An application of regulating male fertility in plants and a method for creating male-sterile plants.

Abstract

This invention discloses an application for regulating male fertility in plants and a method for creating male-sterile plants. The application involves using the TaALIS-7A protein (amino acid sequence as shown in SEQ ID NO:1) or its encoding gene (nucleotide sequence as shown in SEQ ID NO:2) to regulate male fertility in plants. The method includes modifying the plant endogenous gene encoding the TaALIS-7A protein to reduce the biological activity of the protein, thereby creating male-sterile plants. This invention clarifies for the first time the key function of the TaALIS-7A gene in regulating male fertility in wheat, providing a novel gene target. By inhibiting the function of this gene and its homologs through gene editing technology, male-sterile plants with significantly reduced seed setting rate can be created efficiently and stably, laying the foundation for crop hybridization breeding and the utilization of heterosis.

Description

Technical Field

[0001] This application belongs to the field of agricultural biotechnology, specifically to plant molecular genetics and genetic engineering technology, and particularly to the application of a protein TaALIS-7A and its encoding gene in regulating male fertility in plants, and a method for creating male-sterile plants using this gene. Background Technology

[0002] The sexual reproduction process in plants, particularly the normal development of anthers and pollen, is fundamental to crop fruit setting and yield. This process is precisely regulated by a series of genes, and abnormalities in any stage can lead to male sterility, thus severely impacting agricultural production. In major food crops such as wheat, using male-sterile lines for hybridization is a core technological pathway to achieve heterosis and increase yield. Therefore, identifying and demonstrating the key genes controlling male fertility and elucidating their mechanisms of action is of significant theoretical and practical value for the efficient creation of genetically stable male-sterile lines.

[0003] In higher plants, anther development involves complex cell differentiation and metabolic processes. Among these, the synthesis, transport, and distribution of lipid molecules are crucial for pollen wall formation, the stability of the intracellular membrane system, and the storage of energy substances. Studies have shown that mature pollen grains are rich in various lipids, which are key components of structures such as the pollen exwall, organelle membranes, and oil reservoirs. Phospholipids, as the basic framework of biological membranes, are vitally important for maintaining organelle function, regulating vesicle transport, and cell secretion through their dynamic distribution and transport within the membrane bilayer.

[0004] P4-ATPase, also known as phospholipid flipper enzyme, is a key transport protein responsible for flipping phospholipid molecules from one side of a biological membrane to the other. Its proper function typically depends on forming heterodimeric complexes with the CDC50 protein family. In plants, the CDC50 protein is often referred to as ALIS (ALA-interacting subunit), an essential helper subunit of P4-ATPase, playing a crucial role in regulating the correct folding, subcellular localization, and transport activity of P4-ATPase. The vesicle transport system participates in the transport of nutrients and pollen wall precursors from anther tapetum cells to developing microspores, ensuring normal pollen development.

[0005] However, although current technologies have recognized the widespread existence of the ALIS protein family and its interaction with P4-ATPase, systematic research and clear evidence regarding the specific functions and mechanisms of action of this family members in important gramineous crops such as wheat, particularly in anther development and male fertility regulation, remain lacking. Therefore, it is urgent to identify and analyze the key ALIS proteins and their encoding genes in wheat that directly participate in the regulation of anther and pollen development. This will not only help deepen our understanding of the molecular mechanisms of plant reproductive development but also provide new, precise, and effective molecular targets for the genetic engineering creation of male-sterile lines in crops. Summary of the Invention

[0006] The purpose of this application is to provide a new protein and its encoding gene for the application of regulating male fertility in plants, and a method for creating male-sterile plants based on this gene, in order to solve the problem of the lack of new gene targets for regulating male fertility in wheat in the prior art.

[0007] To achieve the above objectives, this application provides the following technical solution:

[0008] In a first aspect, this application provides the application of a protein or its encoding gene in regulating male fertility in plants, wherein the protein is a TaALIS-7A protein, the amino acid sequence of which is shown in SEQ ID NO: 1; or, the encoding gene is a TaALIS-7A gene, the nucleotide sequence of which is shown in SEQ ID NO: 2.

[0009] In a preferred embodiment, the regulation of plant male fertility specifically involves inducing the plant to produce a male sterile phenotype by inhibiting or knocking out the expression or function of the endogenous TaALIS-7A gene.

