CN116529376A

CN116529376A - Fertility-related gene and application thereof in cross breeding

Info

Publication number: CN116529376A
Application number: CN202080106639.1A
Authority: CN
Inventors: 王峥; 李健; 何航; 邓兴旺
Original assignee: Beijing Zhiyu Wheat Biotechnology Co ltd; Institute Of Modern Agriculture Peking University
Current assignee: Beijing Zhiyu Wheat Biotechnology Co ltd; Institute Of Modern Agriculture Peking University
Priority date: 2020-11-24
Filing date: 2020-11-24
Publication date: 2023-08-01
Also published as: WO2022109764A1

Abstract

The invention provides a wheat fertility related gene TaMS8 and an application method thereof, belonging to the technical field of biology. The invention obtains recessive single gene control male sterile material through phenotype screening and genetic analysis of EMS mutagenesis library, then obtains wheat fertility related gene TaMS8 through a Mutmap method, regulates and controls plant fertility through regulating the expression of the gene, is used for producing and maintaining a wheat male sterile line and preparing hybrid seeds, and has important theoretical and practical significance for establishing high-efficiency wheat hybrid seed production technology, researching wheat male sterile mechanism and hybrid vigor.

Description

Fertility-related gene and application thereof in cross breeding

Technical Field

The invention belongs to the technical field of plant biology, in particular to cloning of a plant recessive nuclear sterile gene, a propagation method of a male sterile line thereof and application of the male sterile line in cross breeding, and more particularly relates to cloning of a wheat recessive nuclear male sterile gene and a promoter thereof and application of the wheat recessive nuclear male sterile gene and the promoter thereof in cross breeding.

Background

Heterosis is a natural phenomenon commonly found in the biological world. The hybrid vigor is fully utilized to greatly improve the crop yield and the crop quality, and great economic benefit is obtained in various crops such as rice, corn, sorghum, cotton, rape and the like. Wheat is the most widely cultivated food crop in the world with the strongest adaptability, and about 40% of the population worldwide takes wheat as the main food. Like rice, wheat also has obvious heterosis. However, due to its complex genetic characteristics, hybrid wheat performs far less well than hybrid rice, and theoretical and practical studies of wheat heterosis are far behind those of rice, maize, rape and other crops.

Wheat is a self-pollinated crop, and the core of heterosis utilization is to establish a technical system for efficiently producing wheat hybrid seeds. The key of the wheat heterosis utilizing way seed production technology is that proper female parent of recessive nuclear male sterile line is needed. Male sterility refers to the inability of plants to produce anthers, pollen or male gametes with normal function during sexual reproduction, and the disclosure of the mechanism of male sterility is the basis for improving wheat yield and quality by utilizing heterosis. The wheat genome is large and complex, so that the research on the mechanism of wheat male sterility has been so far limited. Therefore, the application of modern molecular biology and cell biology means to the research of wheat male sterility mechanism has important theoretical and practical significance for strengthening the research and utilization of wheat heterosis.

Wheat is a heterologous hexaploid and three sets of similar but not nearly identical genomes exist, so that single gene-controlled recessive mutants in wheat are significantly fewer than other species. The reported monogenic control wheat recessive nuclear male sterile material has only 2 sites, MS1 and MS5.MS1 is located on the short arm of the wheat 4B chromosome, encoding a 220 amino acid nonspecific lipocalin (non-specific lipid transfer protein [ nsLTP ]) (Wang et al, 2017; tuker et al, 2017). nsLTP is widely found in plants and is characterized by an 8CM domain consisting of 8 cysteine residues (Boutrot et al, 2008). MS1 is a newly evolved protein from Gramineae, and in situ hybridization shows that the gene is specifically expressed in high amounts in microsporocytes of anthers. Lipid binding experiments have shown that MS1 is capable of specifically binding phosphatidic acid (phosphatidic acid [ PA ]) and several types of inositol phospholipids (PIPs). Sequence analysis showed that there was one MS1 homologous gene in each of the ABD genomes of the wheat genome, but both genes were not expressed at all in hexaploid wheat due to methylation modification of the promoter region of the MS1 homologous gene in the AD genome. MS5 is located on the wheat 3A chromosome and also encodes a non-specific lipocalin, but its amino acid sequence is not highly homologous to MS1 except for the 8CM domain which also has 8 cysteine residues (Pallotta et al, 2019). MS5 is maintained only in the wheat family.

In order to obtain more wheat male sterile resources, more than twenty genetically stable recessive single gene controlled male sterile materials are screened out through phenotype screening and genetic analysis of an EMS mutagenesis library. After carrying out allele detection on the materials and the existing MS1 and MS5 mutants, 1 new single-gene control wheat recessive nuclear male sterile line is found, and an allele hybridization detection experiment confirms that the materials are not allelic to an MS1 site and an MS5 site, and the invention names the materials as MS8. The invention clones the wheat TaMS8 gene by utilizing the Mutmap method, and the gene and a male sterile line generated by the mutation of the gene provide necessary elements for constructing a novel hybridization breeding system.

Disclosure of Invention

All references mentioned herein are incorporated herein by reference.

Unless defined to the contrary, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless indicated to the contrary, the techniques used or referred to herein are standard techniques well known to those of ordinary skill in the art. The materials, methods, and examples are illustrative only and not intended to be limiting.

The invention provides a fertility-related gene TaMS8, wherein the nucleotide sequence of the fertility-related gene is selected from one of the sequences of the following groups:

(a) As set forth in SEQ ID NO:1 or 2;

(b) The coding amino acid sequence is shown as SEQ ID NO:3, a nucleotide sequence shown in figure 3;

(c) A DNA sequence capable of hybridizing under stringent conditions to a DNA of the sequence set forth in (a) or (b); or (b)

(d) A DNA sequence having at least 80% (preferably at least 85%) sequence similarity to the sequences of (a) to (c) and having fertility restoration function; or (b)

(e) A DNA sequence complementary to any one of the sequences of (a) - (d).

Those skilled in the art will appreciate that fertility-related genes of the present invention also include homologous gene sequences that are highly homologous to the nucleotide or protein sequences of the TaMS8 gene and have the same fertility control or restoration function. The homologous genes with high homology and fertility regulation function comprise genes capable of hybridizing with the genes with SEQ ID NO:1 or 2. Or the encoded amino acid sequence thereof is identical to the amino acid sequence of SEQ ID NO:3, and the nucleotide sequence with more than 85% of similarity in the amino acid sequence of the protein. As used herein, "stringent conditions" are well known and include, for example, hybridization in a hybridization solution containing 400mM NaCl, 40mM PIPES (pH 6.4) and 1mM EDTA at 53℃to 60℃for 12 to 16 hours, followed by washing with a washing solution containing 0.5 XSSC and 0.1% SDS at 62℃to 68℃for 15 to 60 minutes.

