A gene ZmKO1 for regulating the flowering and pollen shedding of male spikelets in maize and its application in hybrid seed production of male sterile maize

Abstract

The present invention discloses a gene ZmKO1 that regulates the flowering and pollen shedding of maize male spikelets and its application in maize male sterile hybrid seed production, belonging to the field of plant genetic engineering. The present invention provides a gene ZmKO1 that regulates the flowering and pollen shedding of maize male spikelets, and the phenomenon of nuclear male sterility can be caused by mutating the ZmKO1 gene. It is also clarified that based on the ZmKO1 gene site, the CRISPR-Cas9 technology or the mutant gene backcrossing breeding technology can be used to cultivate maize nuclear male sterile lines. The present invention also found that spraying exogenous gibberellins on the male spikelets during the silking period of the sterile line can restore the male fertility of the sterile line, and it can be self-fertilized normally and used as a maintenance line to achieve economical and efficient maintenance of the sterile line. Therefore, the nuclear male sterile line created based on the ZmKO1 gene site has the characteristics of one line with two uses and can be used for maize male sterile hybrid seed production. The present invention also provides a technical system for realizing maize two-line sterile hybrid seed production using ZmKO1 gene mutation.

Description

Technical Field

[0001] The present invention relates to the field of plant genetic engineering, in particular to a gene ZmKO1 for regulating the flowering and pollen shedding of corn male spikelets and its application in corn male sterile hybrid seed production. Background Art

[0002] Corn is one of the world's most important food crops. In the 1830s, the application of hybrid vigor to corn led to rapid increases in corn yield. Hybrid vigor is a ubiquitous genetic phenomenon in the biological world, whereby a hybrid plant outperforms both parent plants in one or more traits. As a monoecious crop with different flowers and different sexes, corn has made the application of hybrid vigor very successful in corn production. Currently, hybrids are the primary corn seed used in production. To ensure hybrid purity, the maternal parent is detasseled during hybrid seed production to prevent self-pollination and ensure that the maternal parent receives only pollen from the paternal line. The quality of detasseling largely determines hybrid yield and purity, making it a critical step in seed production.

[0003] Methods for detasseling corn include manual detasseling, mechanical detasseling, chemical detasseling, and the use of male sterile lines. Potential risks of chemical detasseling agents, including incomplete pollen lethality, reduced female fertility, and environmental pollution, have limited their large-scale application. Currently, manual and mechanical detasseling remain the primary methods used in commercial corn hybrid production. Manual detasseling, due to its high controllability, wide applicability, and ease of operation, is the primary method used in corn hybrid seed production in my country. However, manual detasseling is time-consuming and labor-intensive, and its cost is rising annually. Furthermore, manual detasseling often leads to incomplete and untimely detasseling, impacting seed quality. With the acceleration of urbanization and the continuous outflow of rural labor to urban areas and non-agricultural sectors, the problem of manual detasseling will persist and become increasingly severe. In recent years, many seed companies have also experimented with mechanical detasseling, but the high equipment investment and inconsistent detasseling quality, coupled with significant plant damage and reduced seed yield, have limited its large-scale application. Therefore, detasseling remains a major challenge in corn hybrid seed production in my country.

[0004] Using male sterile lines as female parents for hybrid seed production eliminates the need for detasseling, saving labor, reducing seed production costs, and ensuring hybrid purity. Plant male sterility is categorized into cytoplasmic male sterility (CMS) and genomic male sterility (GMS). However, the CMS sterility seed production system in maize suffers from several challenges that have yet to be effectively overcome. These include low genetic diversity between sterile and restorer lines, susceptibility of sterile cytoplasmic material to specialized pathogen infection, and environmental and genetic influences on the stability of abortion and fertility recovery, limiting its widespread application. GMS, on the other hand, exhibits stable and complete abortion, with abundant genetic resources. Most lines are controlled by a recessive single gene, resulting in simple fertility recovery, making it an ideal sterility type for sterile seed production. However, due to its genomic genetic characteristics, GMS cannot be efficiently propagated, making it difficult to meet seed production requirements and hindering its application in commercial maize hybrid seed production.