[0010] In a further embodiment, the inhibition or knockout of the expression or function of the TaALIS-7A gene is achieved through gene editing or gene silencing techniques. Specifically, the gene editing techniques may include site-specific modification of the TaALIS-7A gene using clusters of regularly spaced short palindromic repeats and their associated protein 9 (CRISPR / Cas9) system, transcription activator-like effector nuclease (TALEN) technology, or zinc finger nuclease (ZFN) technology.

[0011] In a more specific embodiment, the application further includes using a CRISPR / Cas9 system to simultaneously edit the plant’s endogenous TaALIS-7A gene, as well as its homologous genes TaALIS-7B and TaALIS-7D, such editing results in a reduction in the plant’s fruit set rate to less than 25% relative to the unedited wild-type plant, thereby obtaining a stable male-sterile phenotype.

[0012] In a preferred embodiment, the plant is wheat.

[0013] Secondly, this application provides a method for creating male-sterile plants, the method comprising the steps of: reducing the biological activity of TaALIS-7A protein in target plant cells, wherein the amino acid sequence of TaALIS-7A protein is shown in SEQ ID NO: 1.

[0014] In a preferred embodiment, the step of reducing the biological activity of the TaALIS-7A protein specifically involves modifying the plant endogenous TaALIS-7A gene encoding the protein, the nucleotide sequence of which is shown in SEQ ID NO:2.

[0015] In a further embodiment, the step of modifying the TaALIS-7A gene specifically involves using gene editing technology to edit the TaALIS-7A gene and its homologous genes (including the TaALIS-7B and TaALIS-7D genes). The editing introduces insertions, deletions, or base substitutions into the coding or regulatory regions of the gene, thereby causing loss of function or significant downregulation of the expression level of the encoded protein.

[0016] In a more specific embodiment, the male-sterile plant created by the method of this application exhibits at least one of the following phenotypes: shorter spike length compared to wild-type plants; presence of infertile florets; shriveled or smaller grains; and a significantly reduced seed setting rate. Specifically, under self-pollination conditions, the seed setting rate of the male-sterile plant is less than 30% of that of the wild-type plant.

[0017] In a preferred embodiment, the target plant is wheat.

[0018] Compared with the prior art, the beneficial effects of this application are as follows:

[0019] 1. The function of a new gene was clarified: For the first time, the protein TaALIS-7A and its encoding gene TaALIS-7A in wheat were identified and confirmed to play a key role in regulating anther and pollen development and maintaining male fertility, which deepens the understanding of the molecular mechanism of plant reproductive development.

[0020] 2. Provides new molecular targets: It provides a novel, precise and effective gene target (TaALIS-7A and its homologous genes) for creating wheat male sterile lines using modern genetic engineering technology. Compared with traditional mutagenesis or distant hybridization breeding methods, this method is more targeted, more efficient and has a shorter cycle.

[0021] 3. Promoting the utilization of heterosis: The applications and methods provided in this application can efficiently and stably create male-sterile lines of wheat, laying the foundation for wheat hybrid seed production and large-scale utilization of heterosis, and having important practical application value for ensuring food security and increasing crop yield.

[0022] 4. Significant and controllable sterility effect: Experimental data show that different degrees of fertility reduction can be achieved by editing TaALIS-7A and its homologous genes in different combinations. For example, when three homologous genes are edited simultaneously, the seed setting rate of the plants can be reduced to about 20%, exhibiting a significant and stable male sterility phenotype, which meets the needs of breeding production. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a schematic diagram of the structure of the CRISPR / Cas9 gene editing expression vector provided in the embodiments of this application.

[0025] Figure 2 This is a flowchart of a method for creating male-sterile plants provided in an embodiment of this application.

[0026] Figure 3 This is an image showing the agarose gel electrophoresis results of the PCR amplification products of the TaALIS-7A gene coding region provided in the embodiments of this application.

[0027] Figure 4 This is a graph showing the PCR detection results of positive screening of transgenic wheat plants provided in the embodiments of this application.

[0028] Figure 5 This is a diagram showing the results of PCR detection of TaALIS-7A, TaALIS-7B, and TaALIS-7D gene editing types provided in the embodiments of this application.