The homologous genes also include genes homologous to SEQ ID NOs: 1 or 2, and the DNA sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence similarity and fertility control function can be isolated from any plant. The percentage of sequence similarity can be obtained by well known bioinformatics algorithms, including Myers and Miller algorithms, needleman-Wunsch global alignment, smith-Waterman local alignment, pearson and Lipman similarity search, karlin and Altschul algorithms. As is well known to those skilled in the art.

The invention also provides an expression cassette comprising the DNA sequence of the fertility-related gene disclosed by the invention, wherein the nucleotide sequence of the fertility-related gene is selected from one of the following sequences:

(a) As set forth in SEQ ID NO:1 or 2;

(e) A DNA sequence complementary to any one of the sequences of (a) - (d).

Specifically, the fertility-related gene in the above expression cassette is further operably linked to a promoter that drives its expression, including but not limited to a constitutive expression promoter, an inducible promoter, a tissue-specific expression promoter, or a space-time-specific expression promoter. More specifically, the promoter is a pollen specific expression promoter. Preferably, the nucleotide sequence of the pollen specific expression promoter is shown as SEQ ID NO. 10.

The expression cassette further comprises a pollen inactivation gene, wherein the pollen inactivation gene can interfere with the function or formation of male gametes containing the pollen inactivation gene in plants. The pollen inactivation genes include, but are not limited to, barnase genes, amylase genes, DAM methylases, and the like. More specifically, the pollen inactivation gene is a maize a amylase gene, and the nucleotide sequence of the pollen inactivation gene is shown as SEQ ID NO. 14.

The expression cassette of the invention further comprises a screening gene which can be used for screening plants, plant tissue cells or vectors containing the expression cassette. Such screening genes include, but are not limited to, blue grain genes, antibiotic resistance genes, herbicide resistance genes, fluorescent protein genes, and the like. Specifically, the screening genes include, but are not limited to: blue granule gene, chloramphenicol resistance gene, hygromycin resistance gene, streptomycin resistance gene, spectinomycin resistance gene, sulfonamide resistance gene, glyphosate resistance gene, glufosinate resistance gene, bar gene, red fluorescent gene DsRED, mCherry gene, cyan fluorescent protein gene, yellow fluorescent protein gene, luciferase gene, green fluorescent protein gene, and the like.

The invention also discloses a method for regulating and controlling plant fertility, which comprises the step of transferring fertility-related genes into a wheat ms8 mutant to recover male fertility of the wheat ms8 mutant, wherein the nucleotide sequence of the fertility-related genes is selected from one of the following sequences:

(a) As set forth in SEQ ID NO:1 or 2;

(e) A DNA sequence complementary to any one of the sequences of (a) - (d).

The invention also discloses a method for maintaining the male sterile line, which uses a wheat ms8 mutant plant as a transformation receptor material to transform the closely linked 3 target genes into the sterile mutant receptor plant. The 3 target genes are fertility related gene TaMS8, pollen inactivation gene and screening gene respectively. Wherein, fertility-related gene TaMS8 can recover sterile transformation from fertility, pollen inactivation genes can inactivate pollen containing transformed exogenous genes, namely lose insemination capability, screening genes can be used for sorting transgenic seeds or tissues and non-transgenic seeds or tissues, the sorted non-transgenic seeds are used as sterile line production hybrid, and the transgenic seeds are used as maintainer lines to continuously and stably produce sterile lines.

In the above-mentioned method for maintaining male sterile line, the pollen inactivation genes include, but are not limited to, barnase gene, amylase gene, DAM methylase, etc. More specifically, the pollen inactivation gene is corn a amylase gene Zm-AA, and the nucleotide sequence of the pollen inactivation gene is shown as SEQ ID NO. 14. The pollen inactivation gene is linked to a promoter that favors expression of male gametes. More specifically, the promoters that favor male gamete expression include, but are not limited to, the PG47 promoter, zm13 promoter, and the like. The screening gene can be used for screening out plants or vectors containing the expression cassette. Such screening genes include, but are not limited to, blue grain genes, antibiotic resistance genes, herbicide resistance genes, fluorescent protein genes, and the like. Specifically, the screening genes include, but are not limited to: blue granule gene, chloramphenicol resistance gene, hygromycin resistance gene, streptomycin resistance gene, spectinomycin resistance gene, sulfonamide resistance gene, glyphosate resistance gene, glufosinate resistance gene, bar gene, red fluorescent gene DsRED, mCherry gene, cyan fluorescent protein gene, yellow fluorescent protein gene, luciferase gene, green fluorescent protein gene, and the like.

More specifically, the invention also discloses a propagation method of the male sterile line, which comprises the following steps:

(a) Transferring the following vectors into a wheat ms8 male sterile line to obtain a maintainer line containing the following vectors, wherein the vectors comprise: fertility-related gene TaMS8, wherein the fertility-related gene TaMS8 can restore male fertility of wheat ms8 mutant; and a pollen inactivation gene, which, when expressed, interferes with the function or formation of a male gamete in the plant that contains the pollen inactivation gene, such that the active male gamete produced in the plant is free of the vector; and screening genes that can be used for sorting of transgenic seeds or tissue and non-transgenic seeds or tissue.

(b) Selfing a maintainer line plant formed after transferring the vector into the plant, and simultaneously generating a wheat ms8 male sterile line without the vector and a maintainer line seed with the vector; or the pollen of the maintainer line plant is driven to the wheat ms8 male sterile line plant, so that the wheat ms8 male sterile line plant pollinates and breeds the ms8 male sterile line or the allelic sterile line seed thereof.

The invention also discloses a production method of the maintainer line, which comprises the following steps:

(a) Transferring the following vectors into a wheat ms8 male sterile line to obtain a maintenance line of the wheat ms8 nuclear male sterile line or an allelic sterile line thereof, wherein the vectors comprise: fertility-related gene TaMS8, wherein the fertility-related gene TaMS8 can restore male fertility of a wheat ms8 nuclear male sterile line or an allelic sterile line; and a pollen inactivation gene, which, when expressed, interferes with the function or formation of a male gamete in the plant containing the pollen inactivation gene, such that the fertile male gametes produced in the plant are all vector-free; and screening genes that can be used for sorting of transgenic and non-transgenic seeds.

