Gene ZmKO1 for regulating flowering and pollination of maize male flower spikelet and application of gene ZmKO1 in maize male sterility hybrid seed production

Abstract

The invention discloses a gene ZmKO1 for regulating flowering and pollination of corn male flower spikelet and application of the gene ZmKO1 in corn male sterility hybrid seed production, and belongs to the field of plant genetic engineering. The invention provides a gene ZmKO1 for regulating and controlling flowering and pollination of maize male flower spikelet, and the phenomenon of nuclear male sterility can be caused by mutation of the gene ZmKO1. It is also clear that on the basis of the ZmKO1 gene locus, a CRISPR-Cas9 technology or a mutant gene backcross transformation technology is utilized, and cultivation of the maize nuclear male sterile line can be achieved. The invention also finds that in the spinning period of the sterile line, the male flower fertility of the sterile line can be recovered by spraying exogenous gibberellin to tassels, normal selfing propagation can be realized, and the sterile line can be economically and efficiently maintained as a maintainer line. Therefore, the cell nucleus male sterile line created based on the ZmKO1 gene locus has the characteristic of dual purposes and can be used for maize male sterility hybrid seed production, and the invention also provides a technical system for realizing maize two-line method sterile hybrid seed production by utilizing ZmKO1 gene mutation.

Description

Technical Field

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

[0002] Corn is one of the most important food crops in the world. In the 1830s, the application of hybrid vigor to corn promoted the rapid increase of corn yield. Hybrid vigor is a common genetic phenomenon in the biological world, which refers to the phenomenon that the heterozygote is superior to the two parents in one or more traits. As a monoecious crop with different flowers, corn has made the application of hybrid vigor in corn production very successful. At present, the seeds used for corn production are mainly hybrids. In order to ensure the purity of hybrids, in the process of corn hybrid seed production, the female parent needs to be emasculated to prevent the female parent line from self-pollination, so that the female parent line only accepts the male parent line pollen to complete hybrid seed production. The quality of emasculation largely determines the yield and purity of hybrids and is a key link in seed production.

[0003] Maize emasculation methods include manual emasculation, mechanical emasculation, chemical emasculation, and the use of male sterile lines. The potential risks of chemical emasculation agents, including incomplete pollen lethality, reduced female fertility, and ecological environmental pollution, limit their large-scale application. At present, manual and mechanical emasculation are still the main emasculation methods used in commercial corn hybrid production. Artificial emasculation is highly controllable, widely applicable, and simple to operate, and is the main emasculation method used in my country's corn hybrid seed production. However, manual emasculation is time-consuming and labor-intensive, and the cost of emasculation is rising year by year. At the same time, manual emasculation often leads to untimely and incomplete emasculation, which affects the quality of seed production. With the acceleration of urbanization, rural labor continues to flow to cities and towns and non-agricultural industries, and the problem of manual emasculation will exist for a long time and become more and more serious. In recent years, many seed companies have also tried mechanical emasculation, but the equipment investment is large and the emasculation quality is unstable. In addition, mechanical emasculation causes great damage to plants, reduces seed production, and limits the large-scale application of mechanical emasculation. Therefore, emasculation is the main problem of corn hybrid seed production in my country.

[0004] Using male sterile lines as female parents for hybrid seed production, the female parents do not need to be demasculine, which can save demasculine labor, reduce seed production costs, and ensure the purity of hybrids. Plant male sterility is divided into cytoplasmic male sterility (CMS) and genic male sterility (GMS). However, there are several problems that have not been effectively overcome in the maize CMS sterile seed production system, including low genetic diversity between sterile lines and restorer lines, susceptibility of sterile cytoplasmic materials to specialized infection by pathogens, and the stability of abortion and fertility recovery affected by the environment and genetic background, which limits its wide application. GMS genetic resources are rich in stable and thorough abortion, most of which are controlled by recessive single genes, and fertility recovery is simple, which is an ideal sterile type for sterile seed production. However, due to the genetic characteristics of genic nuclei, GMS cannot be efficiently reproduced and it is difficult to meet the needs of seed production, which hinders the application of GMS in commercial hybrid seed production of maize.