[0029] Figure 6 This is a Sanger sequencing comparison diagram of the gene editing status of TaALIS-7A, TaALIS-7B, and TaALIS-7D in the T0 generation transgenic wheat plants provided in the embodiments of this application.

[0030] Figure 7 This is a sequencing peak diagram of the TaALIS-7A gene editing status in the T0 generation edited plants provided in the embodiments of this application.

[0031] Figure 8 This is a sequencing peak diagram showing the TaALIS-7B gene editing status in the T0 generation edited plants provided in this application embodiment.

[0032] Figure 9 This is a sequencing peak diagram of the TaALIS-7D gene editing status in the T0 generation edited plants provided in the embodiments of this application.

[0033] Figure 10 This is a phenotypic comparison diagram of gene-edited plants and wild-type control plants provided in the embodiments of this application, where A is the phenotype of mature plants, B is the morphology of ears, C is the morphology of grains, and D is the statistics of seed setting rate. Detailed Implementation

[0034] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0035] Example 1

[0036] This embodiment details the application of the protein TaALIS-7A and its encoding gene TaALIS-7A provided in this application in regulating male fertility in plants. The effectiveness of this application was verified through a series of molecular biology and genetic transformation experiments, the specific process of which is as follows.

[0037] 1. Cloning and identification of the core functional gene TaALIS-7A

[0038] The core of this application lies in the discovery and verification of the key role of the protein TaALIS-7A (its amino acid sequence is shown in SEQ ID NO: 1) and its encoding gene TaALIS-7A (its nucleotide sequence is shown in SEQ ID NO: 2) in regulating male fertility in wheat.

[0039] To obtain the complete coding sequence of this gene, anthers of the wheat photoperiod-temperature-sensitive male sterile line BS366 in the tetrad stage were used as experimental material. Total RNA was extracted from the anther tissue using the conventional Trizol method, and then the extracted RNA was reverse transcribed into cDNA first strand using a reverse transcription kit, which served as a template for subsequent PCR amplification.

[0040] Based on the known sequence information of the TaALIS-7A gene coding region, a pair of specific primers, namely primer 1, was designed to amplify its complete protein coding sequence (CDS). The sequence of primer 1 is as follows:

[0041] Upstream primer: 5'-atgatgatggacgccgtc-3'

[0042] Downstream primer: 5'-ttagcgacctgcggggtg-3'

[0043] Using cDNA obtained through reverse transcription as a template, PCR amplification was performed using primer 1 described above. The PCR products were then detected by 1% agarose gel electrophoresis. (Refer to...) Figure 3 The electrophoresis results, with reference to the molecular weight marker, showed a clear, single band of approximately 1062 bp, which is consistent with the expected theoretical size of the TaALIS-7A gene coding region.

[0044] The target band was gel-recovered, and the recovered DNA fragment was ligated into the pBM16A cloning vector. The ligation product was transformed into *E. coli* DH5α competent cells and cultured on a selection medium containing ampicillin to screen for positive clones. Single colonies were selected, recombinant plasmids were extracted, and nucleotide sequences were determined using universal primers (e.g., M13F / R) on the vector. Sequencing results, after comparative analysis, confirmed that the cloned fragment was the complete coding sequence of the target gene TaALIS-7A, whose nucleotide sequence is shown in SEQ ID NO: 2, and the amino acid sequence of the protein TaALIS-7A encoded by this sequence is shown in SEQ ID NO: 1.

[0045] 2. Gene function verification: Creating male-sterile wheat through gene editing.

[0046] To verify the function of the TaALIS-7A gene in regulating male fertility, this embodiment uses gene editing technology to knock it out in common wheat and observes phenotypic changes.

[0047] Reference Figure 1 The schematic diagram shows that the T-DNA region of the CRISPR / Cas9 gene editing expression vector constructed in this embodiment contains a Cas9 expression cassette for cleaving target DNA and a gRNA expression cassette for guiding site-specific cleavage. Cas9 expression is driven by the maize ubiquitin (Ubi) promoter.

[0048] Specifically, this embodiment utilizes the CRISPR / Cas9 gene editing system to perform targeted editing of the endogenous TaALIS-7A gene and its homologous genes (TaALIS-7A, TaALIS-7B, and TaALIS-7D) in the wheat variety Fielder. The CRISPR / Cas9 system works by using sgRNA (single guide RNA) to guide the Cas9 endonuclease to a specific target DNA sequence in the genome via base pairing. The Cas9 protein then cleaves the target DNA, causing a double-strand break. The cell's own non-homologous end joining (NHEJ) repair mechanism, when repairing the break, easily introduces random base insertions or deletions, leading to frameshift mutations or loss of gene function, thus achieving gene knockout.