In the above-described production method of the maintainer line, the pollen-inactivating gene includes, but is not limited to, barnase gene, amylase gene, DAM methylase and the like. More specifically, the pollen inactivation gene is corn a amylase gene Zm-AA, and the nucleotide sequence of the pollen inactivation gene is shown as SEQ ID NO. 14. The pollen inactivation gene is linked to a promoter that favors expression of male gametes. More specifically, the promoters that favor male gamete expression include, but are not limited to, the PG47 promoter, zm13 promoter, and the like. The screening gene can be used for screening out plants or vectors containing the expression cassette. Such screening genes include, but are not limited to, blue grain genes, antibiotic resistance genes, herbicide resistance genes, fluorescent protein genes, and the like. Specifically, the screening genes include, but are not limited to: blue granule gene, chloramphenicol resistance gene, hygromycin resistance gene, streptomycin resistance gene, spectinomycin resistance gene, sulfonamide resistance gene, glyphosate resistance gene, glufosinate resistance gene, bar gene, red fluorescent gene DsRED, mCherry gene, cyan fluorescent protein gene, yellow fluorescent protein gene, luciferase gene, green fluorescent protein gene, and the like.

The invention also discloses a breeding method of the maintainer line, which comprises the following steps:

(a) Transferring the following vectors into a wheat ms8 nuclear male sterile line or an allelic sterile line thereof, namely obtaining a maintainer line of the wheat ms8 nuclear male sterile line or the allelic sterile line thereof, wherein the vectors comprise: fertility-related gene TaMS8, wherein the fertility-related gene TaMS8 can restore male fertility of a wheat ms8 nuclear male sterile line or an allelic sterile line; and a pollen inactivation gene, which, when expressed, interferes with the function or formation of a male gamete in the plant containing the pollen inactivation gene, such that the fertile male gametes produced in the plant are all vector-free; and a screening gene that can be used for sorting of transgenic and non-transgenic seeds; and

(b) Selfing the maintainer line plant formed after transferring the vector, namely breeding the wheat ms8 nuclear male sterile line without the vector or the allelic sterile line seed thereof and the maintainer line seed with the vector according to the proportion of 1:1.

The invention also discloses a seed production method, which comprises the following steps:

(a) Introducing the following vectors into a wheat ms8 nuclear male sterile line or an allelic sterile line thereof to obtain a maintainer line of the wheat ms8 nuclear male sterile line or the allelic sterile line thereof, wherein the vectors comprise: fertility-related gene TaMS8, wherein the fertility-related gene TaMS8 can restore male fertility of a wheat ms8 nuclear male sterile line or an allelic sterile line; and pollen inactivation genes which, when expressed, interfere with the function or formation of male gametes in the plant containing the pollen inactivation genes, such that the fertile male gametes produced in the plant are free of the vector.

(b) Selfing the maintainer plant transformed into the vector; and

(c) After selfing, the maintainer line seed containing the vector and the wheat ms8 nuclear male sterile line without the vector or the like are obtained

And (5) positioning sterile line seeds.

In the above-mentioned propagation or maintenance method of male sterile line, production method or propagation method of maintainer line and production method of seed, etc., in the step (a), the carrier containing fertility-related gene TaMS8, pollen inactivating gene and screening gene can be introduced into the ordinary plant so as to obtain transgenic plant containing the described carrier, then the transgenic plant is hybridized with wheat ms8 nuclear male sterile line or its allelic sterile line, and after the oriented breeding process the maintainer line plant whose background is wheat ms8 nuclear male sterile line or its allelic sterile line and containing the described carrier can be obtained.

In the above-described propagation method or maintenance method of male sterile line, production method or propagation method of maintenance line, production method of seed, etc., the nucleotide sequence of fertility-related gene is selected from one of the sequences of the following group:

(a) As set forth in SEQ ID NO:1 or 2;

(e) A DNA sequence complementary to any one of the sequences of (a) - (d).

The fertility-related gene TaMS8 is also operably connected with a pollen specific expression promoter, and can drive the expression of the TaMS8 gene in plant pollen. The promoter for pollen specific expression is selected from one of the group consisting of promoters of fertility controlling genes such as MS26, NP1, MSP1, PAIR2, ZEP1, MELL, PSS1, TDR, UDT1, GAMYB4, PTC1, API5, WDA1, CYP704B2, MS26, MS22, DPW, MADS3, OSC6, RIP1, CSA, AID1, 5126 or Ms 45. More specifically, the nucleotide sequence of the pollen specific expression promoter is shown as SEQ ID NO. 10. The fertility-related gene TaMS8 is further operably linked to a terminator, which may be any one of the disclosed genes, specifically, one of which has a nucleotide sequence shown in SEQ ID NO. 11. In the above-described method for breeding or maintaining male sterile line, method for producing or breeding maintainer line, method for producing seed, etc., the pollen inactivation gene includes, but is not limited to, barnase gene, amylase gene, DAM methylase, etc. More specifically, the pollen inactivation gene is corn a amylase gene Zm-AA, and the nucleotide sequence of the pollen inactivation gene is shown as SEQ ID NO. 14. The pollen inactivation gene is linked to a promoter that favors expression of male gametes. More specifically, the promoters that favor male gamete expression include, but are not limited to, the PG47 promoter, zm13 promoter, and the like.

The above-mentioned method for breeding or maintaining male sterile line, method for producing or breeding maintainer line, method for producing seed and the like of the present invention, wherein the screening gene includes but is not limited to blue grain gene, antibiotic resistance gene, herbicide resistance gene or fluorescent gene. Specifically, the screening genes include, but are not limited to: blue granule gene, chloramphenicol resistance gene, hygromycin resistance gene, streptomycin resistance gene, spectinomycin resistance gene, sulfonamide resistance gene, glyphosate resistance gene, glufosinate resistance gene, bar gene, red fluorescent gene DsRED, mCherry gene, cyan fluorescent protein gene, yellow fluorescent protein gene, luciferase gene, green fluorescent protein gene, and the like.

The invention also provides a pollen specific expression promoter, the nucleotide sequence of which is shown as SEQ ID NO. 10. The sequence SEQ ID NO. 10 is connected with a reporter gene GUS, a vector is constructed to transform rice and wheat, GUS expression activity and expression pattern in a transgenic plant are detected and analyzed, and GUS staining analysis is carried out on roots, stems, leaves and flowers of the transgenic plant, so that the result shows that the promoter provided by the invention drives the GUS gene to express in plant pollen. The SEQ ID NO. 10 provided by the invention is a promoter for pollen specific expression.

The plant pollen specific expression promoter provided by the invention contains a nucleotide sequence shown as SEQ ID NO. 10 in a sequence table, or contains a nucleotide sequence with more than 90% of similarity with the nucleotide sequence shown as SEQ ID NO. 10, or contains 500 and more than 500 continuous nucleotide fragments from the sequence shown as SEQ ID NO. 10, and can drive the expression of the nucleotide sequence operatively connected with the promoter in plant pollen. Expression vectors, transgenic cell lines, host bacteria and the like containing the sequences belong to the protection scope of the invention. Primer pairs that amplify any of the nucleotide fragments of the SEQ ID NO. 10 promoters disclosed herein are also within the scope of the present invention.