[0005] To solve the problem of the inability to efficiently reproduce GMS in corn, Pioneer Company developed a "seed production technology" (SPT) based on recessive genetic male sterility (Wu et al., 2016, Development of a novel recessive genetic male sterility system for hybrid seed production in maize and other cross-pollinating crops. Plant Biotechnol J. 14(3): 1046-54), which achieved efficient reproduction of non-transgenic genetic male sterile lines through transgenic technology. Although SPT technology has achieved efficient reproduction of GMS, there is a transgenic drift frequency of 0.002%-0.518%. The subsequent development of the "multi-control sterility system" can reduce the transgenic drift frequency to 1 / 8-1 / 7 of the SPT level, but it still cannot be completely avoided (Zhang et al., 2018, Construction of a multicontrol sterility system for a maize male-sterile line and hybrid seed production based on the ZmMs7 gene encoding a PHD-finger transcription factor. Plant Biotechnol J. 16(2):459-471). The development of a sterile seed production technology that does not contain transgenic components, is easy to operate, and has better universality is an urgent need for my country's corn hybrid seed production.

[0006] Environment-sensitive genic male sterility (EGMS) lines, whose fertility conversion is primarily controlled by environmental conditions such as temperature or day length, can function as sterile lines under specific conditions. Under other conditions, they can function as maintainer lines for self-fertilization, maintaining the sterile lines. By combining a dual-purpose line with a restorer line, two-line hybrid seed production can be achieved. Currently, two-line seed production has achieved considerable success in rice hybrid seed production, primarily due to the discovery and utilization of a series of environmentally sensitive genic male sterile materials, particularly temperature-sensitive genic male sterile materials. However, two-line seed production in maize is currently only in its exploratory stage. Limited by the limited availability of environmentally sensitive genic male sterile materials, fertility conversion is incomplete and unstable. Therefore, the collection, development, and screening of new materials or genes suitable for two-line maize seed production can accelerate innovation in maize hybrid seed production.

[0007] Earlier, the inventors identified a mutant from a maize mutagenesis population. During the silking and pollination stage, the elongation of the filaments in the male spikelets was blocked, the glumes did not crack, and the anthers could not be exposed to shed pollen, resulting in male sterility. Genetic analysis revealed that the male sterility was controlled by a single recessive nuclear gene, and the mutant was named defective stamenfilament 1 (def1). The mature pollen grains of the def1 mutant showed normal I2-IK staining and morphology, and the fertility of the female spikelets and other plant traits were normal. Spraying gibberellins during the silking stage resulted in normal elongation of the filaments in the def1 spikelets, normal dehiscence of the glumes, and normal exposure of the anthers to shed pollen. The mutant was able to self-fertilize, and its seed set rate was not significantly different from that of the wild type. The DEF1 gene was cloned through map-based cloning and transgenic functional verification. The DEF1 gene locus is located at Zm00001eb385100 (reference genome: Zm-B73-REFERENCE-NAM-5.0), and is annotated as the endo-kaurene oxidase 1 gene, ZmKO1. Currently, there are no reports on the ZmKO1 gene's role in regulating filament elongation, pollen shedding during flowering, or male flower fertility in maize spikelets. There is also no evidence of its application in the creation of maize male sterile lines or sterile hybrid seed production. Summary of the Invention

[0008] The purpose of the present invention is to provide a gene ZmKO1 that regulates the flowering and pollen shedding of maize male spikelets and its application in maize male sterile hybrid seed production, so as to solve the problems existing in the above-mentioned prior art. By using CRISPR-Cas9 technology or mutant gene backcrossing technology, the cultivation of maize nuclear male sterile lines can be achieved, and then the maize male sterile lines can be used to create a maize sterile hybrid seed production method.

