Maize pollen fertility regulation gene ZmPUB33 and encoding protein thereof

Abstract

The invention belongs to the field of gene engineering, and discloses a maize pollen fertility regulation gene ZmPUB33 and an encoding protein thereof. The gene mediates the fertility of pollen by regulating and controlling the development of corn pollen pores, the gene cannot bloom normally after mutation, anther glumes cannot be cracked basically, anther is smaller than a wild type and is almost not exposed, pollen grains are inactive, complete male sterility of plants can be caused, the sterile line can be used for sterile seed production and production of crop hybrids, and the application prospect is wide. And the method has huge application and economic values.

Description

Technical Field

[0001] This invention relates to a gene regulating maize pollen development. ZmPUB33 It and its encoded proteins belong to the field of genetic engineering. Background Technology

[0002] Maize is an important food crop and exhibits significant hybrid vigor. Using male-sterile materials for hybrid seed production can save the cost of manual or mechanical emasculation and ensure seed purity.

[0003] Each corn pollen grain has only one pollen pore, which is the channel for controlling the entry and exit of water. This ensures that the pollen grain remains intact during the process of water loss and rehydration from maturation to fertilization. The pollen pore is also a necessary structure for pollen tube germination. Corn pollen grains with defects in their pollen pores cannot germinate, thus leading to male sterility.

[0004] This invention provides a DNA sequence of a gene that controls maize pollen development and its encoded protein. The loss of function of this gene will cause defects in maize pollen pore development, preventing pollen from germinating. This can generate new male-sterile materials, which have important application value in scientific research and agricultural production. Summary of the Invention

[0005] This invention provides a novel gene for regulating maize pollen development. ZmPUB33 The DNA sequence, cDNA sequence, and amino acid sequence of the functional protein encoded by the gene, the loss of which specifically leads to maize pollen sterility.

[0006] The first objective of this invention is to provide a novel gene that regulates maize pollen development. ZmPUB33 The characteristic is that it is a DNA molecule selected from the following 1) or 2) or 3) or 4) or 5): 1) The DNA molecule shown in SEQ ID NO.1 (cloned from the genomic DNA of maize inbred line Mo17). 2) The DNA molecule shown in SEQ ID NO.2 (cDNA cloned from maize inbred line Mo17); 3) DNA molecules that can affect plant fertility, formed by substitution of one or more bases and / or insertion and / or deletion of one or more bases and insertion / deletion of large nucleotide sequences based on SEQ ID NO.1. 4) A DNA molecule that can hybridize with the DNA molecule of SEQ ID NO. 2 and encode a protein related to plant pollen development, in a solution of 0.1× SSPE (or 0.1× SSC) and 0.1% (w / v) SDS at 65°C and after washing the membrane; 5) A DNA molecule that has more than 85% homology with the DNA molecule of SEQ ID NO. 2 and encodes a protein related to plant pollen development.

[0007] The second objective of this invention is to provide the proteins encoded by the aforementioned genes that regulate plant pollen development.

[0008] In one embodiment, the gene encodes the protein described in either 1) or 2): 1) A protein consisting of the amino acid sequence shown in SEQ ID NO.3 of the sequence listing; 2) A protein whose sequence listing SEQ ID NO.3 has been modified by substitution, deletion, and / or addition of one or more amino acid residues and has activities related to male fertility in plants.

[0009] The third objective of this invention is to provide a recombinant expression vector, expression cassette, transgenic cell line, or recombinant bacteria containing the said gene and / or promoter.

[0010] The fourth objective of this invention is to provide the use of the above-mentioned gene in transgenic improved crops.

[0011] In one embodiment, the gene is used to induce male sterility in crop plants in order to introduce foreign genes to obtain high-quality transgenic crops.

[0012] In another specific implementation, the crop is a self-pollinating or cross-pollinating crop.

[0013] In a more specific implementation, the crops include, but are not limited to, corn, wheat, sorghum, and rice.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The plant floral organ development regulatory gene provided by the present invention ZmPUB33 This gene directly participates in the development of pollen pores. Suppression of its expression leads to pollen pore development defects, ultimately disrupting pollen homeostasis and preventing the pollen grains from filling with starch. Even if starch-filled pollen grains do not fill with starch, the pollen pore defect prevents germination, resulting in male sterility. Through plant biotechnology, this invention will play a significant role in utilizing heterosis in crops and in the production of sterile hybrid seeds.

[0015] Terminology Definition The term "gene that regulates plant pollen development" refers to a nucleotide sequence that encodes a protein, specifically encoding an active polypeptide that regulates plant pollen development, such as the nucleotide sequence of SEQ ID NO.2 from position 1 to 2475 and its degenerate sequence.