"promoter" as used herein refers to a DNA regulatory region which generally comprises a TATA box capable of directing RNA polymerase II to initiate RNA synthesis at the appropriate transcription initiation site for a particular coding sequence. Promoters may also contain other recognition sequences, typically located upstream or 5' of the TATA box, commonly referred to as upstream promoter elements, that function to regulate transcription efficiency. It will be appreciated by those skilled in the art that while nucleotide sequences have been identified for the disclosed promoter regions, other regulatory elements that isolate and identify regions upstream of the TATA box of the particular promoter regions identified herein are within the scope of the invention. Thus, the promoter regions disclosed herein are generally further defined as comprising upstream regulatory elements, such as those elements, enhancers, and the like, for regulating the tissue expression and temporal expression functions of the coding sequence. In the same manner, promoter elements that enable expression in a tissue of interest (e.g., male tissue) can be identified, isolated, and used with other core promoters to verify male tissue-preferred expression. A core promoter refers to the minimal sequence required to initiate transcription, such as the sequence known as the TATA box, which is commonly found in promoters of genes encoding proteins. Thus, alternatively, the upstream promoter of the TaMS8 gene may be used in association with its own or a core promoter from another source.

The core promoter may be any known core promoter, such as the cauliflower mosaic virus 35S or 19S promoter (U.S. Pat. No.5,352,605), the ubiquitin promoter (U.S. Pat. No.5,510,474), the IN2 core promoter (U.S. Pat. No.5,364,780), or the figwort mosaic virus promoter.

The function of the gene promoter can be analyzed by the following method: operably connecting a promoter sequence with a reporter gene to form a transformable vector, transferring the vector into a plant, and confirming the expression characteristics of the reporter gene by observing the expression condition of the reporter gene in each tissue organ of the plant in the process of obtaining transgenic offspring; or subcloning the vector into an expression vector for a transient expression experiment, and detecting the function of the promoter or the regulatory region thereof through the transient expression experiment.

The selection of an appropriate expression vector for testing the function of a promoter or regulatory region will depend on the host and the method of introducing the expression vector into the host, such methods being well known to those of ordinary skill in the art. For eukaryotes, the regions in the vector include regions that control transcription initiation and control processing. These regions are operably linked to a reporter gene comprising the YFP, uidA, GUS gene or luciferase. Expression vectors comprising putative regulatory regions located in genomic fragments may be introduced into intact tissues, such as staged pollen, or into callus for functional verification.

In addition, the promoters of the invention may be linked to nucleotide sequences other than the TaMS8 gene to express other heterologous nucleotide sequences. The promoter nucleotide sequences of the invention, and fragments and variants thereof, may be assembled with heterologous nucleotide sequences in an expression cassette for expression in a plant of interest, more specifically in the male organ of the plant. The expression cassette has suitable restriction sites for insertion of the promoter and heterologous nucleotide sequences. These expression cassettes can be used to genetically manipulate any plant to obtain the desired corresponding phenotype.

The pollen-specific expression promoters disclosed herein can be used to drive expression of heterologous nucleotide sequences encoding enzymes or modified enzymes, amylases, debranching enzymes and pectinases that promote carbohydrate degradation, more specifically, barnase genes, maize a amylase genes, auxin genes, rotB, cytotoxin genes, diphtheria toxin, DAM methylase, or dominant male sterility genes, to obtain a male sterile phenotype in transformed plants.

In certain embodiments, the nucleotide sequences referred to herein that are operably linked downstream of the promoters of the present invention, wherein the "nucleotide sequences" may be structural genes, regulatory genes, antisense genes to structural genes, antisense genes to regulatory genes, or small RNAs capable of interfering with expression of endogenous genes operably linked to the promoters disclosed herein.

The invention also provides a transcription terminator sequence, the nucleotide sequence of which is shown as SEQ ID NO. 11, and has the function of stopping gene transcription expression.

The invention also provides an expression cassette, a vector or an engineering strain, wherein the expression cassette, the vector or the engineering strain comprises the pollen specific expression promoter SEQ ID NO. 10. Specifically, the nucleotide sequence of the fertility-related gene TaMS8 provided by the invention can be constructed to the downstream of the promoter SEQ ID NO. 10 provided by the invention, so that the fertility gene can be driven to express in a transformed recipient plant.

The pollen specific expression promoter provided by the invention can be used for specific expression of exogenous genes in pollen, so that adverse effects caused by continuous expression of the exogenous genes in other tissues of plants are avoided, and the pollen specific expression promoter can also be used for functional analysis and identification of genes related to plant pollen growth and development; can be used for the creation of male sterile lines and maintainer lines; the method can be applied to pollen abortion experiments, so that the biosafety problem caused by plant transgene drift or pollen escape is avoided, and the method has important significance for the creation of plant male sterile lines and maintainer lines.

The nucleotide sequence and promoter sequence or expression cassette of the TaMS8 gene provided by the present invention may be inserted into a vector, plasmid, yeast artificial chromosome, bacterial artificial chromosome or any other vector suitable for transformation into a host cell. Preferred host cells are bacterial cells, in particular bacterial cells for cloning or storing polynucleotides, or for transforming plant cells, such as E.coli, agrobacterium tumefaciens and Agrobacterium rhizogenes. When the host cell is a plant cell, the expression cassette or vector may be inserted into the genome of the transformed plant cell. The insertion may be a localized or random insertion.

The method for transferring the nucleotide sequence, the vector or the expression cassette into a plant or introducing the nucleotide sequence, the vector or the expression cassette into the plant or transforming the plant refers to transferring the nucleotide sequence, the vector or the expression cassette into a receptor cell or a receptor plant by a conventional transgenic method. Any transgenic method known to those skilled in the art of plant biotechnology can be used to transform a recombinant expression vector into a plant cell to produce a transgenic plant of the invention. Transformation methods may include direct and indirect transformation methods. Suitable direct methods include polyethylene glycol-induced DNA uptake, liposome-mediated transformation, introduction using a gene gun, electroporation, and microinjection. The transformation method also includes agrobacterium-mediated plant transformation methods and the like.

Compared with the prior art, the invention has the following beneficial effects: the invention provides a fertility-related gene TaMS8 and a promoter thereof, and a method for breeding and maintaining an ms8 male sterile line by using the gene. The fertility-related gene, the fertility maintenance of the nuclear male sterile line and the propagation method of the sterile line provided by the invention have great production popularization value and application value for the cross breeding production of crops. The fertility gene and the sterile line generated by the mutation of the gene provide resources for wheat hybridization and necessary elements for constructing a third generation hybridization system, and the male sterile line generated by the mutation of the gene is used for producing hybrid seeds and has important significance for breaking through and improving the existing three-line and two-line hybridization technology.