[0009] To achieve the above object, the present invention provides the following solutions:

[0010] The present invention provides the use of the ZmKO1 gene in any of the following:

[0011] (1) Application in regulating the development of filaments in maize spikelets;

[0012] (2) Application in regulating the opening of corn spikelet husks;

[0013] (3) Application in regulating maize male flower fertility;

[0014] (4) Application in maize male sterile hybrid seed production;

[0015] The ZmKO1 gene is the Zm00001eb385100 gene in the reference genome Zm-B73-REFERENCE-NAM-5.0 in the EnsemblPlants database, and its gene number in the NCBI (National Center of Biotechnology Information) database is 100274015.

[0016] More preferably, the nucleotide sequence of the ZmKO1 gene in the embodiments of the present invention is as shown in any one of SEQ ID NOs: 1-3, and the nucleotide sequence of the ZmKO1 gene may also be a DNA sequence that has more than 90% identity with the sequence shown in any one of SEQ ID NOs: 1-3 and has the same function as the sequence shown in any one of SEQ ID NOs: 1-3.

[0017] The present invention provides the use of a protein expressed by the ZmKO1 gene in any of the following:

[0018] (1) Application in regulating the development of filaments in maize spikelets;

[0019] (2) Application in regulating the opening of corn spikelet husks;

[0020] (3) Application in regulating maize male flower fertility;

[0021] (4) Application in maize male sterile hybrid seed production;

[0022] More preferably, in the embodiments of the present invention, the amino acid sequence of the protein is as shown in SEQ ID NO: 4. The protein sequence may also be a derived protein sequence obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 4 and having the same function as the amino acid sequence shown in SEQ ID NO: 4.

[0023] The present invention provides use of a recombinant vector comprising the ZmKO1 gene in any of the following:

[0024] (1) Application in regulating the development of filaments in maize spikelets;

[0025] (2) Application in regulating the opening of corn spikelet husks;

[0026] (3) Application in regulating maize male flower fertility;

[0027] (4) Application in maize male sterile hybrid seed production.

[0028] Preferably, genetic engineering technology or gene editing technology is used to change the DNA sequence or protein sequence of the ZmKO1 gene or regulate the transcriptional expression or protein abundance of the ZmKO1 gene, so as to reduce the filament length of the maize male spikelet and / or close the husk and / or make the male flower sterile.

[0029] More preferably, the gene editing method in the embodiments of the present invention involves inserting or deleting bases in the target sequence of the second exon of the ZmKO1 gene, using the sgRNA sequence 5'-GTCTCTGGTTTACCCCTGAT-3'. For example, base 17 of the target sequence targeted by the sgRNA (i.e., the sequence complementary to the sgRNA) may be deleted, or 16 bases may be deleted consecutively starting at base 6 of the target sequence, or a T may be inserted between bases 16 and 17 of the target sequence. However, the present invention is not limited to these targets; mutations at other positions can also lead to male sterility.

[0030] The present invention provides a method for cultivating a maize male sterile line, the method comprising: (1) using a genetic engineering method to change the transcript sequence or protein sequence of the ZmKO1 gene of a recipient plant or the expression level of the ZmKO1 gene at the transcription level or protein level, and screening a male sterile line from the offspring of the recipient plant; or (2) using a gene editing technology (including but not limited to CRISPR-Cas9 technology) to edit the ZmKO1 gene of the maize recipient plant, and screening a maize male sterile line from the offspring of the recipient plant; or (3) using backcrossing to introduce the ZmKO1 gene that can cause male sterility into maize materials with different genetic backgrounds, and creating maize male sterile lines with different genetic backgrounds.

[0031] The present invention provides a method for producing corn male sterile hybrid seeds using a corn male sterile line cultivated by the method, which is characterized by comprising: using the corn male sterile line as the female parent and a fertile corn inbred line as the male parent, and planting them according to hybrid seed production to achieve corn male sterile hybrid seed production.

[0032] Preferably, when the maize male sterile line is propagated, the fertility of the maize male sterile line is restored by exogenously spraying gibberellin or an agricultural growth regulator containing gibberellin during the silking period, thereby completing self-pollination of the sterile line.

