Application of maize gene ZmABCG20 in regulating crop male fertility

Abstract

This invention provides the application of the maize gene ZmABCG20 in regulating male fertility in crops. The genomic DNA sequence of ZmABCG20 in maize variety B73 is shown in SEQ ID NO:1, and the encoded protein sequence is shown in SEQ ID NO:3. This invention also provides a mutant of the ZmABCG20 gene, zmabcg20-1, and its applications. The mutant gene sequence is shown in SEQ ID NO:7. Furthermore, a molecular marker identification method for this mutant gene is provided. The pollen development control gene, mutant, and molecular markers provided by this invention can be applied to crop hybrid breeding and hybrid seed production.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and molecular breeding, specifically, it relates to the application of the maize gene ZmABCG20 in regulating male fertility in crops. Background Technology

[0002] Male sterility mutations in plants are a very common phenomenon in nature, with male sterility mutants found in at least 617 species across 43 families and 162 genera. Genetically, plant male sterility is divided into three main categories: nuclear male sterility, cytoplasmic male sterility, and nuclear-cytoplasmic interaction male sterility. 1) Nuclear male sterility arises from mutations in nuclear genes, including dominant and recessive mutations, and can occur through sporophyte and gametophyte gene mutations. Dominant and gametophyte gene mutations can only be inherited through female gametes, while recessive mutations can be inherited through both female and male gametes, following Mendel's laws. Several recessive nuclear male sterility genes in sporophytes have been cloned, such as ms2 in Arabidopsis, ms45 in maize, and mil1 in rice (Aarts et al., 1997, The Arabidopsis MALE STERILITY 2 protein shares similarity with reductases in elongation / condensation complexes, Plant Journal, 12:615-623; Albertsen, 2006, Male tissue-preferred regulatory sequences of MS45 gene and method of using same, patent number: US7154024B2; Hong et al., 2012, Somatic and reproductive cell development in rice anther is regulated by a putative glutaredoxin, Plant Cell, 24:577-588); some recessive nuclear male sterility genes in gametophytes have also been cloned, such as two microspore mutants of Arabidopsis, sidecar pollen and gemini pollen (Oh et al., 2010, The SIDECAR POLLEN gene encodes a microspore-specific LOB / AS2domain protein required for the correct timing and orientation of asymmetric cell division, Plant Journal, 64:839-50;Park et al., 1998, The Arabidopsis thaliana gametophytic mutation gemini pollen1 disrupts microspore polarity, division asymmetry and pollen cell fate, Development, 125:3789-99); A sporophytic dominant nuclear sterility gene MS44 was also cloned in maize (Cigan and Albertsen, 1998, Reversible nuclear genetic system for malesterility in transgenic plants, US5750868); 2) Cytoplasmic male sterility is controlled by cytoplasmic genes and does not have a corresponding nuclear restoration gene, belonging to maternal inheritance; 3) Cell nucleus-cytoplasmic interaction male sterility is jointly controlled by cytoplasmic genes and nuclear genes, its essence being the result of incompatibility between cytoplasmic and nuclear genetic material. Sterile cytoplasm is caused by some mutated mitochondrial genes, but has a corresponding nuclear restoration gene that can suppress sterile cytoplasmic genes. A sterile cytoplasmic gene can produce a novel protein that can affect normal mitochondrial function (Chen and Liu, 2014, Malesterility and fertility restoration in crops, Annu Rev Plant Biol, 65:5.1-5.28). Regarding fertility restoration genes, Rf-1, Rf-2, Rf-4, and Rf-5 genes have been cloned in rice (Komori et al., 2004, Map-based cloning of a fertility restorer gene, Rf-1, in rice (Oryzasativa L.), Plant Journal, 37:315-325; Itabashi et al., 2011, The fertility restorer gene, Rf2, for Lead Rice-type cytoplasmic male sterility of rice encodes amitochondrial glycine-rich protein, Plant Journal, 65:359-367).Tang et al., 2014, Therice restorer Rf4 for wild-abortive cytoplasmic male sterility encodes a PPRprotein that functions in reduction of WA352transcripts, Molecular Plant, 7:1497-500; Hu et al., 2012, The rice pentatricopeptide repeat protein RF5restorersfertility in Hong-Lian Cytoplasmic male-sterile lines via a complex with the glycine-rich protein GRP162, Plant Cell, 24:109-22). ;

[0003] Corn has become the world's and my country's largest food crop, an important raw material for feed, food processing, and bioenergy, and one of the most consumed vegetables abroad. Currently, almost all corn grown in China is hybrid. Corn hybridization is mainly achieved through artificial emasculation, which requires a large amount of labor and is costly; moreover, emasculation damages the top leaves of the corn, resulting in seed production losses. Using male-sterile lines for seed production can solve the problems caused by artificial emasculation. However, the cytoplasmic male-sterile lines previously used in corn have some drawbacks: firstly, because cytoplasmic male-sterile lines require specific restorer genes to restore fertility, the utilization rate of germplasm resources is very low, limiting the efficiency of breeding superior varieties; secondly, the fertility of some sterile lines is unstable, and fertility can be restored under certain conditions, affecting the purity of hybrids; finally, due to the single cytoplasmic genotype, corn leaf spot diseases have broken out, directly leading to the withdrawal of cytoplasmic male-sterile technology from the market. Ordinary nuclear male sterility can avoid these problems. If applied to corn, it can not only save the labor costs required for artificial emasculation but also increase seed production.

[0004] Plant ABC protein family is a class of membrane transport proteins located on the cell membrane, responsible for the transmembrane transport of metabolites; ABCG transport proteins are the largest subfamily within this family. ABCG proteins can be mainly divided into two categories based on their structural characteristics: full-size proteins contain two nucleotide-binding domains and two transmembrane regions, which can independently form a complete transmembrane transport structure to complete substrate transport; half-size proteins have only one nucleotide-binding domain and one transmembrane region, and need to bind with another half-size protein molecule to form a complete transport unit (Verrier et al., 2008, Plant ABC proteins – a unified nomenclature and updated inventory. Cell, Trends in Plant Science, 13(4):151-159.). The AtABCG26 gene in Arabidopsis thaliana and the orthologous gene OsABCG15 in rice encode a transmembrane transport protein for the precursor of sporopollenin, a pollen wall component. It is expressed in the anther villous layer, transporting the sporopollenin precursor from villous layer cells to the anther chamber, where it is synthesized into sporopollenin on the pollen cell wall. The mutant atabcg26 exhibits extremely low pollen count and male fertility; the rice osabcg15 mutant is completely male sterile and has no pollen; in addition, the rice OsABCG26 mutant also exhibits complete male sterility, with a phenotype similar to osabcg15 (Zhao et al., 2016, ATP binding cassette G transporters and plant male reproduction. Plant Signal and Behavior, 11(3):e1136764.doi:10.1080 / 15592324.2015.1136764). Through genomic bioinformatics analysis, Pang et al. (Pang et al., 2013, Inventory and general analysis of the ATP-binding cassette (ABC) gene superfamily in maize (Zea May L.). Gene, 2013, 526(2): 411-428) identified 54 ABCG genes in maize, but so far no genes related to male fertility have been found. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the maize gene ZmABCG20 in regulating male fertility in crops.

[0006] Another objective of this invention is to provide a mutant of the maize gene ZmABCG20, zmabcg20-1, and its applications.

[0007] To achieve the objectives of this invention, in a first aspect, this invention provides the application of the maize gene ZmABCG20 in regulating male fertility in crops, wherein the cDNA sequence of the gene ZmABCG20 is as follows:

[0008] i) The nucleotide sequence shown in SEQ ID NO:2;

[0009] ii) A nucleotide sequence of the nucleotide sequence shown in SEQ ID NO:2 that has been substituted, deleted and / or added with one or more nucleotides and expresses a protein with the same function;

[0010] iii) A nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO:2 under stringent conditions and expresses a protein with the same function, wherein the stringent conditions are hybridization at 65°C in 0.1×SSPE containing 0.1% SDS or 0.1×SSC containing 0.1% SDS, followed by washing the membrane with the same solution; or

[0011] iv) Nucleotide sequences that have more than 85% homology with the nucleotide sequences of i), ii) or iii) and express the same functional protein.

[0012] The aforementioned application, where regulation refers to inducing male fertility in crops, includes:

[0013] 1) To enable crops to contain the ZmABCG20 gene; or

[0014] 2) It enables crops to express the protein encoded by the ZmABCG20 gene.

[0015] This invention first involves treating maize cultivar Jingkenuo 2000 seeds (M0 generation) with cobalt-60 radiation mutagenesis, and planting the treated seeds to obtain M1 generation plants. The M1 generation plants are then self-pollinated to produce seeds (M2 generation), which are then planted to create M2 generation plants. Morphological, histological, and genetic identification of the M2 generation plants is performed to screen for sterile plants. Subsequently, gene sequencing and DNA sequence analysis are conducted on the sterile plants for molecular-level verification. Finally, homozygous sterile single plants are obtained and used for hybridization breeding and biotechnology research.

[0016] The maize ZmABCG20 gene (pollen development control gene) provided by this invention exhibits complete male sterility upon mutation. Its nucleotide sequence is shown in SEQ ID NO:1 or SEQ ID NO:4; its coding region DNA sequence is shown in SEQ ID NO:2 or SEQ ID NO:5; and its encoded protein sequence is shown in SEQ ID NO:3 or SEQ ID NO:6.

[0017] Secondly, this invention provides the application of the maize gene ZmABCG20 in the preparation of transgenic plants. For example, a recombinant expression vector carrying the ZmABCG20 gene cDNA or genomic sequence is transferred into wild-type maize callus tissue. The transformed material undergoes co-culture-screening-differentiation-rooting-transgenic seedling hardening and transplanting to obtain transgenic plants. Then, the transgenic maize is crossed with male-sterile maize to restore the fertility of male-sterile maize.

[0018] Thirdly, the present invention provides the application of the maize gene ZmABCG20 in restoring fertility in male-sterile plants, wherein the male-sterile trait is caused by a mutant of the gene.

[0019] Fourthly, this invention provides a method for preparing male-sterile transgenic maize by inhibiting the activity of the maize ZmABCG20 gene. By using techniques such as gene silencing, gene suppression, gene knockout, or directed gene mutation, the protein activity level of the ZmABCG20 gene in maize is reduced in terms of transcription, translation, or post-translation, thereby obtaining male-sterile transgenic maize.

[0020] For example, an RNAi sequence carrying the cDNA sequence of the target gene ZmABCG20 can be operatively linked to a constitutive promoter or a flower organ-specific expression promoter and transferred into plant callus tissue. The transformed material is then subjected to co-culture, screening, differentiation, rooting, and hardening-up of transgenic seedlings and transplanted to obtain male-sterile transgenic maize.

[0021] In one specific embodiment of the present invention, the target DNA sequence of the RNAi action is shown in SEQ ID NO:23.

[0022] Fifthly, the present invention provides the application of biological materials obtained by the above methods in crop improvement breeding and seed production.

[0023] Sixthly, this invention provides the application of the maize gene ZmABCG20 in crop improvement breeding and seed production.

[0024] The aforementioned application allows plants containing or expressing the ZmABCG20 gene, or plants inactivated by the ZmABCG20 gene according to the above method, to be hybridized with the same crop having excellent agronomic traits.

