knockout.

6. The method as described in claim 5, characterized in that: The inhibition of the expression level and / or activity of the TaRomo1 protein in the recipient wheat according to claim 1 is achieved by introducing a substance into the recipient wheat that inhibits the expression level and / or activity of the TaRomo1 protein according to claim 1.

7. A wheat breeding method, comprising the following steps: reducing the content and / or activity of the TaRomo1 protein as described in claim 1 in wheat, thereby causing male sterility in wheat; The reduction of the content and / or activity of the TaRomo1 protein in wheat as described in claim 1 is achieved through site-directed gene editing, RNA interference, homologous recombination, or gene knockout.

8. The method according to any one of claims 5-7, characterized in that: The wheat in question is wheat fielder.

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