[0049] 2.1. Construction and genetic transformation of gene editing vectors

[0050] First, a conserved common sequence that could serve as an editing target was identified in the first exons of the three homologous genes TaALIS-7A, TaALIS-7B, and TaALIS-7D. The protospacer adjacent motif (PAM) recognition sequence of this target was 5'-CCN-3'. Specific guided RNAs (gRNAs) were designed targeting this sequence. The gene encoding the Streptococcus pyogenes Cas9 (SpCas9) protein was cloned into the plant expression vector pWMB110, which utilizes the maize ubiquitin (Ubi) promoter to drive efficient Cas9 expression, resulting in the intermediate plasmid pWMB110-SpCas9. Subsequently, the designed gRNA expression cassette was also cloned into this plasmid, ultimately obtaining a CRISPR / Cas9 gene editing expression vector capable of simultaneously targeting three homologous genes.

[0051] The constructed expression vector was transformed into Agrobacterium C58C1 strain using the triparental hybridization method (or electroporation method), and positive Agrobacterium clones containing the vector were screened for expansion culture for later use.

[0052] Gene editing vectors were introduced into wheat using Agrobacterium-mediated genetic transformation. Healthy Fielder wheat grains were selected and surface-sterilized. The specific sterilization conditions were: treatment with 75% alcohol for 1 minute, followed by treatment with 15% sodium hypochlorite solution for 10 minutes, and finally rinsing with sterile water 5-6 times. Under sterile conditions, wheat embryos were harvested under a microscope, and embryos with a diameter of 2-3 mm were selected as transformation recipients. These embryos were co-cultured with Agrobacterium-mediated transformation, allowing the Agrobacterium's T-DNA region (carrying Cas9 and gRNA expression cassettes) to transfer and integrate into the genome of the wheat embryo cells.

[0053] After a series of tissue culture steps, including co-culture, selection culture, callus induction, and differentiation and regeneration, T0 generation transgenic plants were finally obtained. In this experiment, a total of 113 immature embryos were transformed, and 59 independent T0 generation transgenic plants were successfully obtained.

[0054] 2.2. Molecular identification of transgenic plants

[0055] Genomic DNA was extracted from leaves of T0 generation transgenic plants, and transgenic positive events were screened by PCR. (Refer to...) Figure 4 PCR testing showed that 35 of the obtained plants were positive transgenic plants.

[0056] To identify the editing status of target genes in these positive plants, specific primers were designed to amplify target region fragments of the TaALIS-7A, TaALIS-7B, and TaALIS-7D genes, respectively. (Refer to...) Figure 5 Electrophoretic analysis of the amplification products allowed for a preliminary determination of the gene editing type based on the migration of the bands. The results showed that both homozygous edited (only the band representing editing) and heterozygous edited (both wild-type and edited bands were present) individuals existed in the edited plants.

[0057] To further pinpoint the edit type, the PCR products were subjected to Sanger sequencing. (Refer to...) Figure 6 Sequencing and alignment results clearly showed that different types of mutations occurred at the target sites of the TaALIS-7A, TaALIS-7B, and TaALIS-7D genes in different edited plants, mainly including single or multiple nucleotide insertions and deletions. For example, in the TaALIS-abd-22 plant, TaALIS-7A had a 1-base insertion (+1 bp), TaALIS-7B had a 4-base deletion (-4 bp), and TaALIS-7D also had a 1-base insertion (+1 bp).

[0058] Figures 7 to 9Sequencing peak diagrams of the TaALIS-7A, TaALIS-7B, and TaALIS-7D gene editing sites in selected edited plants are shown. Comparison with wild-type sequences visually confirms the occurrence of gene editing events and specific mutation types. Specifically, the TaALIS-7A gene mainly involves a 1 bp insertion upstream of the target site at the 5'-CCN-3' PAM recognition sequence, along with a 3 bp deletion mutation; the TaALIS-7B gene mainly involves 1 bp and 4 bp insertions, as well as a 1 bp deletion; the TaALIS-7D gene primarily involves a 1 bp insertion (see [link to original text]). Figure 7-9 These insertion or deletion mutations cause frameshifts in subsequent coding sequences, resulting in premature stop codons or nonfunctional truncated proteins, thereby leading to the loss of gene function.