[0016] The term "degenerate sequence" refers to a sequence in which one or more codons in the coding frame 1-2475 of SEQ ID NO.2 are replaced by degenerate codons encoding the same amino acid. Due to codon degeneracy, degenerate sequences with less than 70% homology to the nucleotide sequence 1-2475 of SEQ ID NO.2 can also encode the amino acid sequence encoded by SEQ ID NO.2.

[0017] The "gene regulating plant pollen development" also includes a nucleotide sequence that can hybridize with the nucleotide sequence of SEQ ID NO.2 under moderately stringent conditions, and preferably under highly stringent conditions. Moderately stringent conditions can be hybridization and washing at 65°C in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS (w / v).

[0018] The "gene regulating plant pollen development" also includes a nucleotide sequence that has at least 70% homology with the nucleotide sequence from position 1 to 2475 in SEQ ID NO.2, preferably at least 80%, 82%, 85%, 86%, 88%, or 89% homology, more preferably at least 90%, 91%, 92%, 93%, or 94% homology, and most preferably at least 95%, 96%, 97%, 98%, or 99% homology.

[0019] The "genes regulating plant pollen development" also include genes encoding genes similar to those naturally occurring to regulate corn pollen development. ZmPUB33 Variations of the open reading frame sequence of SEQ ID NO.2 for proteins with the same function as the gene. These variations include (but are not limited to): deletions, insertions and / or substitutions of one or more nucleotides, and additions of several (usually up to 60, preferably up to 30, more preferably up to 10, and most preferably up to 5) nucleotides at the 5' or 3' end.

[0020] The "gene regulating plant pollen development" also includes an amino acid sequence capable of translating a class of amino acid sequences that regulate maize pollen development, such as the amino acid sequence of SEQ ID NO. 3. This class of amino acid sequences also includes variants of SEQ ID NO. 3 that have the same function as the naturally occurring maize pollen development-regulating protein. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids, and the addition of one or more amino acids (typically no more than 20, preferably no more than 10, and more preferably no more than 5) to the C-terminus and / or N-terminus. In the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein; similarly, the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter the function of the protein.

[0021] Furthermore, the full-length nucleotide sequence or fragments of the "gene regulating plant pollen development" can typically be obtained using PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed in this embodiment, especially the open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared using conventional methods known to those skilled in the art can be used as templates to amplify the relevant sequences. When the sequence is long, two or more PCR amplifications are often required, and then the fragments amplified from each amplification are spliced ​​together in the correct order. Once the relevant sequence is obtained, it can be obtained in large quantities using recombination. Typically, it is cloned into a vector, and then the relevant sequence is isolated from proliferating host cells using conventional methods such as cell transformation. In addition, mutations can be introduced into the protein sequence of the embodiment through chemical synthesis. Besides recombination, fragments of the protein in the embodiment can also be produced using solid-phase technology by directly synthesizing polypeptides. In vitro protein synthesis can be performed manually or automatically; fragments of the protein in the embodiment can be chemically synthesized separately and then chemically linked to produce full-length protein molecules. Attached Figure Description

[0022] Figure 1 For normally developing wild-type maize Mo17 and ZmPUB33 Gene mutation ms34-6013 Observation of tassel and anther development, pollen KI-I2 staining, and pollen germination detection. A, Wild-type Mo17; B, ZmPUB33 Gene mutants ms34-6013 .

[0023] Figure 2 for ZmPUB33 Fine mapping and map-based cloning results of the gene: A, ZmPUB33 The gene was based on the genetic mapping results using a population of 144 sterile single plants and 5 pairs of molecular markers; B, ZmPUB33 The gene's physical mapping results show that it was precisely mapped to a 190 kb physical distance region (between markers 7088R1 and 24_33); C, ZmPUB33 A schematic diagram of functional genes within the physical map region where the gene is located; red represents candidate genes. Zm00014a007089 .

[0024] Figure 3 for ZmPUB33 Schematic diagram of gene structure in wild-type B73 and two allelic mutants in the reference genome: A: ZmPUB33 Gene structure of the gene in wild-type B73; B: ZmPUB33 Genes in mutants ms34-6013 Gene structure in; C: ZmPUB33Genes in mutants ms34-6004 The gene structure within. Among them, ms34-6013 Mutant genes and wild-type genes ZmPUB33 In contrast, a two-base deletion of TG at +16073~+16074 in exon 11 leads to a frameshift mutation in the coding region, causing premature termination of gene translation. In the mutant... ms34-6004 Cloned ZmPUB33 The gene coding region sequence is not different from the corresponding wild type, but in ZmPUB33 A large fragment of 5334bp was inserted at position +488 upstream of the gene.

[0025] Figure 4 for ZmPUB33 The coding region (CDS) of the gene in wild-type Mo17 and mutant ms34-6013 Nucleotide sequence comparison.