Reference to the literature

Boutrot F.,Chantret N.,Gautier MF.Genome-wide analysis of the rice and Arabidopsis non-specific lipid transfer protein(nsLtp)gene families and identification of wheat nsLtp genes by EST data mining.BMC Genomics 2008,9:86-104

Pallotta MA,Warner P,Kouidri A,Tucker EJ,Baes M,Suchecki R,Watson-Haigh N,Okada T,Garcia M,Sandhu A,Singh M,Wolters P,Albertsen MC,Cigan AM,Baumann U,Whitford R.Wheat ms5 male-sterility is induced by recessive homoeologous A and D genome non-specific lipid transfer proteins.Plant J 2019,99:673-685

Tucker EJ.,Baumann U.,Kouidri A.,Suchecki R.,Baes M.,Garcia M.,Okada T.,Dong C.,Wu Y.,Sandhu A.,Singh M.,Langridge P.,Wolters P.,Albertsen MC.,Cigan AM.,Whitford R.Molecular identification of the wheat male fertility gene Ms1 and its prospects for hybrid breeding.Nat Commun 2017,8:869

Wang Z,Li J,Chen S,Heng Y,Chen Z,Yang J,Zhou K,Pei J,He H,Deng XW,Ma L.Poaceae-specific MS1 encodes a phospholipid-binding protein for male fertility in bread wheat.Proc Natl Acad Sci U S A.2017,114:12614-12619.

Drawings

FIG. 1 is a phenotype of a wheat ms8 mutant, wherein A is the spike of the full-bloom stage of the parent Ningchun No. (left) and ms8 mutants (right), B and C are anthers of the full-bloom stage of the parent Ningchun No. four and ms8 mutants, respectively, and D and E are the iodine-potassium iodide staining of the mature anthers of the parent Ningchun No. four and ms8 mutants, respectively.

FIG. 2 is a graph showing the distribution of wild-type and mutant SNP index on wheat 1A chromosome.

FIG. 3 shows the results of TaMS8 gene mutmap and map-based cloning, wherein the first behavior is 404 ms8 sterile plant positioning results, the second behavior shows fine positioning results after population expansion, the third behavior shows SNP analysis results in a fine positioning interval, and the fourth behavior shows candidate gene structures and mutation sites.

FIG. 4 shows the genotype and phenotype analysis of the crossover individual selected by TaMS8 gene fine positioning.

FIG. 5 shows the analysis of the expression pattern of TaMS8 gene in different tissues of Chinese spring wheat by semi-quantitative RT-PCR and fluorescent quantitative PCR, and ACTIN is housekeeping gene. Wherein the upper graph is agarose gel electrophoresis graph of RT-PCR products of TaMS8 gene in different organs or tissues; the lower panel shows the quantitative RT-PCR results of TaMS8 gene in different organs or tissues, 0 is meiosis anther, 1 is anther in flower single-core stage, 2 is anther in binuclear stage, 3 is anther in trinuclear stage, palea is palea and palea mixed material, gDNA is genome DNA contrast.

FIG. 6 is a schematic diagram of construction of a plant transformation vector for complementation of ms8 mutant functions, wherein LB and RB are left and right boundaries of T-DNA, respectively, and expression of Bar resistance gene is driven by Ubip (promoter of Ubi gene) and terminated by Nos terminator; taMS8 sequence is shown as SEQ ID NO: shown at 12.

Detailed Description

The following describes in detail the examples of the present invention, which are implemented on the premise of the technical solution of the present invention, and detailed embodiments and specific operation procedures are given, but the scope of protection of the present invention is not limited to the following examples.

Example 1 acquisition and phenotypic analysis of wheat ms8 mutants

In order to obtain more wheat male sterile resources, the invention obtains more than one hundred parts of wheat male sterile materials by carrying out large-scale phenotype screening on 100 ten thousand M2 generation plants of spring wheat variety Ningchun No. four EMS mutagenesis library. And then, through genetic analysis of each material, more than twenty parts of genetically stable recessive single gene controlled male sterile materials are screened out. After carrying out allele detection on the materials and the existing MS1 and MS5 mutants, 1 new single-gene control wheat recessive nuclear male sterile line is found, and an allele hybridization detection experiment confirms that the materials are not allelic to an MS1 site and an MS5 site, and the invention names the materials as MS8.

Genetic analysis results show that in the F2 generation group hybridized with the parent Ningchun No. four, the separation ratio of the fertile strain to the sterile strain is 242:69, indicating that the site is a single gene controlled recessive mutation. The ms8 mutant and the parent thereof are subjected to superficial observation, the vegetative growth phase of the ms8 mutant and the parent thereof have no visible difference, and the ms8 mutant spike type has obvious difference compared with the parent Ningchun No. four until the full bloom phase: the glume protection and exotic and wheat grass opening angles of the ms8 mutant are obviously larger than those of a normal plant, the anther of the parent plant can be cracked normally, pollen grains can be obviously seen to be scattered, and the anther of the ms8 mutant is relatively shrunken and does not crack at all. With iodine-potassium iodide (I) ₂ KI) staining to identify pollen activity, the mature pollen starch granules of the parent plants are deeply colored and take the shape of full spheres, and the ms8 mutant is obviously shrunken and completely uncolored, which indicates that the pollen is not viable, so that no seed is beaten at the later stage (see fig. 1).

Example 2 cloning of wheat MS8 Gene

Since ms8 mutant genetic analysis showed it to be a single-gene controlled recessive mutation, we selected the technique of mutmap, i.e., mutation site mapping, to clone its gene.

Firstly, constructing a hybridization F2 generation group of an MS8 mutant and a parent Ningchun No. four, mixing sterile plant DNA in the F2 generation group into a mutant DNA mixed pool, and mixing single plant DNA with a wild type MS8 locus genotype in the F2 generation group into a wild type DNA mixed pool after tracing a post-representative type of a fertile plant in the F2 generation group. Two pools were sent to sequencing company for DNA sequencing, each pool was used to measure 500G data. Sequencing data of each pool were aligned to a wheat chinese spring reference sequence for mutmap analysis to obtain single nucleotide polymorphic sites (single nucleotide polymorphism, SNP) for both samples. From the distribution of SNP Index on each chromosome, it was found that there was a distinct peak at 400-600 Mb in the 1A chromosome among 21 pairs of chromosomes in wheat, indicating that MS8 is likely to be localized in this region (see FIG. 2).