[0033] The present invention discloses the following technical effects:

[0034] The present invention provides a gene, ZmKO1, that regulates flowering and pollen shedding in maize male spikelets. The nucleotide sequence of the protein coding region of this gene transcript is shown in SEQ ID NO:1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO:4. The present invention has determined that mutations in the ZmKO1 gene can result in the inability of the glumes of male spikelets to open, the anthers to expose and shed pollen, exhibiting nuclear male sterility. The present invention also clarifies that based on the ZmKO1 gene locus, CRISPR-Cas9 gene editing technology or mutant gene backcrossing can be used to cultivate maize nuclear male sterile lines. The present invention also discovered that spraying exogenous gibberellins on the male spikelets during the silking stage of the sterile lines can restore male flower fertility in the sterile lines. The sterile plants can then open their glumes normally, expose their anthers and shed pollen, and can self-reproduce normally, thus serving as maintainer lines for economical and efficient maintenance of the sterile lines. Therefore, the nuclear male sterile line created based on the ZmKO1 gene locus has the characteristics of one line with two purposes. The present invention also provides a technical system for realizing corn two-line sterile hybrid seed production by utilizing ZmKO1 gene mutation. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0036] Figure 1 The phenotypic identification results of the maize male sterile mutant def1 and the wild type WT, among which A is the plant type of the wild type WT and mutant def1; B is the fertility performance of the wild type WT and mutant def1 during the pollen shedding period; C is the performance of the male spikelets of the wild type WT and mutant def1 during the pollen shedding period; D is the difference in filament elongation of the male spikelets of the wild type WT and mutant def1 during the pollen shedding period; E is the I2-KI staining result of the wild type WT anther; F is the I2-KI staining result of the mutant def1 anther; G is the fruiting performance of the wild type WT female spike; H is the fruiting performance of the mutant def1 female spike;

[0037] Figure 2 The def1 mutant gene is located; A is the fine positioning of the def1 mutant; B is the sequence variation characteristics of the key candidate gene Zm00001eb385100;

[0038] Figure 3 Figure 3 shows the promoting effect of gibberellin GA3 on flowering and pollination of male spikelets of mutant def1; A shows flowering and pollination of male flowers and self-fertilization of female spikelets of wild type WT; B shows flowering and pollination of male flowers and self-fertilization of female spikelets of mutant def1 after spraying with 50 mg / L gibberellin GA3;

[0039] Figure 4 To verify the function of the Zm00001eb385100 gene using CRISPR-Cas9 gene editing technology; A is the sgRNA site and sequence information; B is the genomic sequence variation information of the target site of three gene-edited strains; C is the protein sequence variation characteristics of the Zm00001eb385100 gene in the gene-edited strain; D is the male sterility of the gene-edited strain and the fertility restoration of the edited strain by GA3; E is a local amplification of D; F is the allelic determination result;

[0040] Figure 5 This is a backcross breeding program for male sterility in an inbred line using the mutant def1. A is a flowchart for backcross breeding; B is the male flower fertility phenotype of the maize inbred line Jing 92; C is the sterility phenotype of the male sterile line Jing 92A developed through backcross breeding; and D is the male flower flowering and pollen shedding phenotype of Jing 92A after spraying with 50 mg / L gibberellic acid.

[0041] Figure 6 In order to develop two-line sterile hybrid seed production using the dual-purpose male sterile line of maize created by using the ZmKO1 gene, def1 / def1 represents the genotype of the sterile line, and DEF1 / DEF1 represents the genotype of the paternal inbred line. A is the reproductive pattern diagram of the sterile line; B is the sterile hybrid seed production pattern diagram. DETAILED DESCRIPTION

[0042] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0043] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0044] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0045] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0046] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0047] The maize inbred lines B73, Mo17 and Huangzaosi were all obtained from the Maize Research Institute of Sichuan Agricultural University.