[0026] Figure 5 for ZmPUB33 Genes in wild-type Mo17 and mutant ms34-6013 Comparison of the amino acid sequences translated in the text.

[0027] Figure 6 To utilize primer combinations 6013page-F2588 and 6013page-R2638 in ms34-6013 homozygous mutant (genotype) ms34-6013 / ms34-6013 ), heterozygous mutant (genotype) ZmPUB33 / ms34-6013 PCR amplification and 12% polyacrylamide gel electrophoresis results: M, DNA Ladder; 1, 2, 6, 8: ms34-6013 Homozygous mutants; 3, 4, 5, 7: ms34-6013 Heterozygous mutant.

[0028] Figure 7 PCR products amplified using primer combinations 6004-MS34-F668, 6004-ins-F676, and 6004-MS34-R671 were identified by 2% agarose gel electrophoresis of the molecular weight bands specific to the sterility mutation (black arrows indicate specific amplification bands for fertile and sterile genotypes): M, DNA Ladder; 1, 3, 4, 5, 6: homozygous sterile lines (genotype...). ms34- 6004 / ms34-6004 ); 2, 7: Heterozygous fertile plants (genotype) ZmPUB33 / ms34-6004 ).

[0029] Figure 8 for ZmPUB33Expression profile analysis of genes in B73 by qPCR: Root; Stem; Leaf; Cob; Silk; Pollen; S5-13, different developmental stages of anther from premeiosis to pollen maturity.

[0030] Figure 9 Wild-type Mo17 and ms34-6013 , ms34-6004 Observations of mature pollen pores of mutants (top row) and transmission electron microscopy (bottom row). ms34-6013 and ms34-6004 The mutant cannot fill pollen grains with starch, and the pollen pores lack the Z structure. Pollen grains that are partially filled with starch burst due to the lack of the Z structure in the pollen pores. Detailed Implementation

[0031] The following embodiments are provided to facilitate a better understanding of the present invention, but do not limit the scope of its application. All technical and scientific terms used in the following embodiments, unless otherwise specified, have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise indicated, the techniques used or mentioned in this invention are standard techniques recognized by one of ordinary skill in the art. Unless otherwise specified, the test materials are all commonly used in the field of this invention. Unless otherwise specified, the test reagents used in the following embodiments were all purchased from conventional biochemical reagent stores. The materials, methods, and embodiments are illustrative only and not intended to be limiting.

[0032] The male sterility described in this invention specifically refers to abnormal development of male flowers (inability to produce male flowers, anthers, or normal male gametophytes) and loss of fertility caused by functional changes in plant cell nuclear genes; this is commonly known as genic male sterility, rather than cytoplasmic male sterility. The abnormality and recovery of pollen fertility are both controlled by genes within the cell nucleus.

[0033] Therefore, this invention also includes regulating plant fertility using sequences described in the sequence listing, that is, using the gene sequences provided by this invention to influence the function of identical or homologous genes in other plants at the genomic, and / or transcriptomic, and / or proteomic levels to control male flower fertility. For example, methods including, but not limited to, those described below: influencing or altering plant gene function by causing gene expression inhibition or protein function loss through variations in the natural sequence; by introducing an antisense sequence of the gene into the plant or introducing a hairpin structure; or by combining the gene with other sequences (DNA or RNA) to generate new functional DNA or RNA chains; or by inhibiting gene expression or activity through gene editing methods such as CRISPR / Cas9; or any other technical method known to those skilled in the art for influencing plant male flower fertility.

[0034] It is worth mentioning that the present invention includes corn. ZmPUB33 The genes, whose DNA and coding sequences differ in different maize inbred lines under different genetic backgrounds, such as the one cloned in this invention in the wild-type Mo17 background of maize. ZmPUB33 Apart from a few single nucleotide differences, the coding sequence in the Mo17 background is 114 bases shorter than the coding sequence in the B73 background, corresponding to the DNA sequence of the seventh exon. ZmPUB33 Its dominant allele plays a crucial role in male flower fertility in plants, while the loss-of-function recessive allele leads to male sterility. This gene is located on chromosome 7 of maize, and its specific location is shown below. Figure 2 As shown.