[0025] In this invention, the crop is a self-pollinating or cross-pollinating crop, including but not limited to corn, wheat, or rice, with corn being preferred.

[0026] In this invention, the superior agronomic traits include, but are not limited to, increased yield, improved quality, resistance to diseases and pests, stress resistance, and lodging resistance.

[0027] In a seventh aspect, the present invention provides an inhibitor for inhibiting the activity of the ZmABCG20 gene, said inhibitor being selected from at least one of shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA / DNA, low molecular weight compounds, peptides, antibodies, etc.

[0028] Eighthly, the present invention provides an expression cassette, expression vector, or cloning vector containing an inhibitor of the aforementioned nucleic acid molecule.

[0029] Ninthly, the present invention provides a mutant gene zmabcg20-1 of the maize gene ZmABCG20, the nucleic acid sequence of which is:

[0030] i) A mutant gene formed by the deletion of 4 TGCA bases from position 326 to 329 after the start codon of the maize gene ZmABCG2 nucleic acid sequence;

[0031] ii) The nucleotide sequence shown in SEQ ID NO:7;

[0032] iii) A nucleotide sequence of the sequence shown in i) or ii) that has been substituted, deleted and / or added with one or more nucleotides and expresses the same functional protein, and contains a 4-base TGCA deletion at the equivalent position to the gene ZmABCG20.

[0033] iv) A nucleotide sequence that hybridizes to the sequence shown in i) or ii) under stringent conditions and expresses a protein with the same function, and contains a 4-base TGCA deletion at the equivalent position to the gene ZmABCG20; the stringent conditions are hybridization at 65°C in 0.1×SSPE containing 0.1% SDS or 0.1×SSC containing 0.1% SDS, followed by washing the membrane with the solution; or

[0034] v) A nucleotide sequence that has more than 85% homology with i) or ii) and expresses the same functional protein, and contains a 4-base TGCA deletion at the equivalent position to gene ZmABCG20.

[0035] The coding region DNA sequence of the maize gene zmabcg20-1 is shown in SEQ ID NO:8. The amino acid sequence of the protein encoded by the maize gene zmabcg20-1 is shown in SEQ ID NO:9, or an amino acid sequence with equivalent function formed by substitution, deletion, or addition of one or more amino acids.

[0036] In a tenth aspect, the present invention provides the application of gene zmabcg20-1 in regulating maize fertility, the application including:

[0037] 1) Make crops contain the zmabcg20-1 gene; or

[0038] 2) It enables crops to express the protein encoded by the zmabcg20-1 gene.

[0039] The aforementioned application caused maize containing or expressing the mutant zmabcg20-1 gene to exhibit recessive male sterility.

[0040] Eleventhly, this invention provides the application of gene zmabcg20-1 in maize improvement breeding and seed production.

[0041] The aforementioned application involves hybridizing maize containing or expressing the mutant zmabcg20-1 gene with maize exhibiting superior agronomic traits.

[0042] In a twelfth aspect, the present invention provides an expression cassette, expression vector, or cloning vector comprising a nucleic acid sequence containing the gene zmabcg20-1 as described.

[0043] In a thirteenth aspect, the present invention provides engineered bacteria, host cells, or transgenic cell lines containing the gene zmabcg20-1, or the expression cassette, expression vector, or cloning vector.

[0044] In a fourteenth aspect, the present invention provides the use of biological materials containing or expressing the said gene zmabcg20-1 in the preparation of transgenic maize.

[0045] In a fifteenth aspect, the present invention provides a promoter specific to male-flowered or hermaphroditic plant spikelets, said promoter being:

[0046] i) The nucleotide sequence shown in SEQ ID NO:12;

[0047] ii) A nucleotide sequence with one or more nucleotides substituted, deleted, and / or added to the nucleotide sequence shown in SEQ ID NO:12 and having the same function;

[0048] iii) A nucleotide sequence that hybridizes to the sequence shown in SEQ ID NO:12 under stringent conditions, wherein the stringent conditions are hybridization at 65°C in 0.1×SSPE containing 0.1% SDS or 0.1×SSC containing 0.1% SDS, followed by washing the membrane with the same solution; or

[0049] iv) Nucleotide sequences that have more than 85% homology with and have the same function as nucleotide sequences of i), ii) or iii).

[0050] In a sixteenth aspect, the present invention provides an expression cassette, expression vector, or cloning vector comprising a nucleic acid containing the sequence shown in SEQ ID NO:12.

[0051] In a seventeenth aspect, the present invention provides engineered bacteria and transgenic cell lines containing the specific promoter or the expression cassette or vector.

[0052] In an eighteenth aspect, the present invention provides the application of the specific promoter in regulating the expression of downstream genes.

[0053] In a nineteenth aspect, the present invention provides the use of the specific promoter in the preparation of transgenic plants.

[0054] For example, by operatively linking a promoter sequence to a target gene, transforming a target plant with the resulting construct, the promoter drives the target gene to be expressed specifically in male spikelets or in spikelets of hermaphroditic plants.

[0055] In a twentieth aspect, the present invention provides a DNA molecular marker related to male fertility in maize. The DNA molecular marker is located at bases 326-329 after the start codon of the maize gene ZmABCG20 nucleic acid sequence, and the sequence is TGCA. Maize lines with these four base deletions exhibit recessive male sterility.

[0056] In a twentieth aspect, the present invention provides primers for specifically amplifying the DNA molecular marker, comprising:

[0057] Upstream primer 3326_F1: 5′-CCAGACGAGGGCAGACCAG-3′ (SEQ ID NO:10)

[0058] Downstream primer 3326_R1: 5′-GATCTCGCCAGGGTCCACA-3′ SEQ ID NO:11)

[0059] In a twentieth aspect, the present invention provides a detection reagent or kit containing the primers 3326_F1 and 3326_R1.

[0060] In a twentieth aspect, the present invention provides the application of the DNA molecular marker, the primer, or the detection reagent or kit in marker-assisted breeding of maize.

[0061] In a twenty-fourth aspect, the present invention provides the application of the DNA molecular marker, the primer, or the detection reagent or kit in the identification or selection of male-sterile maize germplasm resources. The specific method is as follows:

[0062] Genomic DNA was extracted from the maize sample and PCR amplification was performed using primers 3326_F1 and 3326_R1. The amplification products were detected by electrophoresis. If a characteristic band of 79 bp appeared in the amplification product, the maize sample was fertile and the corresponding ZmABCG20 genotype was wild-type. If a characteristic band of 75 bp appeared in the amplification product, the maize sample was a male-sterile variety and the corresponding ZmABCG20 genotype was zmabcg20-1 mutant. If the amplification product showed two bands of 79 bp and 75 bp, the maize sample was heterozygous.

[0063] In a twentieth aspect, the present invention provides the application of the DNA molecular marker, the primer, or the detection reagent or kit in the ZmABCG20 genotyping of maize.

[0064] The advantages of the ZmABCG20 gene provided by this invention are as follows:

[0065] 1) The discovery that the ZmABCG20 mutation can cause male sterility phenotype in maize is of great significance for the study of the utilization and function of this gene, as well as the study of the regulatory mechanism of male fertility in maize.

[0066] 2) The ZmABCG20 mutation only affects male fertility, causing complete male infertility, but has no effect on female fertility or other agronomic traits. It is suitable for industrial applications such as hybridization breeding, seed production and production.

[0067] 3) ZmABCG20 is expressed only in young male spikelets of plants, exhibiting strong time and tissue specificity. Its promoter can be used to drive the specific expression of any gene in young male spikelets.

[0068] The advantages of the mutant zmabcg20-1 provided by this invention are as follows:

[0069] 1) zmabcg20-1 is the first reported ZmABCG20 mutant, which is of great significance for the utilization and functional study of this gene.

[0070] 2) This mutant only affects male fertility, causing complete male infertility, but has no effect on female fertility or other agronomic traits.

[0071] 3) This mutant is a gene deletion mutation caused by the deletion of 4 bases. There is no potential risk of fertility restoration or genetic instability.

[0072] 4) This mutant involves a 4-base deletion within the gene and will not affect the function of the adjacent genes on both sides of ZmABCG20.

[0073] 5) This mutant is a 4-base deletion mutation, which can be used to design Indel markers for high-throughput detection using conventional PCR and electrophoresis; it can also be designed as a marker for gene chip detection.

[0074] 6) The genetic background of this mutant is that of the current main cultivated varieties in China, and it can be directly used for the breeding of Chinese maize varieties without a long improvement process. Attached Figure Description

[0075] Figure 1 The images are the male flowers of the wild-type mutant and zmabcg20-1 in Example 2 of this invention, as well as the fruit spikes of zmabcg20-1.

[0076] Figure 2 The results of I2-KI staining of anthers and pollen of wild-type and mutant zmabcg20-1 in Example 3 of this invention are shown.

[0077] Figure 3 The results of real-time quantitative PCR of the ZmABCG20 gene in different young spikelets and different tissues of Jingkenuo 2000 in Example 7 of the present invention are shown.

[0078] Figure 4 This is a schematic diagram of the ZmABCG20 gene structure and the mutation site of zmabcg20-1 identified in Examples 6 and 8 of this invention.

[0079] Figure 5 Electrophoresis results of molecular marker identification of the ZmABCG20 gene in the F2 progeny of the zmabcg20-1 mutant after open pollination and self-pollination in Example 9 of this invention.

[0080] Figure 6 This is a schematic diagram of the process for constructing the RNAi vector of the ZmABCG20 gene in Example 10 of the present invention.

[0081] Figure 7 The pollen iodine staining results are those of the control and RNAi male sterile plants in Example 11 of this invention.

[0082] Figure 8 This is a technical roadmap for the crossbreeding and conversion of the zmabcg20-1 sterile gene described in Example 13 of the present invention. Detailed Implementation

[0083] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sambrook et al., Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, 2001), or as recommended by the manufacturer's instructions.

[0084] Example 1: Cobalt-60 radiation-induced mutagenesis library

[0085] In September 2015, cobalt 60 was used in Changsha. 60 Three kilograms of *C. coli* 2000 seeds (M0 generation) were irradiated with a dose of 250 Gy. The irradiated seeds were planted in fields in Yacheng District, Sanya City, Hainan Province in October 2015, and strictly self-pollinated individual plants to harvest M1 generation seeds.

[0086] 5400 M1 generation seed lines were selected, with 50 individual plants planted in each line, and planted in fields in Lingao, Hainan in February 2016. Field traits were carefully observed during the seedling, heading, flowering, and grain-filling stages to screen for various types of mutants, including those with different plant types, ear types, fertility, and yield. Individual plants of each mutant type were harvested and preserved.

[0087] Example 2: M2 generation planting and trait observation

[0088] During the heading and flowering period of the M2 generation, the morphology of the anthers was observed in the field. Anthers exhibiting abnormalities such as pale color, small size, and low pollen quantity were selected for further microscopic examination. Nine plants with fertility abnormalities were found in family number 3326; they could not release pollen normally, but their fruit setting was normal. Figure 1 The mutant's anthers were smaller and lighter yellow than the wild type, with no visible pollen. However, it showed no significant differences from the wild type in vegetative growth, heading stage, and ear type, and was selected as a candidate mutant material for further research. Based on the finally identified mutant gene (see Examples 6, 7, and 8), the mutant was named zmabcg20-1.