[0048] The def1 mutant, a maize spikelet filament elongation defect mutant, is a mutant material collected on July 23, 2015 from a population of maize mutagenesis progeny at the Modern Agriculture Research and Development Base of Sichuan Agricultural University in Chongzhou City, Sichuan Province. During the silking and pollination period, the elongation of the filaments of its spikelets is blocked, the glume of the spikelets does not crack, and the anthers cannot be exposed to shed pollen, exhibiting the characteristics of male sterility. It was identified as a male nuclear sterile mutant and its seeds are deposited at the Maize Research Institute of Sichuan Agricultural University. The applicant has pledged to make the seed resources available to the public for 20 years from the date of application.

[0049] Example 1

[0050] 1. Cloning of the def1 gene, a mutant of maize silk elongation

[0051] In July 2015, the inventors identified a maize spikelet filament elongation defect mutant def1. The overall agronomic traits of the mutant def1 were not significantly different from those of the wild type WT ( Figure 1 Middle A); During the silking and pollination period, the glumes of the WT male flower spikelets can open normally and the anthers can be exposed and shed pollen normally, while the glumes of the def1 male flower spikelets do not open and the anthers cannot be exposed and shed pollen naturally, showing male sterility ( Figure 1 B and C); further, after peeling off the husk of the male spikelet, it was found that the filaments of the male spikelet of the WT plant could elongate normally, while the filaments of the male spikelet of the def1 plant did not elongate ( Figure 1During the silking stage, pollen grains of WT and def1 plants can be stained with I2-KI, indicating that pollen vitality is normal ( Figure 1 E and F). When wild-type WT pollen was used to pollinate the female ears of the mutant def1, the fruit setting rate was not significantly different from that of the self-pollinated ears of the wild-type WT ( Figure 1 G and H).

[0052] The mutant def1 was used as the female parent and mated with maize inbred lines B73, Mo17, and Huangzao 4 to form three F2 populations. The number of flowering and pollen-shedding plants and male sterile plants in the F2 population was counted and the chi-square test was used to determine the number of flowering and pollen-shedding plants and male sterile plants. 2 The test found that the ratio of flowering and pollen-shedding plants to male sterile plants in the three F2 populations all met the segregation ratio of 3:1, indicating that the def1 mutant trait is controlled by a single recessive nuclear gene. Using the F2 trait segregating population of the mutant def1 and the inbred line Mo17, the target site was located between the polymorphic markers idp8 and SNP2 on chromosome 9 using polymorphic molecular marker mapping technology. This interval contains 13 protein-coding genes ( Figure 2 A in the middle). On this basis, through expression analysis and sequence comparison, a key candidate gene Zm00001eb385100 (reference genome is Zm-B73-REFERENCE-NAM-5.0) was screened out. This gene was annotated as the endo-kaurene oxidase 1 gene ZmKO1 (nucleotides are shown in SEQ ID NO: 1, amino acids are shown in SEQ ID NO: 4; cDNA sequence is shown in SEQ ID NO: 2, genomic DNA sequence is shown in SEQ ID NO: 3), which is involved in the synthesis of gibberellins. Compared with the wild type WT, the mutant def1 has a large fragment insertion in the first exon of the ZmKO1 gene, resulting in the replacement of the 19bp sequence in the first exon region with an 82bp sequence at the cDNA level (the mutant allele is named ZmKO1-82), which ultimately leads to frameshift mutations and premature termination in the protein sequence ( Figure 2 Middle B).

[0053] During the silking and pollination stage, spraying gibberellin GA3 can restore the development of filaments in the male spikelets of the mutant def1, and the anthers can be exposed and pollen can be shed normally. The mutant can achieve self-fertilization, and the fruit setting rate is not significantly different from that of the wild type ( Figure 3 Furthermore, the inventors discovered that spraying the mutant with gibberellins GA1, GA4, and GA7, respectively, could also restore its fertility. Exogenous spraying of the mutant with gibberellic acid (75% active ingredient content, manufactured by Shanghai Tongrui Biotechnology Co., Ltd., at a concentration of 25-50 mg / L), a commonly used plant growth regulator in agricultural production, also significantly restored def1 fertility, causing it to flower and shed pollen.