[0035] This gene sequence and its homologous sequences can be obtained from any flowering plant, including but not limited to maize (…). Zea mays ), common wheat ( Triticum aestivum ), sorghum ( Sorghum bicolor ), rice ( Oryza sativa ), short-stalked grass ( Brachypodium distachyon ), millet ( Setaria italica ),barley( Hordeum vulgare ),rye( Secale cereale ), rough goat grass ( Aegilops tauschii Arabidopsis thaliana ( ) Arabidopsis thaliana ), cabbage ( Brassica oleracea ), soybeans Glycine max ),tomato( Lycopersicon esculintum Methods of obtaining include, but are not limited to: through corn ZmPUB33 Gene sequences were retrieved from other plant genome sequence databases and / or cDNA sequence databases and / or protein sequence databases using blastx, blastn, or by amino acid sequence using blastp;ZmPUB33 Primers are designed using the DNA, cDNA, or RNA sequence of a gene as a reference sequence, and the gene is directly obtained from the genomic DNA, cDNA, or RNA of other plants using PCR methods; taking maize as an example... ZmPUB33 Gene sequence-designed probes are used to isolate DNA, cDNA, or RNA fragments containing homologous gene sequences from a genomic library using nucleic acid hybridization.

[0036] “ ZmPUB33 "Gene homologous sequence" refers to the DNA sequence of a plant gene that, after BLASTX comparison analysis with the amino acids in SEQ ID NO.3, has 35% or more Identities and 50% or more Positives. When performing BLASTX, all parameters were set to the default settings shown at http: / / blast.ncbi.nlm.nih.gov / .

[0037] The following description provides a more detailed account, but it is not intended to limit the scope of the invention.

[0038] Example 1. ms34-6013 Phenotypic analysis of sterile lines ms34-6013 and ms34-6004 The sterile line was purchased from MaizeGDB (https: / / maizegdb.org / ), where... ms34-6013 Its genetic background is Mo17. Regarding vegetative growth and ear development, ms34-6013 The sterile line showed virtually no difference from the fertile wild type; regarding tassel development, the Mo17 wild type (WT) could tassel normally, with anthers dehiscing and pollen shedding normally, and could produce seeds normally after self-pollination, while... ms34-6013 Although the sterile line can produce males normally, it cannot flower normally. The anthers and glumes are almost unable to dehisce, and the anthers are smaller and almost not exposed compared to the wild type. Figure 1 Further I2-KI staining of wild-type and mutant pollen revealed that wild-type pollen developed normally, and the pollen grains turned black after staining. Figure 1 A), but the mutant pollen only has a small number of pollen grains filled with starch ( Figure 1 B). From ms34-6013 The pollen extruded from the anthers of the sterile line cannot produce seeds from the time of intercourse to the female ear, indicating that... ms34-6013 Even small amounts of starch-filling pollen grains are not viable. The pollen from mature, fertile plants after dispersal... ms34-6013Pollen from the anthers was placed on corn pollen solid medium (1 mM CaCl2, 1 mM Ca(NO3)2, 1 mM MgSO4, 0.01% (w / v) H3BO3, 18% (w / v) sucrose, 0.8% (w / v) agar, pH 7.0) and incubated at 28℃ for 5-20 min. The pollen was then observed and photographed under a BX-53F microscope (Olympus, Japan). Observations revealed that... ms34-6013 The pollen of the mutant could not germinate, while the pollen of the WT germinated normally under the same conditions. Figure 1 ),show ms34-6013 The mutant's pollen loses its viability, resulting in complete male sterility.

[0039] Example 2. ZmPUB33 Identification of gene mutants and fine mapping of genes Male sterility is caused by mutations in genes controlling the development of male flowers or pollen, leading to male flower abortion. It typically manifests as the absence of male flowers, shriveled and unattached anthers, or the presence of no pollen or only a small amount of abnormally developed pollen in the anthers. Compared to normally developed floral organs, ms34-6013 The mutant anthers were not attached externally, and only a small number of starch-filled pollen grains could be stained after 1% KI-I2 staining, but they could not germinate due to abnormal pollen pore development.

[0040] Using Chang 7-2 as the paternal parent, ms34-6013 Using the male-sterile line as the maternal parent, an F2 segregating population of 1156 individual plants was constructed. Planted in the Hainan Nanfan experimental field in the winter of 2018, fertility was investigated according to the aforementioned phenotypic characteristics. A total of 301 F2 individual plants exhibited male-sterile phenotypes. Chi-square test results showed a segregation ratio of 3:1 between fertile and sterile plants (X²=3.84, P=0.05). All sterile individual plants were preserved by extracting total genomic DNA from leaves. A male-sterile gene pool was constructed from 144 individual plants exhibiting the male-sterile phenotype. Bulk-segregant analysis (BSA) was used to analyze mutation sites. The results showed a high ratio of SNPs between 20-140 Mb on chromosome 7, which is consistent with previous studies. ms34-6013 The locus is located on the long arm of chromosome 7 and is related to ms34-6004 This was an allelic mutation (Reference 1). Based on the marker database information in maizeGDB (http: / / www.maizegdb.org / ), polymorphic SSR molecular markers were selected within the candidate regions (Table 1), and 144 fertile and 144 sterile small populations were further screened. ms34-6013 The gene is located between two markers, 6_24 and 8_40, at approximately 0.78 Mb. Figure 2A).