[0089] Example 3: Microscopic examination and genetic analysis of pollen from sterile mutants

[0090] Pollen fertility was statistically analyzed by measuring the ratio of iodine-stained to unstained pollen. Male flower morphology of zmabcg20-1 was observed under a stereomicroscope; the anthers were smaller and lighter in color than those of the wild type. Figure 2 Flowering florets were collected from the field. Anthers were removed with tweezers and gently squeezed into an iodine-potassium iodide solution (0.6% KI, 0.3% I2, w / w). The anthers were then dropped onto a glass slide, covered with a coverslip, and the pollen iodine staining was observed and photographed under a microscope. Wild-type pollen was abundant and stained blue-black, while no pollen grains were visible in the mutant. Figure 2 ).

[0091] The mutant can produce fruit normally under open pollination. Figure 1 This indicates that the mutant is a male-sterile mutant, and female ear fertility is unaffected. Open-pollinated seeds (F1) of zmabcg20-1 were harvested and sown. All F1 seeds showed normal pollen shedding after heading. Bagging and self-pollination resulted in normal seed production, and F2 seeds were harvested from a single ear. One self-pollinated F2 seed ear was sown in a row. Fertility was assessed after heading; 125 plants showed normal pollen staining with iodine, while 38 plants showed no pollen, conforming to a 3:1 segregation ratio (χ²). 2 =0.30), indicating that the infertility trait is controlled by a single recessive gene.

[0092] Example 4: Leaf Sampling and DNA Extraction

[0093] This study used the CTAB method to extract DNA from maize leaves. The specific method is as follows: Weigh approximately 0.1g of leaves and place them in a centrifuge tube. Add 600μL of CTAB extraction buffer and 5μL of RNase A, vortex to disperse, and incubate at 65℃ for 0.5hr, gently shaking 2-3 times during this period. Add an equal volume of chloroform / Tris-saturated phenol (1:1, v / v), mix well, and gently shake for 10min. Centrifuge at 10000rpm for 20min at 4℃. Transfer the supernatant to a new tube, add 1 / 10 volume of 3M sodium acetate (pH 5.2) and 0.6-1 volume of cold isopropanol. Gently shake to mix until a flocculent precipitate appears. Centrifuge at 10000rpm for 10min at 4℃. Discard the supernatant, wash the precipitate twice with 70% ethanol (v / v), air dry, add 50μL of 1×TE to dissolve the precipitate, and store at -20℃. Detect the DNA concentration using Nanodrop 2000 and dilute to 10ng / L for use as a PCR template.

[0094] Example 5: Preliminary Chromosome Localization of Candidate Genes for Male Infertility

[0095] Based on the IBM 2008 genetic map (www.maizegdb.org), SSR and Indel markers evenly distributed on all maize chromosomes were screened. Polymorphic markers among the parents of Jingkenuo 2000 were also screened. Genotyping was performed on nine zmabcg20-1 plants from Jingkenuo 2000 and M2. The PCR program was as follows: PCR reaction system: 1 μL 10× reaction buffer, 0.25 μL dNTP, 0.25 μL forward primer and 0.25 μL reverse primer, 0.5 U Taq enzyme, 1 μL 10 ng / μL template DNA, and ultrapure water to bring the total volume to 10 μL. The PCR reaction program was: denaturation at 94-98℃ for 1-3 min, followed by the following cycles: denaturation at 95℃ for 20 s, annealing at 53-58℃ for 20 s, extension at 72℃ for 30 s, for 30-40 cycles.

[0096] The reaction products were separated by electrophoresis on a 6% polyacrylamide gel. The polyacrylamide gel electrophoresis method is as follows: (1) Preparation of polyacrylamide gel: 80 mL of 6% PA gel, 250 μL (winter) / 125 μL (summer) of 10% ammonium persulfate, and 80 μL of tetramethylethylenediamine (TEMED). After shaking, pour the gel. Clean the glass plate repeatedly with detergent, wipe it clean with alcohol and let it dry. In the fume hood, coat the concave plate with 2% Repel Silane, then wipe it clean with alcohol and let it dry. Coat the other plate with 1.5 mL of 0.5% Bingding Silane (add 7.5 μL of Bingding Silane and 7.5 μL of glacial acetic acid to a 1.5 mL centrifuge tube, and add 95% ethanol to 1.5 mL). During the operation, prevent the two glass plates from contaminating each other. After drying thoroughly, assemble the glass plates and pour the gel. (2) Pre-electrophoresis: After the gel solidifies, remove the comb and wash off the gel, paying particular attention to cleaning the seams. First, put 1×TBE electrode buffer into the lower tank (cathode) of the electrophoresis tank, place the polymerized gel plate into the electrophoresis tank, and inject 0.5×TBE electrode buffer into the upper tank. Maintain a constant power of 40W-65W and perform pre-electrophoresis for about 30 minutes. Use a pipette to remove the urea and air bubbles precipitated on the gel surface and insert the comb. (3) Electrophoresis: Add 5μl of 5×Loading Buffer to the amplified product, mix, denature at 95℃ for 5 minutes, immediately transfer to ice to cool, and add 1.5-3μl to the sample well; perform electrophoresis at a constant power of 40W-65W until the bromophenol blue reaches the bottom of the electrophoresis tank. Adjust the electrophoresis time according to the molecular weight of the SSR amplified product and the distinguishability of the differential bands. (4) Silver staining: Place a glass plate with the gel in 10% glacial acetic acid fixative and shake at 65 r / min for about 30 min until all xylenenitrile is decolorized; rinse twice with distilled water for 5 min each time; place the rinsed gel plate in freshly prepared staining solution (2 g silver nitrate and 3 mL 37% formaldehyde in 2 L water) and shake at 65 r / min for 30 min; rinse the stained gel plate with distilled water for 5 s and immediately remove it for development; quickly transfer the gel plate to pre-cooled developing solution at 4℃ (30 g sodium hydroxide and 10 mL 37% formaldehyde in 2 L water) and gently shake until bands appear; place the gel plate in 10% glacial acetic acid fixative until no bubbles are generated; rinse twice with distilled water for 2 min each time; allow it to air dry at room temperature and then take a picture to save the image.

[0097] In the identification markers, the Indel marker IDP8150 located on chromosome 9 (forward primer: 5'-TGCTCGCAGGAATAGAAAGC-3'; reverse primer: 5'-GACGCAATCGACAGAGTACG-3') showed a heterozygous band in Jingkenuo 2000, while all nine zmabcg20-1 plants showed the same homozygous band, indicating that the fertility-controlling mutant gene is linked to IDP8150 and located on chromosome 9. Analysis of cloned plant male fertility-controlling genes revealed that the orthologous gene ZmABCG20 (GRMZM2G076526 / Zm00001d046537) of maize Ms45, Arabidopsis thaliana AtABCG26, and rice OsABCG15 is located on chromosome 9. We used these two genes as target genes for sequencing analysis.

[0098] Example 6 Candidate Gene Sequencing

[0099] Primers were designed based on the Ms45 and ZmABCG20 gene sequences of maize inbred line B73 to amplify the genomic DNA of wild-type Jingkenuo 2000 and zmabcg20-1. The amplified products were sequenced and then assembled to obtain complete sequences. The primer pairs used for amplifying maize ZmABCG20 were ZmABCG20_1–3, and the primer pairs used for amplifying Ms45 were Ms45_1–Ms45_4; the sequences are shown in Table 1.

[0100] Table 1. Primer pair sequences used for amplifying maize ZmABCG20 and Ms45.

[0101]

[0102]

[0103] The PCR reaction system consisted of: 1 μL 10× reaction buffer, 0.25 μL dNTPs, 0.25 μL forward primer and 0.25 μL reverse primer, 0.5 U Taq enzyme, 1 μL 10 ng / μL template DNA, and ultrapure water to bring the total volume to 10 μL. The PCR program was as follows: denaturation at 94-98℃ for 1-3 min, followed by the following cycles: denaturation at 95℃ for 20 s, annealing at 53-58℃ for 20 s, extension at 72℃ for 30 s, for 30-40 cycles. After each cycle, a final extension at 72℃ for 3-10 min was performed to terminate the reaction. A 1.5% agarose gel was prepared and electrophoresed at 5 V / cm for 30 min. The PCR products were recovered using a commercially available DNA gel extraction kit.

[0104] The recovered PCR product DNA from both wild-type and mutant strains was sequenced using an ABI 3730 sequencer, with forward and reverse primers used for sequencing. The bidirectional sequencing results were assembled using the common DNA sequence analysis software DNAman 6.0. Analysis showed that the Ms45 gene sequence of the mutant zmabcg20-1 was identical to that of the wild-type Jingkenuo 2000, with no mutations. The full-length nucleotide sequence of the ZmABCG20 gene in the mutant zmabcg20-1 is shown in SEQ ID NO:7, and it lacks 4 bases compared to Jingkenuo 2000.

[0105] ZmABCG20 is a direct homolog of OsABCG15 in rice and AtABCG26 in Arabidopsis thaliana. The mutants of the latter two also exhibit male sterility, and the rice mutant osabcg15 also lacks mature pollen.

[0106] Example 7 Tissue-specific expression of ZmABCG20

[0107] Flower spikes from different stages of the Jingkenuo 2000 maize plant were selected, from stage V7 (when the maize tassel begins to form) to stage V18 (when the maize tassel matures) (How a Corn Plant Develops. Special Report No. 48. Iowa State University of Science and Technology, Cooperative Extension Service, Ames, Iowa. Reprinted 2 / 1996), as well as young roots, stems, leaves, outer and inner glumes of male flowers, and female ears. The samples were transported in liquid nitrogen and stored at -80℃. RNA was extracted from the above tissues using the TRIzol RNA extraction kit (Invitrogen, USA), and immediately reverse transcribed into cDNA using the PrimeScript RT reagent kit (TaKaRa, Dalian) according to the instructions.

[0108] Real-time PCR using PowerUp TM SYBR TM Green Master Mix (Thermo Fisher, USA) was used for amplification and fluorescence detection using a PikoReal 96 real-time PCR instrument (Thermo Fisher, USA). The maize Actin1 gene was selected as an internal reference gene, and the amplification primers were actinI-F and actinI-R (SEQ ID NO:15-16). The amplification primers for ZmABCG20 real-time PCR were ABCG-2F and ABCG-2R (SEQ ID NO:17-18).

[0109] The quantitative real-time PCR reaction system was as follows: 5 μL SYBR Green Mix, 0.5 μL Forward Primer, 0.5 μL Reverse Primer, 1 μL cDNA, and 3 μL ultrapure water. The PCR reaction program was: 95℃ denaturation for 5 min; 95℃ denaturation for 15 s, 60℃ annealing-extension for 1 min, 40 cycles; 60℃ for 30 s. The melting curve was calculated with an initial temperature of 60℃, a final temperature of 95℃, a hold time of 1 s, and a temperature increment of 0.2℃.

[0110] The results of real-time quantitative PCR are shown below. Figure 3 The ZmABCG20 gene is expressed only in young male spikelets during stages V10-V15, with a sharp increase in expression at stage V12, and only trace amounts at other stages. ZmABCG20 expression was not detected in other tissues such as roots, stems, leaves, female spikelets, and the inner and outer glumes of male flowers. Stage V12 corresponds to the uninucleate stage of pollen, during which the exine is forming; this expression tissue and stage are consistent with the function of its homologous genes in Arabidopsis and rice.