[0059] 2.3. Phenotypic Identification of Edited Plants

[0060] Gene-edited plants that have been confirmed by molecular identification were planted under the same conditions as wild-type control Fielder plants, and phenotypic observations were conducted.

[0061] Reference Figure 10 A. During the mature stage, it can be observed that the plant height of gene-edited plants (such as TaALIS-7a-39, TaALIS-ad-21 and TaALIS-7abd-22) is generally lower than that of wild-type Fielder plants.

[0062] Reference Figure 10 B. Observation of the ear morphology revealed that the ears of wheat in the edited plants were significantly shorter, and many small, unfertilized florets appeared on the ears, which is a typical characteristic of male sterility.

[0063] Reference Figure 10 C. Comparing the harvested seeds, the wild-type Fielder seeds were plump and uniform in size, while the seeds of the edited plants were noticeably shriveled and smaller, with some florets failing to produce any seeds at all.

[0064] To quantify the degree of fertility reduction, the seed setting rate per spike was statistically analyzed for plants of different editing types. The seed setting rate was calculated as (number of grains per spike / total number of florets per spike) × 100%. (Refer to...) Figure 10 According to statistics from D, the fruit set rate of wild-type Fielder is close to 100%. In comparison:

[0065] - Plants with a single gene edited (such as TaALIS-7a-39, where only TaALIS-7A was edited) have a reduced seed set rate of about 40%.

[0066] - Plants in which two homologous genes were edited simultaneously (such as TaALIS-ad-21, TaALIS-7A and TaALIS-7D being edited) had a further decrease in seed setting rate to about 30%.

[0067] - Plants (TaALIS-abd-22) in which three homologous genes (TaALIS-7A, TaALIS-7B and TaALIS-7D) were simultaneously edited had a significantly reduced seed set rate of about 20%.

[0068] The phenotypic observations and statistical results above strongly demonstrate that inhibiting or knocking out the function of the TaALIS-7A gene and its homologs can lead to abnormal wheat pollen development, thereby inducing a male sterility phenotype. The more homologs edited, the higher the degree of sterility. This fully illustrates the crucial application value of the TaALIS-7A gene and its encoded protein in regulating male fertility in wheat.

[0069] It should be noted that the applications described in this application are not limited to those implemented using CRISPR / Cas9 technology. Those skilled in the art will understand that other gene editing technologies, such as TALEN or ZFN, or gene silencing technologies, such as RNA interference (RNAi), as long as they can inhibit or knock out the expression or function of the endogenous TaALIS-7A gene in plants, are also within the scope of protection of this application. Furthermore, this application is not limited to wheat; it can also be extended to other monocotyledonous or dicotyledonous plants such as rice, maize, Arabidopsis thaliana, and tomato to regulate their male fertility.

[0070] It should be noted that the applications described in this application are not limited to those implemented using CRISPR / Cas9 technology. Those skilled in the art will understand that, based on the TaALIS-7A gene function disclosed in this application, other technologies capable of gene knockout or expression inhibition, such as TALEN, ZFN, or RNA interference (RNAi) technology, can theoretically also be used to inhibit this gene function to achieve male infertility, and these alternatives also fall within the scope of protection of this application. However, the specific embodiments and experimental data of this application are all obtained based on the CRISPR / Cas9 system.

[0071] Example 2

[0072] This embodiment provides a method for creating male-sterile plants, based on the function of the TaALIS-7A gene disclosed in Example 1. (Refer to...) Figure 2 The flowchart shown illustrates the method, which specifically includes the following steps:

[0073] Step S201: Reduce the biological activity of TaALIS-7A protein in target plant cells.

[0074] This step is the core of this method. TaALIS-7A protein (SEQ ID NO: 1) is a key factor in maintaining normal male fertility in plants. By reducing its effective concentration in cells or causing it to lose its function, male sterility can be induced.

[0075] Step S202: Modify the plant endogenous TaALIS-7A gene encoding the TaALIS-7A protein.