[0041] In the summer of 2019, the mapping population was expanded to 730 sterile plants for fine mapping. InDel molecular markers were further designed for the linkage intervals between the two identified markers (Table 1). ms34-6013 Candidate genes were located within a 190 kb interval between molecular markers 7088R1 and 24_33, with marker 12_19 co-segregating with the sterility phenotype. [[ID=九十九]]Figure 2 B).

[0042]

[0043] The genome sequence of Mo17 was retrieved from Maize GDB. After removing pseudogenes and transposon sequences, there were 3 functional genes in the 190 Kb region. Figure 2 C), from ms34-6013 After cloning the coding sequences of three genes from the cDNA of sterile plants, it was found that only Zm00014a007089 The presence of a base deletion mutation caused premature termination of the reading frame, while the coding sequences of the other two genes showed no mutations. Zm00014a007089 Predicted to be a U-box family gene, the corresponding gene in the maize B73 reference genome (5th edition) is: Zm00001eb312220 This gene is annotated as pub33 in maizeGDB. Therefore, it is named... ZmPUB33 .

[0044] Based on the maize B73 genome sequence information, ZmPUB33 The gene is 18422 bp in length, and its genomic sequence structure is as follows ( Figure 3A): Contains 12 exons and 11 introns. The first base (transcription start site) of the cDNA is designated as nucleotide +1, nucleotides +1 to +210 form the first exon, nucleotides +211 to +706 form the first intron, nucleotides +707 to +947 form the second exon, nucleotides +948 to +1034 form the second intron, nucleotides +1035 to +1153 form the third exon, and nucleotides +1154 to +1462 form the third intron. Nucleotides +1463 to +1584 are exon 4; nucleotides +1585 to +1661 are intron 4; nucleotides +1662 to +1855 are exon 5; nucleotides +1856 to +2165 are intron 5; nucleotides +2166 to +2407 are exon 6; nucleotides +2408 to +8620 are intron 6; nucleotides +8621 to +8734 are exon 7; nucleotides +8735 to +9001 are intron 7; nucleotides +9002 to +9115 are exon 8; nucleotides +9116 to +15514 are intron 8; +15 Nucleotides 515 to +15616 are exon 9, nucleotides 15617 to +15702 are intron 9, nucleotides 15703 to +16131 are exon 10, nucleotides 16132 to +16347 are intron 10, nucleotides 16348 to +17103 are exon 11, nucleotides 17104 to +17539 are intron 11, nucleotides 17540 to +18422 are exon 12, nucleotides 749 to +751 are the translation start codon, and nucleotides 17741 to +18422 are the 3' untranslated region (3'-UTR).

[0045] Example 3. ZmPUB33 Cloning of genes in mutants based on ZmPUB33 Annotation information for the gene in the B73 genome: The full-length gene is 18422 bp and contains 11 introns. Primers listed in Table 2 were used in Mo17 and... ms34-6013 Specific cloning in mutants ZmPUB33Gene exon regions. The amplified products were sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for sequencing. The resulting nucleotide sequences, as shown in SEQ ID NO.1, were obtained after assembly. Sequencing sequences from amplified products using primers 7089-7, 7089-10, and 7089-5 yielded sequences covering exons 1 to 6 and containing introns 1 to 5. Sequencing sequences from amplified products using primer 7089-6 yielded sequences covering exons 7 and 8 and containing intron 7. Sequencing sequences from amplified products using primers 7089-12 and 7089-24 yielded sequences covering exons 9 to 12 and containing introns 9 to 11. Therefore, and ZmPUB33 Compared to the full-length gene, SEQ ID NO.1 lacks two intron regions: the region between exon 6 and exon 7, and the region between exon 8 and exon 9. In other words, the Mo17 gene shown in SEQ ID NO.1 lacks these two intron regions. ZmPUB33 The gene's nucleotide sequence is missing the 6th and 8th intron regions, but contains all exon sequences and all nine other intron sequences.

[0046] By comparing Mo17 and ms34-6013 Sequences cloned from mutants were found to be from ms34-6013 The cloned sequence in the mutant contains a two-base TG deletion at positions +16073 to +16074 of exon 11 in the corresponding B73 genome, resulting in a frameshift mutation in the coding region and causing premature termination of gene translation. Figure 3 B. Figure 4 and Figure 5 Using 7089CDS primers, cDNA from the male spike of Mo17 can be amplified. ZmPUB33 The full-length coding sequence corresponding to the gene was obtained by sequencing as shown in SEQ ID NO.2, and the encoded protein sequence is shown in SEQ ID NO.3.