[0005] To solve the problem of the inability to efficiently reproduce corn GMS, Pioneer Company developed a "seed production technology" (SPT) based on recessive nuclear 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 nuclear 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 "multi-control sterility system" developed subsequently can reduce the transgenic drift frequency to 1 / 8-1 / 7 of SPT, 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 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), whose fertility conversion is mainly controlled by environmental conditions such as temperature or light length, can be used as a sterile line under specific environmental conditions; under other environmental conditions, it can be used as a maintainer line for self-fertilization of sterile lines to achieve the maintenance of sterile lines. Through the combination of one line for two purposes and a restorer line, two-line hybrid seed production can be achieved. At present, the two-line seed production technology has been successfully applied to a certain area in rice hybrid seed production, mainly due to the discovery and utilization of a series of environment-sensitive genic male sterile materials in rice, especially the discovery and utilization of temperature-sensitive genic male sterile materials. However, the two-line seed production technology of corn is currently only in the exploratory stage. Its limiting factor is that there are few environmental genic male sterile materials found in corn, and the fertility conversion is incomplete and unstable. Therefore, collecting, creating and screening new materials or new genes suitable for corn two-line seed production technology can accelerate the innovation of corn hybrid seed production technology.

[0007] In the early stage, the inventors screened a mutant in the corn mutagenesis population. During the silking and pollination period, the elongation of the filaments of the male spikelets was blocked, the glume of the male spikelets did not crack, and the anthers could not be exposed to shed pollen, showing male sterility. Genetic analysis showed 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 mutant def1 had normal I2-IK staining and normal morphology, and the fertility of the female spike and other plant traits were normal. During the silking period, spraying gibberellin can make the filaments of the male spikelets of the mutant def1 elongate normally, the glume cracks normally, and the anthers can be exposed normally to shed pollen, achieving self-fertilization of the mutant, and the fruiting rate is not significantly different from that of the wild type. The cloning of the DEF1 gene was completed through map-based cloning and transgenic function verification. The DEF1 gene locus is Zm00001eb385100 (reference genome is Zm-B73-REFERENCE-NAM-5.0), and the annotation is the endo-kaurene oxidase 1 gene ZmKO1. At present, there is no report on the function of ZmKO1 gene in regulating the elongation of filaments of maize male spikelets, the flowering and pollen shedding of male spikelets, or the fertility of male flowers, nor is there any report on the application of ZmKO1 gene in the creation of maize male sterile lines and sterile hybrid seed production. Summary of the invention

[0008] The purpose of the present invention is to provide a gene ZmKO1 for regulating 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. The CRISPR-Cas9 technology or mutant gene backcrossing technology can be used to cultivate maize nuclear male sterile lines, and then the maize male sterile lines are 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 male spikelets;

[0012] (2) Application in regulating the opening of the husk of maize male spikelets;

[0013] (3) Application in regulating the fertility of maize male flowers;

[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 Ensembl Plants 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 embodiment of the present invention is as shown in any one of SEQ ID NOs: 1-3, and the nucleotide sequence of the ZmKO1 gene can 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 male spikelets;

[0019] (2) Application in regulating the opening of the husk of maize male spikelets;

[0020] (3) Application in regulating the fertility of maize male flowers;

[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, and the protein sequence can also be a derived protein sequence having the same function as the amino acid sequence shown in SEQ ID NO: 4 by replacing and / or deleting and / or adding one or more amino acid residues.

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

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

[0025] (2) Application in regulating the opening of the husk of maize male spikelets;

[0026] (3) Application in regulating the fertility of maize male flowers;

[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 that the filament length of the maize male spikelet is reduced and / or the husk is closed and / or the male flower is sterile.