Primers were designed and molecular validation was performed for SNPs within this segment. The position of the SNP index peak is approximately at 480-490 Mb of the wheat 1A chromosome, 5 high-reliability SNPs are selected in the section, the SNPs are respectively named as 1A-1A-5, and other 4 SNPs except 1A-4 are developed into CAPs (Cleaved Amplified Polymorphic Sequences) molecular markers. The results of the verification in the parent and mutant showed that these 5 SNPs were all truly present. Linkage analysis was performed on 5 SNP markers and MS8 sites using 404 MS8 sterile strains isolated from F2 generation populations, and MS8 was located within a range of 3.65Mb between 481,704,309bp (1A-1) -485,352,404bp (1A-3), at a distance of 1A-1.12 cM, and at a distance of 1A-3 DYZ8.25 cM (see FIG. 3).

To further pinpoint MS8, we first screened 2160F 2 generation populations with two CAPs markers 1A-1 (481, 704,309 bp) and 1A-5 (488, 109,008 bp) to obtain a total of 37 exchange individuals, and then genotyping the 37 strains with the CAPs markers 1A-3 (485, 352,404 bp) to obtain 21 exchange individuals between 1A-1 and 1A-3. The results of phenotype observation and genotype identification were combined with 21 crossover individuals, and the location interval of the MS8 site was further narrowed down to a range of 1.33Mb between 481,704,309bp (1A-1) -483,032,932bp (1A-2) (see FIG. 3), and 13 crossover individuals remained in this range. New SNP markers 1A-6 (482, 337,315 bp), 1A-13 (482, 081,042 bp), 1A-16 (482, 661,640 bp), 1A-17 (482, 830, 470 bp) and 1A-20 (482, 184,426 bp) were developed between 1A-1 and 1A-2, and after genotyping these 13 exchange individuals, the MS8 locus was reduced to a 646Kb region between 1A-20 (482, 184,426 bp) and 1A-17 (482, 830, 470 bp) in combination with its MS8 locus genotype (deduced from the phenotypic characterization results of its offspring) (see FIGS. 3 and 4). 13 coding genes are annotated in the 646Kb genome sequence, SNPs in this section are analyzed, and only 1 SNP 1A-6 falls in the gene coding section, resulting in missense mutation, suggesting that the gene in which the SNP is located is likely to be the MS8 gene, which we call TaMS8.1A-6, wherein the nucleotide at position 1664 of the genomic DNA sequence after the ATG gene is mutated from G to a, wherein the mutated genomic sequence is as set forth in SEQ ID NO:23, the CDS sequence after mutation is shown as SEQ ID NO:24, resulting in a mutation of amino acid 208 of the encoded protein sequence from glycine (Gly) to aspartic acid (Asp), the amino acid sequence being as set forth in SEQ ID NO:25 (see fig. 3).

In the Chinese spring variety of wheat, the genomic DNA sequence of the coding region of the TaMS8 gene is shown as SEQ ID NO. 1, the CDS sequence is shown as SEQ ID NO. 2, and the protein sequence is shown as SEQ ID NO. 3.

EXAMPLE 3 expression specificity of TaMS8 Gene

In order to analyze the expression pattern of TaMS8 gene, semi-quantitative RT-PCR primers TaMS8-RTF and TaMS8-RTR (SEQ ID NOS: 4 and 5) and fluorescent quantitative PCR primers TaMS8-QF and TaMS8-RTR (SEQ ID NOS: 5 and 6) were designed using the cDNA sequence of the gene as a template, and semi-quantitative RT-PCR primers ACTIN-RTF and ACTIN-RTR (SEQ ID NOS: 7 and 8) and fluorescent quantitative PCR primers ACTIN-QF and ACTIN-RTR (SEQ ID NOS: 8 and 9) were designed using the wheat ACTIN gene as an internal control. Extracting total RNA from each tissue material of Chinese spring wheat, synthesizing cDNA template, and detecting the expression of TaMS8 gene by semi-quantitative RT-PCR method and fluorescent real-time quantitative RT-PCR method. As a result, as shown in FIG. 5, the peak of gene expression was significantly reduced in the meiotic and mononuclear anthers, to the binuclear anthers, and lower in the trinuclear stage, consistent with semi-quantitative RT-PCR, and relatively low in other tissues and organs such as root, stem, leaf, endo/palea, pistil and pre-meiotic spikes of various sizes.

The primer sequences used were as follows:

TaMS8-RTF:5’-ACTGCACAGACCACCATTGAGATT-3’(SEQ ID NO:4)

TaMS8-RTR:5’-ATCAAGTAGCGCGCAGACATTG-3’(SEQ ID NO:5)

TaMS8-QF:5’-CGACGACAAGAAGAAGGTTGAGGAG-3’(SEQ ID NO:6)

ACTIN-RTF:5’-TCAGCCATACTGTGCCAATC-3’(SEQ ID NO:7)

ACTIN-RTR:5’-CTTCATGCTGCTTGGTGC-3’(SEQ ID NO:8)

ACTIN-QF:5’-TTCCAGCCATCTTTCATTG-3’(SEQ ID NO:9)

example 4 complementation of transgenic Functions of wheat ms8 mutant

To verify the function of the TaMS8 gene, we constructed a stable transformation vector for functional complementation of wheat ms8 mutants. The binary expression vector pCAMBIA1300 is taken as a framework, and a hygromycin resistance expression frame driven by a plant resistance screening gene 35S promoter on the pCAMBIA1300 is replaced by a Bar resistance gene expression frame driven by a Ubi promoter on a pAHC20 vector. On this basis, a 6783bp TaMS8 genomic sequence (shown in SEQ ID NO:12, which contains a 2907bp promoter sequence (SEQ ID NO: 10), a 2988bp genomic sequence (SEQ ID NO: 1) and a 888bp terminator sequence (SEQ ID NO: 11)) was inserted into the multiple cloning site by the in-fusion method, thereby forming a plant expression vector p1300-TaMS8 (see FIG. 6).

And transforming heterozygous plant young embryo of the wheat ms8 mutant by using agrobacterium-mediated genetic transformation technology and using a functional complementary vector p1300-TaMS8 vector to obtain 45 transgenic positive plants. As the genotype of the young embryo serving as the transformation explant can be in a wild type, heterozygous or homozygous mutation state of the MS8 locus, 9 transgenic positive plants are found to be in a MS8 homozygous mutation genotype by adjacent SNP marker genotype identification, pollen fertility of the 9 transgenic positive plants is observed, and pollen is found to be fertile. The above results indicate that mutation of the TaMS8 gene results in the ms8 mutant male sterile phenotype.