[0054] Furthermore, the CRISPR-Cas9 gene site-directed editing technology was used to edit the second exon of the ZmKO1 gene using the maize inbred line B104 as the gene editing receptor ( Figure 4 Three different editing types of non-transgenic homozygous mutant lines were screened, namely KO#1 with 1 bp insertion (T base inserted between the 16th and 17th bases of the target sequence "GTCTCTGGTTTACCCCTGAT" of the second exon of the ZmKO1 gene), KO#2 with 1 bp deletion (T base deleted at the 17th base of the target sequence), and KO#3 with 16 bp deletion (16 bp bases were deleted continuously starting from the 6th base of the target sequence). Figure 4 Middle B), all three mutation types lead to frameshift mutation and premature termination of ZmKO1 gene protein translation ( Figure 4 Field phenotypic observations revealed that compared to the wild-type B104, the male spikelets of the three strains, KO#1, KO#2, and KO#3, were unable to flower and shed pollen. However, after spraying with gibberellin GA3, they became male fertile, exhibiting the same characteristics as the def1 mutant ( Figure 4 At the same time, the mutant def1 was hybridized with the inbred line B104 and the edited line KO#1 for allelic determination. The male flowers of the F1 plants hybridized with def1 and B104 flowered and shed pollen normally, while the male flowers of the F1 plants hybridized with def1 and KO#1 had closed glumes and could not flower and shed pollen. Figure 4 Middle (F) indicates that the male sterility mutation site of KO#1 line is allelic to def1, and the ZmKO1 gene is the DEF1 gene.

[0055] 2. Using the ZmKO1 gene to create a maize male sterile line

[0056] There are two methods for creating maize male sterile lines using the ZmKO1 gene. The first is to use the def1 mutant to backcross and breed the ZmKO1-82 gene to complete the creation of different maternal sterile lines. First, based on the def1 mutant ZmKO1-82 gene sequence, a polymorphic molecular marker that can identify the def1 mutant ZmKO1-82 gene is developed. Through backcrossing combined with molecular marker-assisted selection and phenotypic selection, the sterile line is quickly completed through backcross breeding ( Figure 5 By this method, the present invention converts the inbred line Jing 92 ( Figure 5 B) was transformed into the sterile line Jing 92A ( Figure 5 Middle C), spraying gibberellic acid can promote the flowering and pollination of the sterile line Jing 92A ( Figure 5 D) to achieve self-fertilization of the Jing 92A sterile line. The second method is to quickly cultivate sterile lines using CRISPR-Cas9 gene site-directed editing technology. Figure 4As shown in Figure A, the second exon "GTCTCTGGTTTACCCCTGAT" sequence was used as sgRNA to construct a gene editing vector, transform Agrobacterium EHA105 competent cells, and genetic transformation of the inbred line B104 was carried out through Agrobacterium-mediated embryogenic callus. Three types of edits were identified in the T0 generation ( Figure 4 B and C) were backcrossed with B104, and the homozygous mutant non-transgenic lines of the three editing types were obtained through identification of editing site genotypes, transgenic screening and self-fertilization. They showed male sterility, and fertility returned to normal after spraying with gibberellin GA3 or gibberellic acid ( Figure 4 D and E).

[0057] 3. Using the ZmKO1 gene to create a maize male sterile line for two-line sterile hybrid seed production

[0058] The application of maize nuclear male sterile lines in commercial hybrid seed production depends on whether the lines can be propagated economically and efficiently. The male sterile lines created in this invention based on the ZmKO1 gene can effectively restore their fertility by exogenously spraying gibberellins (such as GA3, GA1, GA4, or GA7) or agricultural growth regulators containing gibberellins. The male flowers of the sterile lines bloom and shed pollen normally, and the sterile lines can be used as maintainer lines to achieve economical and efficient propagation of sterile seeds. Figure 6 In commercial hybrid production, under natural growth conditions, it can be used as a sterile line, with the sterile line as the female parent and the fertile inbred line as the male parent, to achieve sterile seed production of corn hybrids ( Figure 6 Therefore, the maize male sterile line created using the ZmKO1 gene can be used for maize two-line sterile hybrid seed production.