[0029] More preferably, the gene editing method in the embodiment of the present invention is: inserting or deleting bases in the target sequence of the second exon of the ZmKO1 gene, and the sgRNA sequence for gene editing is 5'-GTCTCTGGTTTACCCCTGAT-3'. For example: deleting the 17th base of the target sequence targeted by the sgRNA (i.e., the sequence complementary to the sgRNA), or continuously deleting 16bp bases from the 6th base of the target sequence, or inserting a T base between the 16th and 17th bases of the target sequence. However, the present invention is not limited to the above-mentioned target points, and 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 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 corn male sterile hybrid seed production using a corn male sterile line cultivated by the method, which is characterized by comprising: using the corn male sterile line as a female parent and a fertile corn inbred line as a male parent, and planting them according to hybrid seed production to achieve corn male sterile hybrid seed production.

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

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

[0034] The present invention provides a gene ZmKO1 for regulating the flowering and pollen shedding of maize male spikelets, the nucleotide sequence of the protein coding region of the 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 determines that the mutation of the ZmKO1 gene can cause the male spikelet husk to be unable to open, the anther to be unable to expose and shed pollen, and exhibits the phenomenon of nuclear male sterility. The present invention also clarifies that based on the ZmKO1 gene site, the CRISPR-Cas9 gene editing technology or the mutant gene backcrossing breeding technology can be used to cultivate maize nuclear male sterile lines. The present invention also finds that during the silking period of the sterile line, spraying exogenous gibberellin on the male spike can restore the fertility of the male flowers of the sterile line, the husk of the sterile plant can open normally, the anther can be exposed and shed pollen, and it can be self-fertilized normally, and as a maintenance line, the economical and efficient maintenance of the sterile line can be achieved. Therefore, the nuclear male sterile line created based on the ZmKO1 gene locus has the characteristics of one line with two uses. 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 drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

[0036] Figure 1 The phenotypic identification results of the maize male sterile mutant def1 and the wild type WT, where 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 pollination period; C is the performance of male spikelets of the wild type WT and mutant def1 during the pollination period; D is the difference in filament elongation of male spikelets of the wild type WT and mutant def1 during the pollination 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 The promoting effect of gibberellin GA3 on flowering and pollination of male spikelets of mutant def1; A is the flowering and pollination performance of male flowers and the self-pollination and fruiting of female spikelets of wild type WT; B is the flowering and pollination performance of male flowers and the self-pollination and fruiting of female spikelets of mutant def1 after spraying 50 mg / L gibberellin GA3;

[0039] Figure 4 To verify the function of 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 position of three gene-edited strains; C is the protein sequence variation characteristics of Zm00001eb385100 gene in the gene-edited strain; D is the male sterility performance of the gene-edited strain and the fertility restoration of the edited strain by GA3; E is the local amplification of D; F is the result of allelic determination;

[0040] Figure 5 To use the mutant def1 to carry out backcross breeding of male sterility traits in inbred lines; A is the backcross breeding process; B is the male flower fertility phenotype of the maize inbred line Jing 92; C is the sterility phenotype of the sterile line Jing 92A cultivated by backcross breeding; D is the male flower flowering and pollen shedding phenotype of Jing 92A after spraying 50 mg / L gibberellic acid;

[0041] Figure 6 In order to carry out two-line sterile hybrid seed production using the maize male sterile dual-purpose line created by using the ZmKO1 gene, def1 / def1 represents the genotype of the sterile line, DEF1 / DEF1 represents the genotype of the paternal inbred line, among which 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0044] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content 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 of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary 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 from the Maize Research Institute of Sichuan Agricultural University.

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

[0049] Example 1

[0050] 1. Cloning of 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 husks of the WT male flower spikelets can open normally, and the anthers can be exposed and shed pollen normally, while the husks 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, 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 1Middle D); During 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 wild-type WT self-pollinated ears ( 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. 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 segregation 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 by polymorphic molecular marker positioning 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, and 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 inserted 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 was named ZmKO1-82), which ultimately led to frameshift mutations and premature termination of the protein sequence ( Figure 2 (middle B).