EXAMPLE 5 construction and functional analysis of TaMS8 Gene promoter expression vectors

The genomic DNA of the variety four of Ningchun from wheat was used as a template to amplify a 2907bp TaMS8 gene promoter fragment (SEQ ID NO: 10) and was ligated into the pCAMBIA1300 vector of the Bar gene driven by Ubi promoter by the resistance gene engineering technique of in-fusion, and then a GUS reporter gene (SEQ ID NO: 11) was inserted after 2907bp TaMS8 gene promoter (SEQ ID NO: 10) by the in-fusion technique (see FIG. 8). Transforming the plasmid into wheat young embryo by using agrobacterium-mediated genetic transformation technology to obtain 15 transgenic positive plants. GUS staining analysis results of different tissues and organs of the transgenic positive plant are consistent with the expression pattern analysis results of the TaMS8 gene, which shows that the TaMS8 gene promoter is an anther specific expression promoter.

Example 6 application of TaMS8 Gene in New-generation wheat crossbreeding technology

The invention applies the wheat ms8 male sterile mutant and the TaMS8 gene to the new-generation cross breeding technology of wheat. The core of the new generation cross breeding technology is that a wheat recessive nuclear male sterile mutant is used as a transformation receptor material, and fertility restoration genes, pollen inactivation genes and seed marker genes are transformed into the male sterile mutant together to form a transgenic strain with a restoration line and a maintainer line integrated. The maintainer line is used to pollinate the sterile line, thereby propagating the sterile line.

The invention takes TaMS8 gene as fertility restoration gene, takes corn gene (ZmBT 1-ZmAA) as pollen inactivation gene, takes blue grain gene as seed marker gene, and constructs three expression cassettes together on pCAMBIA1300 vector of Bar gene driven by resistance gene modified Ubi promoter.

1) Based on pCAMBIA1300 vector of the Bar gene driven by the Ubi promoter modified by the resistance gene;

2) TaMS8 gene expression cassette, target gene TaMS8 and promoter and terminator thereof are all from wheat variety Ningchun No. IV, and full-length nucleotide sequence is shown in SEQ ID NO:12, wherein the promoter sequence of the TaMS8 gene is shown as SEQ ID NO:10, the terminator sequence of which is shown in SEQ ID NO:11, the genomic DNA sequence is shown in SEQ ID NO:1, the nucleotide sequence of which encodes a protein with the amino acid sequence shown in SEQ ID NO:3 is shown in the figure;

3) Gene expression cassette PG47: zmBT1-ZmAA-IN2-1, the target gene is ZmAA, the transit peptide is ZmBT1, the open reading frame of ZmBT1-ZmAA (the nucleotide sequence of which is shown as SEQ ID NO: 14) is connected with the downstream of a promoter PG47 (the nucleotide sequence of which is shown as SEQ ID NO: 15) and the upstream of a terminator IN2-1 (the nucleotide sequence of which is shown as SEQ ID NO: 16).

4) The blue granule marker gene expression cassette is disclosed in patent WO2019090496A1. The target genes were 3215bp ThMYB1 genomic sequence (comprising 1952bp promoter sequence (shown as SEQ ID NO: 17), 822bp genomic sequence (shown as SEQ ID NO: 18) and 441bp terminator sequence (shown as SEQ ID NO: 19)) and 4422bp ThR1 genomic sequence (comprising 2084bp promoter sequence (shown as SEQ ID NO: 20), 1720bp CDS sequence (shown as SEQ ID NO: 21) and 618bp terminator sequence (shown as SEQ ID NO: 22)).

And transforming the heterozygote plant immature embryo of the wheat ms8 mutant by using an agrobacterium-mediated genetic transformation technology to obtain 15 transgenic positive plants with the genotype of ms8 homozygous mutation. The pollen activity of the transgenic positive plants is detected by iodine-potassium iodide staining, and the result shows that the sterile pollen proportion of a plurality of transgenic plants is about 50%, which shows that firstly, the fertility restorer gene acts to restore the male sterile phenotype of the ms8 mutant, and secondly, the pollen lethal gene inactivates half of the pollen containing the transgene. In the single harvested T1 generation transgenic plant seeds, a normal color seed to blue seed ratio of about 1:1, i.e., the seed carrying the exogenous transgene, exhibits a blue grain phenotype due to the seed marker gene. The results show that the three expression cassettes of fertility restorer gene, pollen inactivation gene and seed marker gene can all function correctly and can be successfully applied to the new-generation wheat cross breeding technology.

Claims

A fertility-related gene TaMS8, characterized in that the nucleotide sequence of said fertility-related gene TaMS8 is selected from one of the sequences of the group consisting of:

(a) As set forth in SEQ ID NO:1 or 2;

(b) The coding amino acid sequence is shown as SEQ ID NO:3, a nucleotide sequence shown in figure 3;

(c) A DNA sequence capable of hybridizing under stringent conditions to a DNA of the sequence set forth in (a) or (b); or (b)

(d) A DNA sequence having at least 80% (preferably at least 85%) sequence similarity to the sequences of (a) to (c) and having fertility restoration function; or (b)

(e) A DNA sequence complementary to any one of the sequences of (a) - (d).
An expression cassette, expression vector or engineering bacterium, characterized in that the expression cassette, expression vector or engineering bacterium comprises the fertility-related gene of claim 1.
The application of fertility-related genes, expression cassettes, expression vectors or engineering bacteria in regulating plant fertility is characterized in that the fertility-related genes, expression cassettes, expression vectors and engineering bacteria contain one of the nucleotide sequences shown as follows:

(a) As set forth in SEQ ID NO:1 or 2;

(b) The coding amino acid sequence is shown as SEQ ID NO:3, a nucleotide sequence shown in figure 3;

(c) A DNA sequence capable of hybridizing under stringent conditions to a DNA of the sequence set forth in (a) or (b); or (b)

(d) A DNA sequence having at least 80% (preferably at least 85%) sequence similarity to the sequences of (a) to (c) and having fertility restoration function; or (b)

(e) A DNA sequence complementary to any one of the sequences of (a) - (d).
A method for regulating plant fertility by over-expressing, inhibiting or mutating fertility-related genes in plants to affect expression levels thereof, thereby regulating plant fertility, comprising the steps of: the nucleotide sequence of the fertility-related gene TaMS8 is selected from one of the sequences of the following groups:

(a) As set forth in SEQ ID NO:1 or 2;

(b) The coding amino acid sequence is shown as SEQ ID NO:3, a nucleotide sequence shown in figure 3;

(c) A DNA sequence capable of hybridizing under stringent conditions to a DNA of the sequence set forth in (a) or (b); or (b)

(d) A DNA sequence having at least 80% (preferably at least 85%) sequence similarity to the sequences of (a) to (c) and having fertility restoration function; or (b)