[0111] Example 8: Transcript Sequence Analysis of ZmABCG20

[0112] In the Gramene database, the ZmABCG20 gene has two gene annotation numbers, GRMZM2G076526 and Zm00001d046537, with a total of 8 predicted transcripts. To determine the coding region of this gene, cDNA obtained from the young male spikelets of Jingkenuo 2000 was amplified using primers covering the full length of the ZmABCG20 coding region, specifically ZmABCG20_T1–T4 (sequences shown in Table 1). The products were isolated and sequenced according to the method described in Example 6. The sequencing results show that the ZmABCG20 coding region, as shown in SEQ ID NO:5, is consistent with GRMZM2G076526-T001 (SEQ ID NO:2).

[0113] Comparison of the mutant genome, the Jingkenuo 2000 genome, and the cDNA sequence revealed a deletion of 4 TGCA bases in the second exon of zmabcg20-1 at base 246 of the coding region (326 bases from the start codon in the genome sequence). This deletion resulted in a frameshift mutation after amino acid residue 82 in the translated protein, prematurely terminating translation after amino acid residue 100. The coding region sequence of the mutant gene zmabcg20-1 is shown in SEQ ID NO:8, and the encoded protein sequence is shown in SEQ ID NO:9. The genome, coding region, and protein sequences of ZmABCG20 from B73 are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; the genome, coding region, and protein sequences of ZmABCG20 from Jingkenuo 2000 are shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively. The structure of the ZmABCG20 gene and the mutation site of zmabcg20-1 are shown in […]. Figure 4 .

[0114] Example 9: Functional marker identification of the ZmABCG20 genotype in the F2 population

[0115] Based on the sequences flanking the mutation site obtained in Example 6, a pair of gene-specific primers were designed: forward primer 3326_F1, whose nucleotide sequence is shown in SEQ ID NO:10; and reverse primer 3326_R1, whose nucleotide sequence is shown in SEQ ID NO:11.

[0116] If the product size amplified using the above primer pairs is 79 bp, it indicates that the genotype of the plant being tested is wild-type; if the product size is 75 bp, it indicates that the plant being tested is a zmabcg20-1 mutant; if the product size is two bands of 79 bp and 75 bp, it indicates that the ZmABCG20 gene of the plant being tested is a heterozygous genotype of wild-type and zmabcg20-1 mutant.

[0117] In the F2 lines obtained in Example 3, wild-type and mutant phenotype plants were randomly selected, and leaf DNA was extracted. This DNA, along with the genomic DNA of Jingkenuo 2000, was amplified using the primer pairs described above. The PCR reaction system consisted of: 1 μL 10× reaction buffer, 0.25 μL dNTPs, 0.25 μL forward primer and 0.25 μL reverse primer, 0.5 U Taq enzyme, 1 μL 10 ng / μL template DNA, and ultrapure water to bring the total volume to 10 μL. The PCR reaction program was: denaturation at 94-98℃ for 1-3 min, followed by the following cycles: denaturation at 95℃ for 20 s, annealing at 53-58℃ for 20 s, extension at 72℃ for 30 s, for 30-40 cycles. The amplified products were separated by 6% polyacrylamide gel electrophoresis at a constant power of 40 W for 1 hour. After silver nitrate staining, the electrophoretic images were photographed and recorded.

[0118] See results Figure 5 The amplification product size of the wild-type control was 79 bp; the amplification product size of the sterile plants in the F2 ear rows was 75 bp; the amplification product size of all fertile plants was 79 bp, or a heterozygous band of 79 bp + 75 bp, but there was no homozygous 75 bp band. This result indicates that the mutation site described in Example 6 co-segregates with the recessive nuclear male sterility gene.

[0119] Example 10: Construction of the RNAi vector for ZmABCG20

[0120] To verify the function of the ZmABCG20 gene, an RNAi vector for this gene was constructed in this embodiment. The vector construction process is as follows: Figure 6 As shown, the specific method is as follows:

[0121] 1. The highly specific cDNA fragment SEQ ID NO:23 from ZmABCG20 was selected as the RNAi target sequence. From the V12 phase cDNA obtained in Example 7, the forward fragment 17N19-1 of the RNAi stem-loop structure was amplified using primer pairs 17N19-F1 (SEQ ID NO:21) and 17N19-R1 (SEQ ID NO:22); the reverse fragment 17N19-2 of the RNAi stem-loop structure was amplified using primer pairs 17N19-F2 (SEQ ID NO:19) and 17N19-R2 (SEQ ID NO:20).

[0122] 2. The intermediate vector used was pBSK-RTM (provided by Chengdu Haochen Biotechnology Co., Ltd., the vector pBSK-RTM was modified from plasmid pBSK. It contains introns of the Arabidopsis RTM1 gene with the sequence shown in SEQ ID NO:24, such as...). Figure 6As shown, the left side of the intron contains SacI and NotI restriction sites, and the right side contains XbaI and BamHI restriction sites. The pBSK-RTM and forward fragments were double-digested with SacI and NotI, ligated, and transformed into *E. coli*. Eight transformants were selected for PCR verification, and two positive transformants were selected for plasmid extraction and sequencing verification, yielding the pBSK-17N19-1 vector.

[0123] 3. Using pBSK-17N19-1 as a template, clone the reverse fragment 17N19-2 of the 17N19 gene.

[0124] pBSK-17N19-1 and its reverse fragment 17N19-2 were digested with XbaI and BamHI, ligated, and transformed into E. coli. Eight transformants were selected for PCR verification. One positive transformant was selected, and its plasmid was extracted and sequenced, confirming that it was the target vector pBSK-17N19R.

[0125] The pBSK-17N19R vector was digested with BamHI and SacI, and the target fragment containing the forward fragment + RTM + reverse fragment was recovered. Simultaneously, the pCambia3301ky plasmid (provided by Chengdu Haochen Biotechnology Co., Ltd., where a 35S promoter was inserted upstream of the multiple cloning site of the pCambia3301 plasmid to obtain pCambia3301ky) was digested with BamHI and SacI. The target fragment and pCambia3301ky were ligated and transformed into *E. coli*. One PCR-positive transformant was selected, and the plasmid was extracted and digested with BamHI and SacI. Electrophoresis showed the large fragment (forward fragment + RTM + reverse fragment) and the pCambia3301ky plasmid backbone band, indicating that the target fragment was correctly ligated into the pCambia3301ky multiple cloning vector, resulting in the vector pCambia3301-17N19R. The RNAi vector construction was complete.

[0126] Example 11 Genetic transformation and phenotypic identification of transformed plants

[0127] The components of MS and N6 media are as follows:

[0128]

[0129]

[0130] The culture media used in the remaining steps are as follows:

[0131] YEB culture medium: 5.0 g / L yeast, 10.0 g / L peptone, 5.0 g / L NaCl, 50.0 mg / L kanamycin and 25.0 mg / L rifampin, pH 6.8;

[0132] Infection solution: N6 basic medium was prepared with 2,4-D 1.0 mg / L, L-proline 700 mg / L, hydrolyzed casein 100 mg / L, inositol 120 mg / L, sucrose 68 g / L, glucose 36 g / L, acetylsyringone 100 μmol / L, pH 5.2.

[0133] Co-culture medium: N6 basic medium, supplemented with proline 1.38 g / L, hydrolyzed casein 500 mg / L, inositol 120 mg / L, 2,4-D 2.0 mg / L, agar 0.7%, sucrose 3%, acetylsuccinone 100 μmol / L, cysteine ​​200 mg / L, AgNO3 0.85 mg / L, pH 6.0;

[0134] Recovery medium: N6 basic medium supplemented with proline 1.38 g / L, hydrolyzed casein 500 mg / L, inositol 120 mg / L, 2,4-D 2.0 mg / L, agar 0.7%, sucrose 3%, AgNO3 0.85 mg / L, cephalosporin 400 mg / L, pH 5.8;

[0135] First round of screening medium: N6 basic medium supplemented with 2,4-D 1.0 mg / L, L-proline 700 mg / L, hydrolyzed casein 100 mg / L, mannitol 20 g / L, inositol 120 mg / L, agar 0.7%, sucrose 3%, cephalosporin 400 mg / L, AgNO3 0.85 mg / L, diammonium phosphate 0.3 mg / L, pH 5.8;

[0136] Second round of screening medium: Based on the first round of screening medium, the concentration of diammonium phosphate was increased to 0.6 mg / L;

[0137] Differentiation medium: 1 mg / L kinetin, 100 mg / L hydrolyzed casein, 200 mg / L cephalosporin, 0.7% agar, 3% sucrose, pH 5.8 were added to the basic MS medium.

[0138] Rooting medium: 1 / 2 MS basic medium with 100 mg / L hydrolyzed casein, 700 mg / L L-proline, 0.2 mg / L IBA, 0.7% agar, 3% sucrose, pH 5.8.

[0139] Genetic transformation and regeneration of plants were performed using the following steps:

[0140] 1) Preparation of infection material: Take corn ears 10-13 days after self-pollination of corn inbred line B104, pick out the young embryos, soak them in 75% alcohol for 15 seconds, disinfect them by soaking in 2.5% sodium hypochlorite for 10 minutes, and rinse them with distilled water 3-5 times.

[0141] 2) Infection of immature embryos: Single colonies of genetically engineered Agrobacterium are picked from the plate and inoculated into YEB medium. The culture is carried out at 28℃ and 220 rpm with shaking for 20-36 hours. When the bacteria reach the logarithmic growth phase, they are centrifuged at 4℃ and 3000 rpm for 10 minutes. The bacterial cells are collected by centrifugation and resuspended in the infection solution until OD≈0.5, at which point they are ready for infection. 150 selected immature embryos are immersed in the infection solution for 5-10 minutes, and the infection solution is gently blotted dry with filter paper.

[0142] 3) Co-culture and recovery culture: After the embryos have been infected, they are transferred to co-culture medium and cultured in the dark at 22°C for 3 days. Then they are transferred to recovery medium and cultured in the dark at 28°C for 7 days.

[0143] 4) Screening: Transfer the callus obtained after treatment in step 3) to screening medium and incubate in the dark at 28°C. Screen twice, with each round lasting 3 weeks.

[0144] 5) Differentiation and rooting: The selected resistant callus tissues were transferred to differentiation medium and cultured in the dark at 25℃ for 7 days; then cultured under alternating conditions of 25℃, 16h light (light intensity 2000 lux) and 8h dark. When the seedlings grew to about 5cm in length, they were transferred to rooting medium and cultured in the light at 28℃ for 15 days.

[0145] 6) Hardening off and transplanting: Transplant the rooted seedlings into small flowerpots filled with nutrient soil and cultivate them under 28℃ light for 10 days; then transplant the seedlings into a greenhouse (natural light, daytime temperature 32℃, nighttime temperature 28℃) for cultivation.

[0146] The seedlings that had undergone the above steps were identified by primer PCR, and 5 of them were confirmed to be positive for transformation, carrying the ZmABCG20RNAi fragment designed in Example 9.