[0047]

[0048] Using the same primer combination from allelic mutants ms34-6004 Cloned Zm000 No differences were found in the gene sequence compared to B73 due to sequence insertions or deletions that altered the reading frame. However, when the promoter region of the gene was amplified using primer combinations 6004-MS34-F668 (SEQ ID NO.30: 5'-GGAACCCTGCTCAATCCTACG-3') and 6004-MS34-R671 (SEQ ID NO.31: 5'-TCCCTTGTACAATAGACCTGCAGC-3'), it was found that... ​ 6004It carries an unknown fragment of about 5Kb. ​ C), further sequencing revealed that, ​ mutant ​ A 5334bp sequence (SEQ ID NO.32) was inserted at position +488 of the gene. Sequence alignment analysis revealed that this inserted sequence contained another gene. Zm00001eb392640 The full-length sequence.

[0049] Based on the above results, ZmPUB33 Two allelic mutants of the gene ms34-6013 , ms34-6004 In genes Zm00001eb312220 Structural variations causing gene loss of function exist at different positions, and both allelic mutants exhibit similar male sterility phenotypes, proving that... Zm00001eb312220 for ZmPUB33 Genes (as referred to in the following examples) ZmPUB33 All genes refer to genes Zm00001eb312220 ).

[0050] Example 4. Identification of homozygous recessive sterile plants at the seedling stage using diagnostic markers. ZmPUB33 The locus is a homozygous recessive mutant genotype (e.g. ms34-6013 / ms34-6013 Only single plants carrying the mutant gene (genotype 1) can express the male sterility trait. Therefore, the preservation of the sterility gene can only be achieved using heterozygous fertile single plants carrying the mutant gene (genotype 1). ZmPUB33 / ms34-6013 Using this as the male parent, pollination of male-sterile single plants will result in approximately half of the offspring exhibiting the male-sterile phenotype. Therefore, based on... ms34-6013 exist ZmPUB33 Sequence mutations at the site can be identified using the following primer combinations: 6013page-F2588 (SEQ ID NO.33: 5'-GCAAAGTCCATCATAAGACTG-3') and 6013page-R2638 (SEQ ID NO.34: 5'-CAATGGTGCACGGCATAC-3'), as diagnostic markers for identification at the seedling stage. ZmPUB33 The homozygous recessive genotype at the locus is ms34-6013 / ms34-6013 A single plant. This diagnostic marker is in ZmPUB33 Homozygous recessive sterile plants (genotype) ms34-6013 / ms34-6013 It can specifically amplify a 98bp band, and heterozygous fertile plants (genotype) ZmPUB33 / ms34-6013 It can simultaneously detect bands of two molecular weights, 98bp and 96bp. Figure 6 (The black arrow marks the stripe). In ms34-6013 / ms34-6013 × ZmPUB33 / ms34-6013 Among the hybrid offspring, those selected using diagnostic markers ms34-6013 / ms34-6013 All homozygous genotype plants exhibited male sterility upon phenotypic analysis, further proving... Zm00001eb312220 that is ZmPUB33 Candidate genes.

[0051] ZmPUB33 site ms34-6004 Allelic mutations are homozygous recessive mutant genotypes (e.g.) ms34-6004 / ms34- 6004 Only single plants carrying the mutant gene (genotype 1) can express the male sterility trait. Therefore, the preservation of the sterility gene can only be achieved using heterozygous fertile single plants carrying the mutant gene (genotype 1). ZmPUB33 / ms34-6004 As the male parent, it pollinates male-sterile single plants, and about half of the offspring will exhibit the male-sterile phenotype. According to... ms34-6004 exist ZmPUB33 Sequence mutations at the site can be identified using the following primer combination: 6004-MS34-F668 (SEQ ID NO.30: 5'-GGAACCCTGCTCAATCCTACG-3'), 6004-ins-F676 (SEQ ID NO.35: 5'-AGATCGCCTGGCCAAAGAAG-3') / 6004-MS34-R671 (SEQ ID NO.31: 5'-TCCCTTGTACAATAGACCTGCAGC-3'), as diagnostic markers for seedling identification. ZmPUB33 The homozygous recessive genotype at the locus is ms34-6004 / ms34-6004 A single plant. This diagnostic marker is in ZmPUB33 Homozygous recessive sterile plants (genotype) ms34-6004 / ms34-6004 It can specifically amplify a 558bp band, and heterozygous fertile plants (genotype) ZmPUB33 / ms34-6004 It can simultaneously detect bands of two molecular weights, 558bp and 299bp. Figure 7 (The black arrow marks the stripe). In ms34-6004 / ms34-6004 × ZmPUB33 / ms34-6004 Among the hybrid offspring, those selected using diagnostic markers ms34-6004 / ms34-6004 All homozygous genotype plants exhibited male sterility upon phenotypic analysis, further proving... Zm00001eb312220 that is ZmPUB33 Candidate genes.