[0076] This is the preferred approach to achieve step S201. Stable, heritable functional repression can be achieved by directly modifying the gene encoding this protein (SEQ ID NO: 2) at the genome level.

[0077] Step S203: Use gene editing technology to perform site-specific modification of the target gene.

[0078] Gene editing technology is a precise and efficient means to achieve step S202. In this embodiment, the CRISPR / Cas9 system is preferably used. The specific operation is as described in Example 1, including designing gRNA targeting specific sites (such as exons) of the TaALIS-7A gene, constructing a vector containing Cas9 and a gRNA expression cassette, and introducing it into the target plant through genetic transformation.

[0079] Step S204: Co-edit the TaALIS-7A gene and its homologs.

[0080] For allopolyploid crops like wheat, their genomes often contain functionally redundant homologous genes. To obtain a thorough and stable male sterility phenotype, this method preferably involves simultaneous editing of the TaALIS-7A gene and its homologs (TaALIS-7B and TaALIS-7D). By introducing insertions, deletions, or base substitutions into the coding regions or key regulatory regions of these genes, the encoded proteins collectively lose their function or their expression levels are significantly downregulated.

[0081] Step S205: Screen and obtain plants with male sterility phenotype.

[0082] After the above gene modification and plant regeneration processes, the obtained transgenic plants need to be screened. The screening criteria are based on male sterility-related phenotypic characteristics. According to the research in this application, male-sterile plants created using this method typically exhibit one or more of the following identifiable phenotypes:

[0083] - Changes in spike morphology: The spike length is significantly shorter compared to the wild type.

[0084] - Abnormal floret development: A large number of empty, shriveled, and non-fertile florets appear.

[0085] - Decreased grain quality: Even if there are a few firm grains, they appear shriveled and smaller.

[0086] - Significantly reduced seed setting rate: Under self-pollination conditions, the seed setting rate per spike is significantly lower than that of wild-type plants. For example, in wheat, the seed setting rate per spike can be less than 30% of that of wild-type plants, or even lower, such as below 25%.

[0087] By following the above methods and steps, genetically stable male-sterile plant materials, especially male-sterile wheat, can be created efficiently, providing a foundation for subsequent hybridization breeding work.

[0088] Those skilled in the art should understand that the above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

[0089] sequence list

[0090] <110> applicant

[0091] <120> An application of regulating male fertility in plants and a method for creating male-sterile plants.

[0092] <160> 2

[0093] <170> PatentIn version 3.5

[0094] <210> 1

[0095] <211> 353

[0096] <212> PRT

[0097] <213> Wheat (Triticum aestivum)

[0098] <400> 1

[0099] MMMDAVAAGT SNGGSGADGD AARRNNTRMP KYSKFTQQEL PACKPILTPK WVVSVFFLVG

[0100] VVFVPVGVVS LLAAQDVVEI IDRYDHACVP PNMTDNKLAY IQNETIPKDC TRTLTVTKEM

[0101] KQPIYVYYQL DNFYQNHRRY VKSRNDAQLR DYKKSNTTTS CDPERFTADG KPIVPCGLIA

[0102] WSLFNDTYSF TRGKDNLTVD KKDISWKSDR EHKFANNVYP SNFQNGALIG GKKLNSSIPL

[0103] SEQEDLIVWM RTAALPTFRK LYGRIYVDLK ANDTIIVRLS NNYNTYSFGG KKKLVLSTAT

[0104] WLGGKNDFLG FAYLIVGGLC IFLAFAFTLL YLIKPRKLGD HNYLSWNRHP AGR

[0105] <210> 2

[0106] <211> 1062

[0107] [[ID=...]]<212> DNA

[0108] <213> Wheat (Triticum aestivum)