[0147] When the transformed plants flowered, anthers from both control and positively transformed plants were collected, and pollen fertility was assessed using the iodine staining method described in Example 3. The results were as follows: Figure 7 As shown, the wild-type inbred line B104 has normal pollen and is fertile, while a transformed plant, numbered R02, exhibits male sterility and lacks pollen, consistent with the zmabcg20-1 mutant phenotype. This result indicates that knocking out ZmABCG20 does indeed cause male sterility.

[0148] Based on the results of Examples 1-10, the zmabcg20-1 mutant phenotype and mutant gene are consistent with the ZmABCG20 homologous gene mutants in Arabidopsis and rice; the ZmABCG20 gene is specifically expressed in maize male florets and young spikelets, but not expressed in other stages and tissues; the sterility phenotype co-segregates with the zmabcg20-1 mutant gene; knockout of the ZmABCG20 gene leads to the same male sterility phenotype as zmabcg20-1. These results demonstrate that ZmABCG20 is an essential gene for male fertility development in maize; its loss of function can lead to the male sterility phenotype in maize; and the male sterility phenotype of the zmabcg20-1 mutant is caused by a point mutation in the ZmABCG20 gene described in Example 6.

[0149] Example 12: Cloning of the ZmABCG20 promoter

[0150] Using primers pZmABCG20_F (sequence shown in SEQ ID NO:13) and pZmABCG20_R (sequence shown in SEQ ID NO:14) to amplify maize genomic DNA, a DNA fragment of 1653 bp in size can be obtained, of which 1634 bp is upstream of the start codon ATG (SEQ ID NO:12). Analysis of this sequence using the online transcriptional element analysis tool PlantCARE (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ) revealed CAAT-box and TATA-box at +1237 and +1385, respectively. Additionally, several hormone-response elements were identified: abscisic acid response element ABRE (GCAACGTGTC, +1236); jasmonic acid response elements TGACG_motif (TGACG, +655) and CGTCA_motif (CGTCA, +765); and gibberellin response elements GRAE_motif (TCTGTTG, +513; AAACAGA, +1397). A circadian rhythm regulatory element, CAANNNNATC, was found at +973. The abundance of transcriptional and regulatory elements indicates that this region is the promoter region of ZmABCG20.

[0151] Example 13 Hybridization and Transformation of Mutant Genes

[0152] The mutants obtained by this invention and the functional markers of the mutant genes described in Example 9 can be used in various marker-assisted selection methods. Taking backcrossing as an example, they can be used according to... Figure 8 The steps involved transferring the sterility gene zmabcg20-1 into other maize genetic backgrounds through hybridization:

[0153] ① Hybridization:

[0154] F1 seeds were obtained by crossing zmabcg20-1 mutant strain as the female parent with recipient maize material as the male parent.

[0155] ② First round of intercourse:

[0156] F1 plants were obtained after sowing F1 seeds. F1 plants were then crossed with recurrent parents to obtain BC1 seeds.

[0157] ③ BC1 infertility gene selection (prospect selection):

[0158] Sow BC1 seeds to obtain no less than 500 seedlings. Collect leaves from each seedling during the seedling stage and extract DNA according to the method described in Example 4. Amplify and electrophore the DNA using the primer pair (3326_F1, 3326_R1) in Example 9. Select heterozygous seedlings for further planting and discard homozygous wild-type seedlings.

[0159] ④ BC1 background selection:

[0160] A set of molecular markers (including but not limited to SSR, INDEL, SNP, EST, RFLP, AFLP, RAPD, SCAR, etc.) that are polymorphic between the mutant zmabcg20-1 and the recurrent parent and are evenly distributed on the genome are used to identify the single plants selected in step ③. Materials with high similarity to the recurrent parent (e.g., greater than 88% similarity, or 2% selection rate) are selected.

[0161] ⑤ Second round of cross: Use the single plant selected in step ④ as the male parent to pollinate the recurrent parent to obtain BC2 seeds;

[0162] ⑥ BC2 foreground and background selection: Repeat steps ③ to ④ for the selected materials, and select BC2 generation plants with a similarity to the recurrent parent that is higher than the selection criteria (such as similarity greater than 98%, or 2% selection rate, etc.).

[0163] ⑦ Self-pollination to obtain BC2F2 seeds: Self-pollinate the BC2 plants selected in step ⑥ to obtain BC2F2 seeds;

[0164] ⑧ Foreground selection of BC2F2: Sow the BC2F2 seeds obtained in step ⑦ to obtain more than 500 seedlings. Collect leaves during the seedling stage and extract DNA according to the method described in Example 4. Amplify and electrophore using the primer pair (3326_F1, 3326_R1) in Example 9. Select single plants with homozygous mutant and heterozygous banding for continued cultivation, and remove single plants with homozygous wild type.

[0165] ⑨ Background selection and application of BC2F2: The single plants selected in step ⑧ are subjected to background screening according to the method in step ④, selecting single plants with 100% background homozygosity. If the genotype of the selected single plant is a homozygous mutant, then this single plant is our final target material, which can be further hybridized with the recurrent parent to preserve material, or hybridized with other maize materials. If the selected single plant is heterozygous, it can be directly used for germplasm preservation, or sterile plants can be obtained through self-pollination for hybridization breeding or seed production.