[0059] SEQ ID NO: 1 is as follows:

[0060]

[0061] SEQ ID NO: 2 is as follows:

[0062]

[0063] SEQ ID NO: 3 is as follows:

[0064]

[0065] SEQ ID NO: 4 is as follows:

[0066] MESLVAALPAGGAAAAAAFGGLVAAAALAGKVGLVGSKKHLNAPPAVSGLPLIGNLHQLKEKKPHQTFTKWAEIYGPIYTIRTGSSTVVVLNSAQVAKEAMIAKFSSISTRKLSKALSALTRDKTMV ATSDYGDFHKMIKRYIMTFMLGTSGQKQFRDTRNMVDNMLNTFHTLLMDDPNSPLNFREVFKNELFRLSLVQALGEDVSSIYVEEYGKVISKEEIYKATVVDMMMCAIEVDWRDFFPYLSWIPNRT FETRVLTTEARRTTVMQALIKQQKERIARGETRISYLDFLLAENTLTDEQLLMLVWEAVIEAADTTLVTTEWAMYEIAKHPEKQEYLYQEIQKVCGNKTVTEDHLPELPYLNAVFHETMRRHSPVPL VPPRLVHENTNLAGYEVPAGTEIIINLYGCNMNKNDWAEPEEWKPERFLDGRFEAVDMHKTMAFGAGRRACAGSMQAMNISCTAIGRFVQEFAWRLEEGDEDKVDTIQLTTNRLYPLHVYLAPRGRK.

[0067] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Use of the ZmKO1 gene in any of the following: (1) Application in inhibiting the flowering and pollen shedding of corn spikelets; (2) Application in producing male sterility in maize; (3) Application in maize male sterile hybrid seed production; The ZmKO1 gene is the Zm00001eb385100 gene in the reference genome Zm-B73-REFERENCE-NAM-5.0 in the EnsemblPlants database, and its gene number in the NCBI database is 100274015.

2. Use of the protein expressed by the ZmKO1 gene according to claim 1 in any of the following: (1) Application in inhibiting the flowering and pollen shedding of corn spikelets; (2) Application in producing male sterility in maize; (3) Application in maize male sterile hybrid seed production.

3. Use of the recombinant vector comprising the ZmKO1 gene according to claim 1 in any of the following: (1) Application in inhibiting the flowering and pollen shedding of corn spikelets; (2) Application in producing male sterility in maize; (3) Application in maize male sterile hybrid seed production.

4. The use according to any one of claims 1 to 3, characterized in that Genetic engineering technology is used to change the DNA sequence or protein sequence of the ZmKO1 gene or regulate the transcriptional expression or protein abundance of the ZmKO1 gene, so that the male spikelets of corn cannot bloom and shed pollen, making the corn plants male sterile for corn male sterile hybrid seed production.

5. A method for cultivating a maize male sterile line, characterized in that: The method comprises: (1) using genetic engineering methods to change the transcript sequence or protein sequence of the ZmKO1 gene described in claim 1 or the expression level of the ZmKO1 gene transcription level or protein level in the recipient plant, and screening out male sterile lines from the offspring of the recipient plant; or (2) using gene editing technology to perform gene editing on the ZmKO1 gene described in claim 1 in the corn recipient plant, and screening out corn male sterile lines from the offspring of the recipient plant; or (3) using backcrossing to introduce the ZmKO1 gene described in claim 1 that can cause male sterility into corn materials with different genetic backgrounds, and creating corn male sterile lines with different genetic backgrounds.

6. A method for producing corn male sterile hybrid seeds using the corn male sterile line cultivated by the method of claim 5, characterized in that: include: Corn male sterile hybrid seed production can be achieved by using corn male sterile line as the female parent and fertile corn inbred line as the male parent and planting them according to hybrid seed production.

7. The method according to claim 6, wherein When the maize male sterile line reproduces, the fertility of the maize male sterile line is restored by exogenously spraying gibberellin or an agricultural growth regulator containing gibberellin during the silking period, thereby completing the self-pollination reproduction of the sterile line.

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