[0109] <400> 2

[0110] atgatgatgg acgccgtcgc ggccggcacg agcaacggcg gatccggggc cgatggggac

[0111] gccgccaggc ggaacaacac caggatgccc aagtattcca agttcacgca gcaagagctg

[0112] ccggcctgca agccgatcct tactccaaaa tgggttgtct cggtgttttt ccttgtcggc

[0113] gtcgtctttg tcccagttgg tgtcgtttcg ctactagccg cacaagatgt tgttgagatc

[0114] attgatcggt atgatcatgc atgtgtccca cctaacatga ctgataacaa gcttgcgtac

[0115] atccagaatg agactatacc caaagactgc acaaggactc tcacggttac aaaggagatg

[0116] aaacagccaa tttatgtgta ctaccagctt gataacttct atcagaatca taggaggtat

[0117] gtgaagagcc gaaatgatgc acagctaaga gattacaaga agtcaaatac gacaacctca

[0118] tgtgaccctg agaggttcac ggctgatgga aaaccaattg ttccttgtgg tctgattgct

[0119] tggagtttgt ttaatgacac atatagcttc actcgtggta aagacaactt gacagtagac

[0120] aagaaggaca tctcctggaa aagtgatagg gagcacaaat ttgccaacaa tgtctaccca

[0121] agcaacttcc agaatggtgc gctcataggt ggaaagaagc ttaactcaag tatcccgctg

[0122] agcgaacagg aggatcttat tgtttggatg cggactgcag cgcttcctac attcagaaag

[0123] ttatatggga ggatatacgt tgatctcaag gcgaatgata ccataatagt gaggctgagt

[0124] aacaactaca atacatatag cttcggtggc aagaagaagt tggtcctttc cactgcaacc

[0125] tggctgggag gaaagaatga ttttcttgga ttgcgtacc ttatagttgg tggactctgt

[0126] atttcttgg catttgcatt caccttgcta tacttgataa agccaaggaa actgggagat

[0127] cacaactacc tgtcctggaa caggcacccc gcaggtcgct aa

Claims

1. The application of a protein or its encoding gene in regulating male fertility in plants, characterized in that, The protein is TaALIS-7A protein, and the amino acid sequence of the TaALIS-7A protein is shown in SEQ ID NO: 1; or, the encoding gene is the TaALIS-7A gene, and the nucleotide sequence of the TaALIS-7A gene is shown in SEQ ID NO:

2.

2. The application according to claim 1, characterized in that, The regulation of plant male fertility specifically involves inducing the plant to produce a male sterile phenotype by inhibiting or knocking out the expression or function of the endogenous TaALIS-7A gene.

3. The application according to claim 2, characterized in that, The inhibition or knockout of the expression or function of the TaALIS-7A gene is achieved through gene editing technology or gene silencing technology; the gene editing technology includes site-specific modification of the TaALIS-7A gene using clustered regularly spaced short palindromic repeat sequences and their associated protein 9 (CRISPR / Cas9) system, transcription activator-like effector nuclease (TALEN) technology or zinc finger nuclease (ZFN) technology.

4. The application according to claim 3, characterized in that, The application further includes using the CRISPR / Cas9 system to simultaneously edit the plant's endogenous TaALIS-7A gene, as well as the TaALIS-7B and TaALIS-7D genes, which are homologous to the TaALIS-7A gene, such that the editing results in a reduction of the plant's fruit set rate to less than 25% relative to the unedited wild-type plant, thereby obtaining the male-sterile phenotype.

5. The application according to claim 1, characterized in that, The plant in question is wheat.

6. A method for creating male-sterile plants, characterized in that, The method includes the step of reducing the biological activity of TaALIS-7A protein in target plant cells, wherein the amino acid sequence of TaALIS-7A protein is shown in SEQ ID NO:

1.

7. The method according to claim 6, characterized in that, The step of reducing the biological activity of the TaALIS-7A protein specifically involves modifying the plant endogenous TaALIS-7A gene encoding the TaALIS-7A protein, the nucleotide sequence of which is shown in SEQ ID NO:

2.

8. The method according to claim 7, characterized in that, The step of modifying the TaALIS-7A gene specifically involves using gene editing technology to edit the TaALIS-7A gene and its homologous genes, including the TaALIS-7B and TaALIS-7D genes. The editing introduces insertions, deletions, or base substitutions into the coding or regulatory regions of the gene, thereby causing the encoded protein to lose its function or its expression level to be significantly downregulated.

9. The method according to claim 8, characterized in that, The male-sterile plant created by the method exhibits at least one of the following phenotypes: shorter spike length compared to the wild type; presence of sterile florets; shriveled or smaller grains; and a significantly reduced seed setting rate, specifically, the seed setting rate of the male-sterile plant under self-pollination conditions is less than 30% of the seed setting rate of the wild type plant per spike.

10. The method according to claim 6, characterized in that, The target plant is wheat.

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