[0166] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention. sequence list <110> Hainan Bolian Rice Gene Technology Co., Ltd. <120> Application of maize gene ZmABCG20 in regulating crop male fertility <130> KHP171115359.3 <160> twenty four <170> SIPOSequenceListing 1.0 <210> 1 <211> 2366 <212> DNA <213> Corn (Zea mays) <400> 1 atggagatca gcgacgagca gaggatgcaa gtggagtgcc agcgccttcc gccttcctgg 60 caaggcaacg gatcagacgc cgatgtcgaa gtggatcatc atctgtggcc gtcaaaagat 120 ggccctcttc caatattcct taaggtgtta gttgtgctac ctcactcctg ttttttttct 180 tttaggctga cgtgtgcaat gacttgctga actccaaaac ccagtttgag aacgtggagt 240 acagggtgaa gatgaccttg aagaaccccc tcacagcggc gagagtggcg tttgcgtccc 300 agacgagggc agaccagggc agcagctgca agcacatcct caagggcatc gctgggagtg 360 tggaccctgg cgagatcctg gcgctgatgg gtccatctgg cagcggcaag accaccttgc 420 tcaagatcct ggggggcagg cttggtggcg gcgtcaaggg ccacataacc tacaacgaca 480 ctccctacag cccctgcctc aaaaggaggt actgaaactg taatagctag caaacaagaa 540 tttttcactt cacttaataa tgaagatttt ttcactaatc tgtgctgtgc gctgcaggat 600 cggatttgtg actcaggacg acgtcctctt cccacagctg acggtggagg agaccctcgt 660 gttcgccgcc ttcttgaggc tccctgcttg catgtccaag cagcagaagc gcgacagggt 720 agacgccatc atcgccgagt tgaatctaga gaggttgcct ttcattccat ttcttgtatt 780 tatcggggaa tttgagtttt cgcctgtatg caaatgcaag catgcatgca tacatgttgt 840 gtggatcatc atttgaattg gtttaggtgc cggcacacca agatcggggg agcgttcgtg 900 aggggggtgt caggaggcga gaggaagagg accagcatcg ggaacgagat cctcgtcgac 960 ccgtcgctgc tcctcctcga cgagcccacc tccggcctcg actccacgtc ggcgagcaag 1020 ctcatcttta tcctccagcg cctggccaag gtacattaat ttgcgcgcag ctagcaacgc 1080 caccgacgag acgacgatca tgcacgttgc taattccatc gatgctggac gcatggtcgc 1140 agacgcggag gacgatcatc acgacgatcc accagccgtc gagccggatg ttccacatgt 1200 tcgacaagct gctgctcatc tccgacgggc acgccatcta ccacggcaag gcccgggact 1260 gcatgcacca cttctcctcg ctgggcttcg tcccggagat ccccatgaac ccggccgagt 1320 tcctgctgga cctcgccacc ggcaacctcg acgacatcag cgtccccgag gcgctgcgcg 1380 gctcgccgga cccgcaggag ttcaggtccc aggtcatcag gcacctgcag ctcaagtacc 1440 gggcgggcgc cgaggctccc gcggggagaa ggacgcccac tgagcagctg cgtctggcgg 1500 tgcgggcgca taacaaggac cgccgccggc ggagcatcgg ctggctccag cagttcgccg 1560 tgctgtcccg gcgcacgttc cgggagcgcg catccgacta cctggacaag atgcggctcg 1620 cgcaggccgt cggcgtggcg ctcctgctgg gtctcctctg gtggaagtcc cagaccggga 1680 acgaggccca gctgcgggac caggtcggtc tcatcttcta catctgcatc ttctggacgt 1740 cgtcgtcgct cttcggctcc gtctacgtgt tccccttcga gaagctgtac ctggtcaagg 1800 agcgcaaggc ggacatgtac cggctgagcg cctactacgc cagcagcacg ctgtgcgacg 1860 ccgtgccgca cgtcgtgtac ccggtgctct tcatggccat cctctacttc atggccggcc 1920 tccgccgcac cgtgccgtgc ttcttcctca cgctcctcgc cacgctgctc atcgtgttca 1980 ccagccaggg caccggggag ctgctgggcg ccgccatcct cagcgtcaag agggcggggg 2040 tcatggcgtc gctcgtgctc atgctcttcc tcctcaccgg cggctactac gtccagcaca 2100 tccccaagtt catccgctgg ctcaagtacg tctccttcat gcactacggc ttcaacctgc 2160 tgctcaaagc gcagtaccac ggccacctca cgtacaactg tgccagccgg ggcggctgcc 2220 agcgcctgca gtcgtcgccg tcgttcggca ccgtggacct cgacggcggc atgcgcgagg 2280 tctggatcct gctcgccatg gcgctcgcat accgactcct cgcctacttc tgcctcctca 2340 agcggatcag cctcacgccc ttgtga 2366 <210> 2 <211> 1992 <212> DNA <213> Zea mays <400> 2 atggagatca gcgacgagca gaggatgcaa gtggagtgcc agcgccttcc gccttcctgg 60 caaggcaacg gatcagacgc cgatgtcgaa gtggatcatc atctgtggcc gtcaaaagat 120 ggccctcttc caatattcct taagtttgag aacgtggagt acagggtgaa gatgaccttg 180 aagaaccccc tcacagcggc gagagtggcg tttgcgtccc agacgagggc agaccagggc 240 agcagctgca agcacatcct caagggcatc gctgggagtg tggaccctgg cgagatcctg 300 gcgctgatgg gtccatctgg cagcggcaag accaccttgc tcaagatcct ggggggcagg 360 cttggtggcg gcgtcaaggg ccacataacc tacaacgaca ctccctacag cccctgcctc 420 aaaaggagga tcggatttgt gactcaggac gacgtcctct tcccacagct gacggtggag 480 gagaccctcg tgttcgccgc cttcttgagg ctccctgctt gcatgtccaa gcagcagaag 540 cgcgacaggg tagacgccat catcgccgag ttgaatctag agaggtgccg gcacaccaag 600 atcgggggag cgttcgtgag gggggtgtca ggaggcgaga ggaagaggac cagcatcggg 660 aacgagatcc tcgtcgaccc gtcgctgctc ctcctcgacg agcccacctc cggcctcgac 720 tccacgtcgg cgagcaagct catctttatc ctccagcgcc tggccaagac gcggaggacg 780 atcatcacga cgatccacca gccgtcgagc cggatgttcc acatgttcga caagctgctg 840 ctcatctccg acgggcacgc catctaccac ggcaaggccc gggactgcat gcaccacttc 900 tcctcgctgg gcttcgtccc ggagatcccc atgaacccgg ccgagttcct gctggacctc 960 gccaccggca acctcgacga catcagcgtc cccgaggcgc tgcgcggctc gccggacccg 1020 caggagttca ggtcccaggt catcaggcac ctgcagctca agtaccgggc gggcgccgag 1080 gctcccgcgg ggagaaggac gcccactgag cagctgcgtc tggcggtgcg ggcgcataac 1140 aaggaccgcc gccggcggag catcggctgg ctccagcagt tcgccgtgct gtcccggcgc 1200 acgttccggg agcgcgcatc cgactacctg gacaagatgc ggctcgcgca ggccgtcggc 1260 gtggcgctcc tgctgggtct cctctggtgg aagtcccaga ccgggaacga ggcccagctg 1320 cgggaccagg tcggtctcat cttctacatc tgcatcttct ggacgtcgtc gtcgctcttc 1380 ggctccgtct acgtgttccc cttcgagaag ctgtacctgg tcaaggagcg caaggcggac 1440 atgtaccggc tgagcgccta ctacgccagc agcacgctgt gcgacgccgt gccgcacgtc 1500 gtgtacccgg tgctcttcat ggccatcctc tacttcatgg ccggcctccg ccgcaccgtg 1560 ccgtgcttct tcctcacgct cctcgccacg ctgctcatcg tgttcaccag ccagggcacc 1620 ggggagctgc tgggcgccgc catcctcagc gtcaagaggg cgggggtcat ggcgtcgctc 1680 gtgctcatgc tcttcctcct caccggcggc tactacgtcc agcacatccc caagttcatc 1740 cgctggctca agtacgtctc cttcatgcac tacggcttca acctgctgct caaagcgcag 1800 taccacggcc acctcacgta caactgtgcc agccggggcg gctgccagcg cctgcagtcg 1860 tcgccgtcgt tcggcaccgt ggacctcgac ggcggcatgc gcgaggtctg gatcctgctc 1920 gccatggcgc tcgcataccg actcctcgcc tacttctgcc tcctcaagcg gatcagcctc 1980 acgcccttgt ga 1992 <210> 3 <211> 663 <212> PRT <213> Zea mays <400> 3 Met Glu Ile Ser Asp Glu Gln Arg Met Gln Val Glu Cys Gln Arg Leu 1 5 10 15 Pro Pro Ser Trp Gln Gly Asn Gly Ser Asp Ala Asp Val Glu Val Asp 20 25 30 His His Leu Trp Pro Ser Lys Asp Gly Pro Leu Pro Ile Phe Leu Lys 35 40 45 Phe Glu Asn Val Glu Tyr Arg Val Lys Met Thr Leu Lys Asn Pro Leu 50 55 60 Thr Ala Ala Arg Val Ala Phe Ala Ser Gln Thr Arg Ala Asp Gln Gly 65 70 75 80 Ser Ser Cys Lys His Ile Leu Lys Gly Ile Ala Gly Ser Val Asp Pro 85 90 95 Gly Glu Ile Leu Ala Leu Met Gly Pro Ser Gly Ser Gly Lys Thr Thr 100 105 110 Leu Leu Lys Ile Leu Gly Gly Arg Leu Gly Gly Gly Val Lys Gly His 115 120 125 Ile Thr Tyr Asn Asp Thr Pro Tyr Ser Pro Cys Leu Lys Arg Arg Ile 130 135 140 Gly Phe Val Thr Gln Asp Asp Val Leu Phe Pro Gln Leu Thr Val Glu 145 150 155 160 Glu Thr Leu Val Phe Ala Ala Phe Leu Arg Leu Pro Ala Cys Met Ser 165 170 175 Lys Gln Gln Lys Arg Asp Arg Val Asp Ala Ile Ile Ala Glu Leu Asn 180 185 190 Leu Glu Arg Cys Arg His Thr Lys Ile Gly Gly Ala Phe Val Arg Gly 195 200 205 Val Ser Gly Gly Glu Arg Lys Arg Thr Ser Ile Gly Asn Glu Ile Leu 210 215 220 Val Asp Pro Ser Leu Leu Leu Leu Asp Glu Pro Thr Ser Gly Leu Asp 225 230 235 240 Ser Thr Ser Ala Ser Lys Leu Ile Phe Ile Leu Gln Arg Leu Ala Lys 245 250 255 Thr Arg Arg Thr Ile Ile Thr Thr Ile His Gln Pro Ser Ser Arg Met 260 265 270 Phe His Met Phe Asp Lys Leu Leu Leu Ile Ser Asp Gly His Ala Ile 275 280 285 Tyr His Gly Lys Ala Arg Asp Cys Met His His Phe Ser Ser Leu Gly 290 295 300 Phe Val Pro Glu Ile Pro Met Asn Pro Ala Glu Phe Leu Leu Asp Leu 305 310 315 320 Ala Thr Gly Asn Leu Asp Asp Ile Ser Val Pro Glu Ala Leu Arg Gly 325 330 335 Ser Pro Asp Pro Gln Glu Phe Arg Ser Gln Val Ile Arg His Leu Gln 340 345 350 Leu Lys Tyr Arg Ala Gly Ala Glu Ala Pro Ala Gly Arg Arg Thr Pro 355 360 365 Thr Glu Gln Leu Arg Leu Ala Val Arg Ala His Asn Lys Asp Arg Arg 370 375 380 Arg Arg Ser Ile Gly Trp Leu Gln Gln Phe Ala Val Leu Ser Arg Arg 385 390 395 400 Thr Phe Arg Glu Arg Ala Ser Asp Tyr Leu Asp Lys Met Arg Leu Ala 405 410 415 Gln Ala Val Gly Val Ala Leu Leu Leu Gly Leu Leu Trp Trp Lys Ser 420 425 430 Gln Thr Gly Asn Glu Ala Gln Leu Arg Asp Gln Val Gly Leu Ile Phe 435 440 445 Tyr Ile Cys Ile Phe Trp Thr Ser Ser Ser Leu Phe Gly Ser Val Tyr 450 455 460 Val Phe Pro Phe Glu Lys Leu Tyr Leu Val Lys Glu Arg Lys Ala Asp 465 470 475 480 Met Tyr Arg Leu Ser Ala Tyr Tyr Ala Ser Ser Thr Leu Cys Asp Ala 485 490 495 Val Pro His Val Val Tyr Pro Val Leu Phe Met Ala Ile Leu Tyr Phe 500 505 510 Met Ala Gly Leu Arg Arg Thr Val Pro Cys Phe Phe Leu Thr Leu Leu 515 520 525 Ala Thr Leu Leu Ile Val Phe Thr Ser Gln Gly Thr Gly Glu Leu Leu 530 535 540 Gly Ala Ala Ile Leu Ser Val Lys Arg Ala Gly Val Met Ala Ser Leu 545 550 555 560 Val Leu Met Leu Phe Leu Leu Thr Gly Gly Tyr Tyr Val Gln His Ile 565 570 575 Pro Lys Phe Ile Arg Trp Leu Lys Tyr Val Ser Phe Met His Tyr Gly 580 585 590 Phe Asn Leu Leu Leu Lys Ala Gln Tyr His Gly His Leu Thr Tyr Asn 595 600 605 Cys Ala Ser Arg Gly Gly Cys Gln Arg Leu Gln Ser Ser Pro Ser Phe 610 615 620 Gly Thr Val Asp Leu Asp Gly Gly Met Arg Glu Val Trp Ile Leu Leu 625 630 635 640 Ala Met Ala Leu Ala Tyr Arg Leu Leu Ala Tyr Phe Cys Leu Leu Lys 645 650 655 Arg Ile Ser Leu Thr Pro Leu 660 <210> 4 <211> 2360 <212> DNA <213> Zea mays <400> 4 atggagatca gcgacgagca gaggatgcaa gtggagtgcc agcgccttcc gccttcctgg 60 caaggcaacg gatcagacgc cgatgtcgaa gtggatcatc atctgtggcc gtcaaaagat 120 ggccctcttc caatattcct taaggtgtta gttgtgctac ctcactcctg ttttttttct 180 tttaggctga cgtgtgcaat gacttgctga actccaaaac ccagtttgag aacgtggagt 240 acagggtgaa gatgaccttg aagaaccccc tcacagcggc gagagtggcg tttgcgtccc 300 agacgagggc agaccagggc agcagctgca agcacatcct caagggcatc gctgggagtg 360<所给原文中此处标签重复,应为 ,按照规则修改后翻译如下 tggaccctgg cgagatcctg gcgctgatgg gtccatctgg cagcggcaag accaccttgc 420 tcaagatcct ggggggcagg cttggtggcg gcgtcaaggg ccacataacc tacaacgaca 480 需注意,原文中 标签重复,按照规则修改后进行了翻译。