[0052] Example 5. ZmPUB33Transcriptional level analysis of genes in B73 Roots, stems, leaves, cob, silks, mature pollen, and anthers at different developmental stages were collected from maize inbred line B73 at the tasseling and silking stage. Anther samples of different lengths were collected according to their length; for each sample, 20 fresh anthers of similar length were collected, of which 3 were fixed in FAA solution (Coolaber, China) and their specific developmental stage was determined by resin semi-thin sectioning experiments, and the remaining 17 anthers were immediately frozen in liquid nitrogen for RNA extraction.

[0053] The anthers fixed for resin sectioning were dehydrated using a gradient of ethanol (50%, 70%, 90%, 100%) for 15-30 minutes per step. During dehydration, the anthers could be preserved long-term in 70% ethanol. To facilitate later embedding, 0.1% eosin could be added to 90% ethanol for staining. To ensure thorough dehydration, the material needed to be dehydrated 2-3 times in anhydrous ethanol. Resin replacement was then performed, with the anthers placed sequentially in ethanol-to-Spurr resin solutions at volume ratios of 3:1, 1:1, and 1:3 for 2-4 hours, and finally in pure resin overnight. After resin replacement, the anthers were placed in a mold, 200 µL of Spurr resin was added, and the mold was placed in an oven at 70°C for overnight polymerization. The mold was then trimmed, and sections were prepared using a Leica microtome to a thickness of 2 µm. The sections were then picked up with forceps, placed in sterile water in the center of a glass slide, and incubated overnight at 42°C. Immerse the glass slide containing the fixed sample in 0.1% toluidine blue staining solution for 1 minute, then rinse with deionized water, place on a slide stage, and dry before use for microscopic observation; alternatively, it can be mounted for long-term preservation. Analyze the resin sections and determine the specific developmental stage of each sample based on the cytological characteristics of 14 different developmental stages of maize (Stage 1-Stage 14: S1-S14).

[0054] Total RNA was extracted from the above-mentioned root, stem, leaf, cob, filament, mature pollen samples, and maize anthers at different developmental stages (S5-S12, Reference 2) using Trizol reagent (Invitrogen, USA). cDNA was then synthesized using 5X All-in-One RT Master Mix (ABM, Canada). Quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed using TB Green™ PreMix Ex Taq™ (TaKaRa, Japan) on a QuantStudio 5 Real-Time PCR System (ABI, USA). The amplification primers were: qPUB33-F (SEQ ID NO.36: 5'-TGCAGCTTAGGTTATCAAGG-3') and qPUB33-R (SEQ ID NO.37: 5'-GAAAACGATGATGTAGCAGA-3'). ZmUBI2 was used as the reference gene, and its amplification primers were: UBI2-F (SEQ ID NO.38: 5'-CGACAACGTGAAGGCGAAGA). -3') and UBI2-R (SEQ ID NO.39: 5'-ACCGAGATACAGGTACAGC-3'); each developmental stage included three biological replicates, and each sample had three technical replicates; data were used in 2 -ΔΔCt The methods were analyzed, and the quantitative results are presented in the form of mean ± standard deviation (Means ± SD).

[0055] ZmPUB33 Gene expression was high during the early stage of rachis and anther development (S6), and maintained at a moderate level during the middle and late stages of anther development. Figure 8 ).

[0056] Example 6. ZmPUB33 The effect of gene mutation on pollen pore development The pollen pore is the location where corn pollen germinates. (For analysis...) ZmPUB33 The effect of gene mutations on pollen fertility, specifically on the B73 wild-type. ms34-6013 and ms34-6004Pollen pores of the mutant sterile plants were analyzed using scanning electron microscopy (SEM). Anthers of wild-type and mutant plants at maturity (S13) were excised and immediately fixed in FAA (Coolaber, China) solution, with the volume of the fixative not less than 20 times the volume of the studied material. For mutant anthers, perforations in the anther wall were made using a dissecting needle to improve fixative penetration, or repeated vacuuming was performed until the anthers sank to the bottom of the fixative. After fixation at room temperature for 2 hours, the material was stored at 4°C, or sequentially dehydrated in 50%, 60%, 70%, 80%, 90%, and 100% ethanol, maintaining each gradient for 15 minutes. The material could also be stored overnight in 70% ethanol. After dehydration, the samples were subjected to critical point drying with carbon dioxide and then gold-plated for observation. Findings ms34-6013 and ms34-6004 The appearance of pollen pores in the mutant sterile plants was not significantly different from that in the wild type. Figure 9 Top row).