(e) A DNA sequence complementary to any one of the sequences of (a) - (d).
The method of claim 4, wherein the mutation comprises a substitution, deletion, or addition of one or more nucleotides in the nucleotide sequence of the fertility-related gene.
The method according to any one of claims 4-5, wherein said "mutation" comprises, but is not limited to, a method such as a gene mutation caused by physical or chemical means, including a mutagenesis caused by treatment with a mutagen such as EMS, or by gene silencing means such as RNAi or by gene editing, including, but not limited to, a gene editing method such as ZFN, TALEN, and/or CRISPR/Cas 9.
The method of claim 4, wherein the method comprises restoring the ms8 male sterile line to fertility by complementing the male sterile phenotype caused by mutation of the TaMS8 gene with the nucleotide sequence of the TaMS8 gene.
The method of claim 7, wherein the sequence of the TaMS8 gene mutation is as set forth in SEQ ID NO:23 or 24, and the amino acid is shown as SEQ ID NO: 25.
Use of the method according to any one of claims 4-8 for regulating plant fertility.
A method of producing or propagating a male sterile line, the method comprising the steps of:

(a) Transferring into the ms8 male sterile line a vector comprising: fertility-related gene TaMS8, wherein the fertility-related gene TaMS8 can restore male fertility of an ms8 male sterile line; and a pollen inactivation gene, which, when expressed, interferes with the function or formation of a male gamete in the plant containing the pollen inactivation gene, such that the fertile male gametes produced in the plant are all vector-free; and a screening gene that can be used for sorting of transgenic and non-transgenic seeds; and

(b) Selfing a maintainer plant formed after transferring the vector into the plant, and simultaneously generating a ms8 male sterile line seed without the vector and a maintainer line seed with the vector; or pollinating the ms8 sterile line plant with the pollen of the maintainer line plant, so that the ms8 sterile line pollinates and breeds the ms8 sterile line seed.
The production or propagation method according to claim 10, wherein the nucleotide sequence of fertility-related gene TaMS8 is selected from one of the sequences of the group consisting of:

(a) As set forth in SEQ ID NO:1 or 2;

(b) The coding amino acid sequence is shown as SEQ ID NO:3, a nucleotide sequence shown in figure 3;

(c) A DNA sequence capable of hybridizing under stringent conditions to a DNA of the sequence set forth in (a) or (b); or (b)

(d) A DNA sequence having at least 80% (preferably at least 85%) sequence similarity to the sequences of (a) to (c) and having fertility restoration function; or (b)

(e) A DNA sequence complementary to any one of the sequences of (a) - (d).
Production or propagation method according to claim 11, wherein said fertility-related gene TaMS8 is driven by a pollen-specific expression promoter, preferably the nucleotide sequence of said pollen-specific expression promoter is as shown in seq id No. 10.
A method of production or propagation as claimed in any one of claims 10 to 12 wherein said pollen inactivating genes include, but are not limited to, barnase genes, amylase genes, DAM methylases and the like, preferably said pollen inactivating genes are maize alpha amylase genes, more preferably the nucleotide sequences of which are set forth in SEQ ID NOs: 14.
A method of production or propagation according to claim 13 wherein the pollen inactivation gene is linked to a promoter which favors expression of male gametes, preferably the promoter is the PG47 promoter or Zm13 promoter.
Propagation method according to any one of claims 10-14, wherein said screening genes include, but are not limited to, blue grain genes, antibiotic resistance genes, herbicide resistance genes, fluorescent protein genes, etc., preferably said screening genes include, but are not limited to: blue granule gene, chloramphenicol resistance gene, hygromycin resistance gene, streptomycin resistance gene, spectinomycin resistance gene, sulfonamide resistance gene, glyphosate resistance gene, glufosinate resistance gene, bar gene, red fluorescent gene DsRED, mCherry gene, cyan fluorescent protein gene, yellow fluorescent protein gene, luciferase gene, green fluorescent protein gene, and the like.
A method of producing or propagating a maintainer line, the method comprising the steps of:

(a) Transferring the following vectors into the ms8 male sterile line, namely obtaining a maintainer line of the ms8 male sterile line, wherein the vectors comprise: fertility-related gene TaMS8, wherein the fertility-related gene TaMS8 can restore male fertility of an ms8 male sterile line; and a pollen inactivation gene, which, when expressed, interferes with the function or formation of a male gamete in the plant containing the pollen inactivation gene, such that the fertile male gametes produced in the plant are all vector-free; and a screening gene that can be used for sorting of transgenic and non-transgenic seeds; and

(b) Selfing the maintainer line plant which is formed after being transferred into the vector, and simultaneously generating a ms8 male sterile line seed without the vector and a maintainer line seed with the vector.
The production or propagation method according to claim 16, wherein the nucleotide sequence of fertility-related gene TaMS8 is selected from one of the sequences of the group consisting of:

(a) As set forth in SEQ ID NO:1 or 2;

(b) The coding amino acid sequence is shown as SEQ ID NO:3, a nucleotide sequence shown in figure 3;

(c) A DNA sequence capable of hybridizing under stringent conditions to a DNA of the sequence set forth in (a) or (b); or (b)

(d) A DNA sequence having at least 80% (preferably at least 85%) sequence similarity to the sequences of (a) to (c) and having fertility restoration function; or (b)

(e) A DNA sequence complementary to any one of the sequences of (a) - (d).
Production or propagation method according to claim 17, wherein said fertility restorer gene TaMS8 is driven by a pollen-specific expression promoter, preferably the nucleotide sequence of said pollen-specific expression promoter is as shown in SEQ ID NO. 10.
A method of production or propagation as claimed in any one of claims 16 to 18 wherein said pollen inactivating genes include, but are not limited to, barnase genes, amylase genes, DAM methylases and the like, preferably said pollen inactivating genes are maize alpha amylase genes, more preferably the nucleotide sequences of which are set forth in SEQ ID NOs: 14.
A method of production or propagation according to claim 19 wherein the pollen inactivation gene is linked to a promoter which favors expression of male gametes, preferably the promoter is the PG47 promoter or Zm13 promoter.
Propagation method according to any one of claims 16-20, wherein said screening genes include, but are not limited to, blue grain genes, antibiotic resistance genes, herbicide resistance genes, fluorescent protein genes, etc., preferably said screening genes include, but are not limited to: blue granule gene, chloramphenicol resistance gene, hygromycin resistance gene, streptomycin resistance gene, spectinomycin resistance gene, sulfonamide resistance gene, glyphosate resistance gene, glufosinate resistance gene, bar gene, red fluorescent gene DsRED, mCherry gene, cyan fluorescent protein gene, yellow fluorescent protein gene, luciferase gene, green fluorescent protein gene, and the like.