实际使用时请确认原文标签是否准确。ctccctacag cccctgcctc aaaaggaggt actgaaactg taatagctag caaacaagaa 540 tttttcactt cacttaataa tgaagaattt tcattaatct gtgctgtgcg ctgcaggatc 600 ggatttgtga ctcaggacga cgtcctcttc ccacagctga cggtggagga gaccctcgtg 660 ttcgccgcct tcttgaggct ccctgcctgc atgtccaagc agcagaagcg cgacagggtc 720 gacgccatca tcgccgagtt gaatctagag aggttgcctt tcattccatt tcttgtattt 780 atcggggaat ttgagttttc gcctgtatgc aagcatgcat gcatacatgt ggtgtggatc 840 accatttgaa ttggtttagg tgccggcaca ccaagatcgg gggagcgttc gtgagggggg 900 tgtcaggagg cgagaggaag aggaccagca tcgggaacga gatcctcgtc gacccgtcgc 960 tgctcctcct cgacgaaccc acctccggcc tcgactccac atcggcgagc aagctcatct 1020 ttatcctcca gcgcctggcc aaggtacatt tgcgcgcagc tagcaacgcc accgacgaga 1080 cgatacgacg atcatgcacg ttgctaattc gatcgatgct ggacgcatgg tcgcagacgc 1140 ggaggacgat catcacgacg atccaccagc cgtcgagccg gatgttccac atgttcgaca 1200 agctgctgct catctccgac gggcacgcca tctaccacgg caaggcccgg gactgcatgc 1260 accacttctc ctcgctgggc ttcgtcccgg agatcccat gaacccggcc gagttcctgc 1320 tggacctcgc caccggcaac ctcgacgaca tcagcgtccc cgaggcgctg cgcggctcgc 1380 cggacccgca ggagttcagg tcccaggtca tcaggcacct gcagctcaag taccgggcgg 1440 gcgccgaggc tcccgcgggg agaaggacgc ccacggagca gctgcgtctt gctgtgcggg 1500 cgcataacaa ggaccgccgc cggcggagca tcggctggct ccagcagttc gccgtgctgt 1560 cccggcgcac gttccgggag cgcgcgtccg actacctgga caagatgcgg ctcgcgcagg 1620 ccgtcggcgt ggcgctcctg ctgggtctcc tctggtggaa gtcccagacc gggaacgagg 1680 cccagctgcg ggaccaggtg gggctcatct tctacatctg catcttctgg acgtcgtcgt 1740 cgctcttcgg ctccgtctac gtgttcccct tcgagaagct gtacctggtg aaggagcgca 1800 aggcggacat gtaccggctg agcgcctact acgccagcag cacgctgtgc gacgccgtgc 1860 cgcacgtcgt gtacccggtg ctcttcatgg ccatcctcta cttcatggcc ggcctccgcc 1920 gcaccgtgcc gtgcttcttc ctcacgctcc tcgccacgct gctcatcgtg ttcaccagcc 1980 agggcaccgg ggagctgctg ggcgccgcca tcctcagcgt caagagggcg ggggtcatgg 2040 cgtcgctcgt gctcatgctc ttcctcctca ccggcggcta ctacgtccag cacatcccca 2100 agttcatccg ctggctcaag tacgtctcct tcatgcacta cggcttcaac ctgctgctca 2160 aagcgcagta ccacggccac ctcatgtaca actgtgccag ccggggcggc tgccagcgcc 2220 tccagtcgtc gccgtcgttc ggcaccgtgg acctcgacgg cggcatgcgc gaggtctgga 2280 tcctgctcgc catggcgctc gcataccgac tcctcgccta cttctgcctc ctcaagcgga 2340 tcagcctcac gcccttgtga 2360 <210> 5 <211> 1992 <212> DNA​​​​​​​​​​​aagaaccccc tcacagcggc gagagtggcg tttgcgtccc agacgagggc agaccagggc 240 agcagctgca agcacatcct caagggcatc gctgggagtg tggaccctgg cgagatcctg 300 gcgctgatgg gtccatctgg cagcggcaag accaccttgc tcaagatcct ggggggcagg 360 cttggtggcg gcgtcaaggg ccacataacc tacaacgaca ctccctacag cccctgcctc 420 aaaaggagga tcggatttgt gactcaggac gacgtcctct tcccacagct gacggtggag 480 gagaccctcg tgttcgccgc cttcttgagg ctccctgcct gcatgtccaa gcagcagaag 540 cgcgacaggg tcgacgccat catcgccgag ttgaatctag agaggtgccg gcacaccaag 600 atcgggggag cgttcgtgag gggggtgtca ggaggcgaga ggaagaggac cagcatcggg 660 aacgagatcc tcgtcgaccc gtcgctgctc ctcctcgacg aacccacctc cggcctcgac 720 tccacatcgg cgagcaagct catctttatc ctccagcgcc tggccaagac gcggaggacg 780 atcatcacga cgatccacca gccgtcgagc cggatgttcc acatgttcga caagctgctg 840 ctcatctccg acgggcacgc catctaccac ggcaaggccc gggactgcat gcaccacttc 900 tcctcgctgg gcttcgtccc ggagatcccc atgaacccgg ccgagttcct gctggacctc 960 gccaccggca acctcgacga catcagcgtc cccgaggcgc tgcgcggctc gccggacccg 1020 caggagttca ggtcccaggt catcaggcac ctgcagctca agtaccgggc gggcgccgag 1080 gctcccgcgg ggagaaggac gcccacggag cagctgcgtc ttgctgtgcg ggcgcataac 1140 aaggaccgcc gccggcggag catcggctgg ctccagcagt tcgccgtgct gtcccggcgc 1200 acgttccggg agcgcgcgtc cgactacctg gacaagatgc ggctcgcgca ggccgtcggc 1260 gtggcgctcc tgctgggtct cctctggtgg aagtcccaga ccgggaacga ggcccagctg 1320 cgggaccagg tggggctcat cttctacatc tgcatcttct ggacgtcgtc gtcgctcttc 1380 ggctccgtct acgtgttccc cttcgagaag ctgtacctgg tgaaggagcg caaggcggac 1440 atgtaccggc tgagcgccta ctacgccagc agcacgctgt gcgacgccgt gccgcacgtc 1500 gtgtacccgg tgctcttcat ggccatcctc tacttcatgg ccggcctccg ccgcaccgtg 1560 ccgtgcttct tcctcacgct cctcgccacg ctgctcatcg tgttcaccag ccagggcacc 1620 ggggagctgc tgggcgccgc catcctcagc gtcaagaggg cgggggtcat ggcgtcgctc 1680 gtgctcatgc tcttcctcct caccggcggc tactacgtcc agcacatccc caagttcatc 1740 cgctggctca agtacgtctc cttcatgcac tacggcttca acctgctgct caaagcgcag 1800 taccacggcc acctcatgta caactgtgcc agccggggcg gctgccagcg cctccagtcg 1860 tcgccgtcgt tcggcaccgt ggacctcgac ggcggcatgc gcgaggtctg gatcctgctc 1920 gccatggcgc tcgcataccg actcctcgcc tacttctgcc tcctcaagcg gatcagcctc 1980 acgcccttgt ga 1992 <210> 6 <211> 663 <212> PRT <213> Zea mays <400> 6 Met Glu Ile Ser Asp Glu Gln Arg Met Gln Val Glu Cys Gln Arg Leu 1 5 10 15 Pro Pro Ser Trp Gln Gly Asn Gly Ser Asp Ala Asp Val Glu Val Asp 20 25 30 His His Leu Trp Pro Ser Lys Asp Gly Pro Leu Pro Ile Phe Leu Lys 35 40 45 Phe Glu Asn Val Glu Tyr Arg Val Lys Met Thr Leu Lys Asn Pro Leu 50 55 60 Thr Ala Ala Arg Val Ala Phe Ala Ser Gln Thr Arg Ala Asp Gln Gly 65 70 75 80 Ser Ser Cys Lys His Ile Leu Lys Gly Ile Ala Gly Ser Val Asp Pro 85 90 95 Gly Glu Ile Leu Ala Leu Met Gly Pro Ser Gly Ser Gly Lys Thr Thr 100 105 110 Leu Leu Lys Ile Leu Gly Gly Arg Leu Gly Gly Gly Val Lys Gly His 115 120 125 Ile Thr Tyr Asn Asp Thr Pro Tyr Ser Pro Cys Leu Lys Arg Arg Ile 130 135 140 Gly Phe Val Thr Gln Asp Asp Val Leu Phe Pro Gln Leu Thr Val Glu 145 150 155 160 Glu Thr Leu Val Phe Ala Ala Phe Leu Arg Leu Pro Ala Cys Met Ser 165 170 175 Lys Gln Gln Lys Arg Asp Arg Val Asp Ala Ile Ile Ala Glu Leu Asn 180 185 190 Leu Glu Arg Cys Arg His Thr Lys Ile Gly Gly Ala Phe Val Arg Gly 195 200 205 Val Ser Gly Gly Glu Arg Lys Arg Thr Ser Ile Gly Asn Glu Ile Leu 210 215 220 Val Asp Pro Ser Leu Leu Leu Leu Asp Glu Pro Thr Ser Gly Leu Asp 225 230 235 240 Ser Thr Ser Ala Ser Lys Leu Ile Phe Ile Leu Gln Arg Leu Ala Lys 245 250 255 Thr Arg Arg Thr Ile Ile Thr Thr Ile His Gln Pro Ser Ser Arg Met 260 265 270 Phe His Met Phe Asp Lys Leu Leu Leu Ile Ser Asp Gly His Ala Ile 275 280 285 Tyr His Gly Lys Ala Arg Asp Cys Met His His Phe Ser Ser Leu Gly 290 295 300 Phe Val Pro Glu Ile Pro Met Asn Pro Ala Glu Phe Leu Leu Asp Leu 305 310 315 320 Ala Thr Gly Asn Leu Asp Asp Ile Ser Val Pro Glu Ala Leu Arg Gly 325 330 335 Ser Pro Asp Pro Gln Glu Phe Arg Ser Gln Val Ile Arg His Leu Gln 340 345 350 Leu Lys Tyr Arg Ala Gly Ala Glu Ala Pro Ala Gly Arg Arg Thr Pro 355 360 365 Thr Glu Gln Leu Arg Leu Ala Val Arg Ala His Asn Lys Asp Arg Arg 370 375 380 Arg Arg Ser Ile Gly Trp Leu Gln Gln Phe Ala Val Leu Ser Arg Arg 385 390 395 400 Thr Phe Arg Glu Arg Ala Ser Asp Tyr Leu Asp Lys Met Arg Leu Ala 405 410 415 Gln Ala Val Gly Val Ala Leu Leu Leu Gly Leu Leu Trp Trp Lys Ser 420 425 430 Gln Thr Gly Asn Glu Ala Gln Leu Arg Asp Gln Val Gly Leu Ile Phe 435 440 445 Tyr Ile Cys Ile Phe Trp Thr Ser Ser Ser Leu Phe Gly Ser Val Tyr 450 455 460 Val Phe Pro Phe Glu Lys Leu Tyr Leu Val Lys Glu Arg Lys Ala Asp 465 470 475 480 Met Tyr Arg Leu Ser Ala Tyr Tyr Ala Ser Ser Thr Leu Cys Asp Ala 485 490 495 Val Pro His Val Val Tyr Pro Val Leu Phe Met Ala Ile Leu Tyr Phe 500 505 510 Met Ala Gly Leu Arg Arg Thr Val Pro Cys Phe Phe Leu Thr Leu Leu 515 520 525 Ala Thr Leu Leu Ile Val Phe Thr Ser Gln Gly Thr Gly Glu Leu Leu 530 535 540 Gly Ala Ala Ile Leu Ser Val Lys Arg Ala Gly Val Met Ala Ser Leu 545 550 555 560 Val Leu Met Leu Phe Leu Leu Thr Gly Gly Tyr Tyr Val Gln His Ile 565 570 575 Pro Lys Phe Ile Arg Trp Leu Lys Tyr Val Ser Phe Met His Tyr Gly 580 585 590 Phe Asn Leu Leu Leu Lys Ala Gln Tyr His Gly His Leu Met Tyr Asn 595 600 605 Cys Ala Ser Arg Gly Gly Cys Gln Arg Leu Gln Ser Ser Pro Ser Phe 610 615 620 Gly Thr Val Asp Leu Asp Gly Gly Met Arg Glu Val Trp Ile Leu Leu 625 630 635 640 Ala Met Ala Leu Ala Tyr Arg Leu Leu Ala Tyr Phe Cys Leu Leu Lys 645 650 655 Arg Ile Ser Leu Thr Pro Leu 660 <210> 7 <211> 2356 <212> Ms <213> Zea mays <400> 7 atggagatca gcgacgagca gaggatgcaa gtggagtgcc agcgccttcc gccttcctgg 60 caggcaacg gatcagacgc cgatgtcgaa gtggatcatc