[0053] During the silking and pollination period, spraying 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 fruiting rate is not significantly different from that of the wild type ( Figure 3 ). Furthermore, the inventors found that spraying gibberellins GA1, GA4 and GA7 respectively can also restore the mutant fertility. Exogenous spraying of gibberellic acid (active ingredient content 75%, produced by Shanghai Tongrui Biotechnology Co., Ltd., spraying concentration can be selected from 25 to 50 mg / L), a plant growth regulator commonly used in agricultural production, on the mutant can also significantly restore def1 fertility and make it bloom 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 types of edited non-transgenic homozygous mutant strains were screened, namely KO#1 with 1bp 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 1bp deletion (T base deleted at the 17th base of the target sequence), and KO#3 with 16bp deletion (16bp bases were continuously deleted starting from the 6th base of the target sequence). Figure 4 B), all three mutation types lead to frameshift mutation and premature termination of ZmKO1 gene protein translation ( Figure 4 (C). Field phenotypic observations revealed that compared with the wild-type B104, the male spikelets of the three strains KO#1, KO#2, and KO#3 were unable to bloom and shed pollen, but after spraying with gibberellin GA3, they became male fertile, showing the same characteristics as the def1 mutant ( Figure 4 D and E). 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 bloomed and shed pollen normally, while the male flowers of the F1 plants hybridized with def1 and KO#1 had closed glume and could not bloom and shed pollen ( Figure 4 F) in the middle shows 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 ZmKO1 gene to create male sterile maize line

[0056] There are two methods to create maize male sterile lines using the ZmKO1 gene. The first is to use the mutant def1 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, and the sterile line is quickly completed through backcrossing combined with molecular marker-assisted selection and phenotypic selection ( 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 C), spraying gibberellic acid can promote 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 through CRISPR-Cas9 gene site-directed editing technology. Figure 4As shown in A, the second exon "GTCTCTGGTTTACCCCTGAT" sequence was used as sgRNA, constructed into a gene editing vector, transformed into Agrobacterium EHA105 competent cells, and genetic transformation of the inbred line B104 was performed through Agrobacterium-mediated embryogenic callus. Three types of editing were identified in the T0 generation ( Figure 4 B and C) were backcrossed with B104, and the homozygous mutant non-transgenic lines of the above three editing types were obtained through editing site genotype identification, transgenic screening and self-fertilization. They showed male sterility, and the fertility returned to normal after spraying gibberellin GA3 or gibberellic acid ( Figure 4 D and E).

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

[0058] The premise of whether the maize nuclear male sterile line can be applied to commercial hybrid seed production is whether the sterile line can be propagated economically and efficiently. The male sterile line created by the present invention based on the ZmKO1 gene can effectively restore its fertility by spraying gibberellins (such as GA3, GA1, GA4, or GA7) or agricultural growth regulators containing gibberellins. The male flowers of the sterile line bloom and shed pollen normally, and can be used as a maintenance line to achieve economic and efficient propagation of sterile line seeds ( Figure 6 In the 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 (B) Therefore, the maize male sterile line created by using the ZmKO1 gene can realize the 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 only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of ZmKO1 gene in any of the following: (1) Application in regulating the development of filaments in maize male spikelets; (2) Application in regulating the opening of the husk of maize male spikelets; (3) Application in regulating the fertility of maize male flowers; (4) 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 regulating the development of filaments in maize male spikelets; (2) Application in regulating the opening of the husk of maize male spikelets; (3) Application in regulating the fertility of maize male flowers; (4) 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 regulating the development of filaments in maize male spikelets; (2) Application in regulating the opening of the husk of maize male spikelets; (3) Application in regulating the fertility of maize male flowers; (4) 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 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 male flower spikelet of corn and / or close the husk and / or make the male flower sterile.

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 of the recipient plant or the expression level of the transcription level or protein level of the ZmKO1 gene, and screening out male sterile lines from the offspring of the recipient plant; or (2) using gene editing technology to edit the ZmKO1 gene of 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 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 line is used as the female parent, and fertile corn inbred line is used as the male parent. The two are planted according to hybrid seed production to achieve corn male sterile hybrid seed production.

7. The method according to claim 6, characterized in that When the male sterile corn line is propagated, the fertility of the male sterile corn line is restored by spraying gibberellin or an agricultural growth regulator containing gibberellin in the silking period, thereby completing the self-pollination propagation of the sterile line.

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