atctgtggcc gtcaagat 120 ggccctctc caatattcct taaggtgtta gttgtgctac ctcactcctg ttttttct 180 tttaggctga cgtgtgcaat gacttgctga actccaaac ccagtttgag aacgtggagt 240 acagggtgaa gatgaccttg aagaaccccc tcacagcggc gagagtggcg ttgcgtccc 300 agacgaggggc agaccaggggc agcagcagca catcctcaag ggcatcgctg ggagtgtgga 360 ccctggcgag atcctggcgc tgatgggtcc atctggcagc ggcagacca ccttgctcaa 420 gatcctgggg ggcaggcttg gtggcggcgt caagggccac atacctaca acgacactcc 480 ctacagcccc tgcctcaaa ggaggtactg aactgtaat agctagcaa caagaatttt 540 tcacttcact taataatgaa gattttcat taatctgtgc tgtgcgctgc aggatcggat 600 ttgtgactca ggacgacgtc ctctcccac agctgacggt ggaggagacc ctcgtgttcg 660 ccgccttctt gaggctccct gcctgcatgt ccaagcagca gaagcgcgac agggtcgacg 720 ccatcatcgc cgagttgaat ctagagaggt tgcctttcat tccatttctt gtatttatcg 780 gggaatttga gttttcgcct gtatgcaagc atgcatgcat acatgtggtg tggatcacca 840 tttgaattgg tttaggtgcc ggcacaccaa gatcggggga gcgttcgtga ggggggtgtc 900 aggaggcgag aggaagagga ccagcatcgg gaacgagatc ctcgtcgacc cgtcgctgct 960 cctcctcgac gaacccacct ccggcctcga ctccacatcg gcgagcaagc tcatctttat 1020 cctccagcgc ctggccaagg tacatttgcg cgcagctagc aacgccaccg acgagacgat 1080 acgacgatca tgcacgttgc taattcgatc gatgctggac gcatggtcgc agacgcggag 1140 gacgatcatc acgacgatcc accagccgtc gagccggatg ttccacatgt tcgacaagct 1200 gctgctcatc tccgacgggc acgccatcta ccacggcaag gcccgggact gcatgcacca 1260 cttctcctcg ctgggcttcg tcccggagat ccccatgaac ccggccgagt tcctgctgga 1320 cctcgccacc ggcaacctcg acgacatcag cgtccccgag gcgctgcgcg gctcgccgga 1380 cccgcaggag ttcaggtccc aggtcatcag gcacctgcag ctcaagtacc gggcgggcgc 1440 cgaggctccc gcggggagaa ggacgcccac ggagcagctg cgtcttgctg tgcgggcgca 1500 taacaaggac cgccgccggc ggagcatcgg ctggctccag cagttcgccg tgctgtcccg 1560 gcgcacgttc cgggagcgcg cgtccgacta cctggacaag atgcggctcg cgcaggccgt 1620 cggcgtggcg ctcctgctgg gtctcctctg gtggaagtcc cagaccggga acgaggccca 1680 gctgcgggac caggtggggc tcatcttcta catctgcatc ttctggacgt cgtcgtcgct 1740 cttcggctcc gtctacgtgt tccccttcga gaagctgtac ctggtgaagg agcgcaaggc 1800 ggacatgtac cggctgagcg cctactacgc cagcagcacg ctgtgcgacg ccgtgccgca 1860 cgtcgtgtac ccggtgctct tcatggccat cctctacttc atggccggcc tccgccgcac 1920 cgtgccgtgc ttcttcctca cgctcctcgc cacgctgctc atcgtgttca ccagccaggg 1980 caccggggag ctgctgggcg ccgccatcct cagcgtcaag agggcggggg tcatggcgtc 2040 gctcgtgctc atgctcttcc tcctcaccgg cggctactac gtccagcaca tccccaagtt 2100 catccgctgg ctcaagtacg tctccttcat gcactacggc ttcaacctgc tgctcaaagc 2160 gcagtaccac ggccacctca tgtacaactg tgccagccgg ggcggctgcc agcgcctcca 2220 gtcgtcgccg tcgttcggca ccgtggacct cgacggcggc atgcgcgagg tctggatcct 2280 gctcgccatg gcgctcgcat accgactcct cgcctacttc tgcctcctca agcggatcag 2340 cctcacgccc ttgtga 2356 <210> 8 <211> 303 <212> DNA <213> Zea mays <400> 8 atggagatca gcgacgagca gaggatgcaa gtggagtgcc agcgccttcc gccttcctgg 60 caaggcaacg gatcagacgc cgatgtcgaa gtggatcatc atctgtggcc gtcaaaagat 120 ggccctcttc caatattcct taagtttgag aacgtggagt acagggtgaa gatgaccttg 180 aagaaccccc tcacagcggc gagagtggcg tttgcgtccc agacgagggc agaccagggc 240 agcagcagca catcctcaag ggcatcgctg ggagtgtgga ccctggcgag atcctggcgc 300 tga 303 <210> 9 <211> 100 <212> PRT <213> Zea mays <400> 9 Met Glu Ile Ser Asp Glu Gln Arg Met Gln Val Glu Cys Gln Arg Leu 1 5 10 15 Pro Pro Ser Trp Gln Gly Asn Gly Ser Asp Ala Asp Val Glu Val Asp 20 25 30 His His Leu Trp Pro Ser Lys Asp Gly Pro Leu Pro Ile Phe Leu Lys 35 40 45 Phe Glu Asn Val Glu Tyr Arg Val Lys Met Thr Leu Lys Asn Pro Leu 50 55 60 Thr Ala Ala Arg Val Ala Phe Ala Ser Gln Thr Arg Ala Asp Gln Gly 65 70 75 80 Ser Ser Ser Thr Ser Ser Arg Ala Ser Leu Gly Val Trp Thr Leu Ala 85 90 95 Arg Ser Trp Arg 100 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <400> 10 ccagacgagg gcagaccag 19 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <400> 11 gatctcgcca gggtccaca 19 <210> 12 <211> 1634 <212> DNA <213> Zea mays <400> 12 taccacctgt ataaaggttc tcgatatgac tggtctgaaa ttgtcagcac tacaccaaat 60 gaaggtacag cctgcaatat cattgtattc tattctcttc tgttctccaa actagaagaa 120 ttgattttca taacgtccgt aggggcgaca acgatgatta tatgcctata ttaagtaaaa 180 atcaatatgc tgtataaata aggccgcaca caaattagaa gactataaat agtacccatc 240 aaaataagta aaacgtgcac ggtgcaaatg ctatccactt ttttttaccc tactgctgac 300 taattattga tgtattacag attgtgactg caatatctac agttgatgat ttgaattacc 360 ctgaaaagac tgagacctat tatatagtaa acgctccata catattttct gcatgttgga 420 aggtgtgctt cttgagatta ctacaaatct taacactttt tttttcctgt aaatcttctc 480 taacgtttca tttgatttta ggtcgtgaag cctctgttgc aagagagaac aaggaagaaa 540 gttcatgtgt tgcgtggctg cgggagagac gagcttctac aggtaacgaa gatcgattac 600 tgtttacatg gcccttgatt tttaccctta aagcgtcttc tggcttaaag cctttgacga 660 tcacagtgca atgacatggg ggcagttctg gaacatgcag atcatggact actcctccct 720 cccccatttc tgcagacagg agggctcggg ctcatccaaa cattcgtcag gcgacgccga 780 taactgcttc tctctcgacc acccgttcca ccaggagctc tacagcttca tccaggagca 840 ggcgctgaac caggagctca tcaagcaggg ctccttgcac gtgaaaatcc ccgagcagga 900 ccctgaggac gcaaagatcg tggaggtcat cgaggccgag ttccacaagc ttggcgtgca 960 gaacgggtcc gccaatggca tcgaccaagc atagcacctt tgtctaataa ggccagcctg 1020 attcaaccga gacgttgctt gccacttctg atgcaactaa tcctactgtt taattttttt 1080 ggtggggtgg gcccaccccc attgctgttt cctgggggag gggggggggg gggggggtga 1140 ttggtattgg tggtatacat agcaaagtca tttgcagatc catccatgat gcaggactga 1200 gatagacgat atcacctgtg taaacttgca gatttgcaat gtgtcctcat ctcatatgta 1260 tgctgagttg agatgaaatg aataaatcat cctacaagtt ttttttgagt catcttcaga 1320 attgtgttgt gctcagtcag aaaagtaacc atgcgtttct gtgccgattc ttgctagccc 1380 taattaatat aaggcgaaac agataatgcc tatccattgg agggattaga ggaaggttct 1440 ccccttagaa agattgtggg tcaccaaaca aagggtaaac gaaccgaacc gaaccgtgca 1500 tatcaacaac attacaacaa agtaagcatg ctcttggttg gcagtagtta ccacttgata 1560 aaaagaagag ggagaccatg tgctttggct tcctctctga gccatctttc ttgctctagc 1620 cttaggcctt agca 1634 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <400> 13 taccacctgt ataaaggttc tcga 24 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <400> 14 gctcgtcgct gatctcca 18 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <400> 15 atgtttcctg ggattgccga t 21 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <400> 16 gcatcacaag ccagtttaac c 21 <210> 17 <211> twenty one <212> DNA <213> Artificial Sequence <400> 17 accttgaaga accccctcac a 21 <210> 18 <211> 19 <212> DNA <213> Artificial Sequence <400> 18 ggtccacact cccagcgat 19 <210> 19 <211> 29 <212> DNA <213> Artificial Sequence <400> 19 cgggatccga gaacgtggag tacagggtg 29 <210> 20 <211> 28 <212> DNA <213> Artificial Sequence <400> 20 gctctagacc tccttttgag gcaggggc 28 <210> twenty one <211> 28 <212> DNA <213> Artificial Sequence <400> twenty one cgagctcgag aacgtggagt acagggtg 28 <210> twenty two <211> 32 <212> DNA <213> Artificial Sequence <400> 22 atttgcggcc gccctccttt tgaggcaggg gc 32 <210> 23 <211> 282 <212> DNA <213> Zea mays <400> 23 gagaacgtgg agtacagggt gaagatgacc ttgaagaacc ccctcacagc ggcgagagtg 60 gcgtttgcgt cccagacgag ggcagaccag ggcagcagct gcaagcacat cctcaagggc 120 atcgctggga gtgtggaccc tggcgagatc ctggcgctga tgggtccatc tggcagcggc 180 aagaccacct tgctcaagat cctggggggc aggcttggtg gcggcgtcaa gggccacata 240 acctacaacg acactcccta cagcccctgc ctcaaaagga gg 282 <210> 24 <211> 128 <212> DNA <213> Arabidopsis thaliana <400> 24 acgttgtaag tctatttttg actcttcttt tttctccgtc acaatttcta cttccaacta 60 aaatgctaag aacatggtta taactttttt tttataactt aatatgtgat ttggacccag 120 cagataga 128

Claims

1. By inhibiting corn ZmABCG20 A method for preparing male-sterile transgenic maize by activating gene activity, characterized in that, Will carry targeted genes ZmABCG20 The cDNA sequence RNAi sequence is operatively linked to a constitutive promoter or a flower organ-specific expression promoter and transferred into maize callus tissue. The transformed material undergoes co-culture-screening-differentiation-rooting-transgenic seedling hardening and transplanting to obtain male-sterile transgenic maize. The target DNA sequence for RNAi is shown in SEQ ID NO:

23.

2. The application of the biomaterials obtained by the method of claim 1 in crop improvement breeding and seed production; in, The crop in question is corn.

3. The application according to claim 2, characterized in that, Make include or express the ZmABCG20 Genetically modified corn, or corn obtained by the method of claim 1. ZmABCG20 Genetically inactivated corn is hybridized with the same crop that has excellent agronomic traits; The crop in question is corn.

4. The application according to claim 3, characterized in that, The superior agronomic traits include increased yield, improved quality, resistance to diseases and pests, stress resistance, and lodging resistance.

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