[0057] For in-depth analysis ZmPUB33 The effect of gene mutations on pollen pore development, and thus pollen fertility, is particularly relevant to the B73 wild-type. ms34-6013 and ms34-6004The pollen pore structure of the mutant sterile plant was analyzed by transmission electron microscopy (TEM). The specific method was as follows: Wild-type and mutant anthers at maturity (S13) were collected and placed in freshly prepared 3% glutaraldehyde fixative. The glutaraldehyde was aspirated, and the anthers were rinsed five times with 0.1 mol / L PBS for 30 min each time. The rinsed anthers were then refixed with 2% osmium tetroxide fixative for 2-3 h. The osmium tetroxide-fixed anthers were rinsed three times with 0.1 mol / L PBS for 15 min each time. The anthers were then dehydrated in a gradient of 50%, 60%, 70%, 85%, 95%, and 100% ethanol. The anthers were then incubated overnight at 4°C with shaking in 100% ethanol. The dehydrated anthers were replaced with anhydrous acetone for 1-2 hours, followed by multiple replacements with resin, with low-speed shaking for 2-3 hours after each replacement, until the anthers were completely submerged in 100% resin. The mixture was then dried overnight in a 30°C oven. The resin-infiltrated anthers were embedded in silicone molds, and the embedding plates were placed in a 37°C oven for 2 hours, a 45°C oven for 2 hours, and a 65°C oven overnight for resin polymerization. Using an ultramicrotome (Leica EM UC6), the anther samples were cut into 70nm ultrathin sections and placed in a water bath. The ultrathin sections were then removed using a copper mesh, which was placed on filter paper to dry, thus fixing the ultrathin sections onto the mesh. First, the ultrathin sections on the copper mesh were stained with 2% (m / v) uranium acetate for 15 min. The copper mesh was then rinsed five times with double-distilled water for 5 min each time. Next, the copper mesh was stained with 2.6% (m / v) lead citrate for 15 min, followed by rinsing with double-distilled water five times for 5 min each time. After drying, the copper mesh was observed and photographed using a transmission electron microscope (Hitachi H-7500). Observation revealed a complete "Z" structure below the pore operculum of the wild-type pollen, while... ms34-6013 and ms34-6004 In mutant sterile plants, the pollen grains without starch filling lack a "Z" structure below the pollen pore cap, while pollen grains with partial starch filling experience pollen pore rupture due to the absence of the "Z" structure. Figure 9 (Bottom row) ms34-6013 and ms34-6004 Defects in the development of pollen pores in mutant sterile plants are the cause of pollen failure to germinate, resulting in complete sterility.

[0058] References 1. Trimnell, MR et al. (1999) New chromosome 7L male-sterile mutantms34. Maize Genet. Coop. Newsletter 73:49; 2. Tian Youhui, Wan Xiangyuan. Research methods of cell biology and molecular genetics of maize anther development. China Biotechnology Journal, 2018, 38: 88-99.

Claims

1. Zea mays ZmPUB33 application of the gene in regulating maize pollen development, characterized in that, Suppress the ZmPUB33 Gene expression and / or activity can induce male sterility in maize plants; ZmPUB33 The gene corresponds to the 5th edition of the maize B73 reference genome. Zm00001eb312220, SEQ ID NO.1 is the one described in Maize Mo17. ZmPUB33 The full-length gene does not contain the DNA sequences of introns 6 and 8; SEQ ID NO. 2 is as described in Mo17. ZmPUB33 The coding region sequence of the gene, SEQ ID NO.3 is the amino acid sequence encoded by this gene in Mo17.

2. Use according to claim 1, characterized in that, Methods of inhibiting the expression and / or activity of the gene, including any of gene editing, RNA interference, T-DNA insertion. ZmPUB33 Methods of inhibiting the expression and / or activity of the gene, including any of gene editing, RNA interference, T-DNA insertion.

3. The method of claim 1 ZmPUB33 Use of the genes in the improvement of corn, characterized in that, The following aspects are included: (1) inhibiting the expression and / or activity of said ZmPUB33 gene, obtaining Zmpub33 a maize male sterile line; (2) the obtained Zmpub33 The male sterile line is crossed and backcrossed with other target materials, so that the target materials obtain Zmpub33 male sterile gene and male sterile trait; (3) comprising Zmpub33 Maize male sterile lines comprising male sterility genes are used in hybrid corn breeding, seed production and corn production.

4. The method of claim 1 ZmPUB33 mutant gene of the gene ms34-6013, characterized in that the mutant gene ms34-6013 In claim 1 ZmPUB33 a deletion of two bases TG at position +16073~+16074 of the 11th exon of the gene, resulting in ms34-6013 a frameshift mutation in the coding region, causing premature termination of translation of the gene.

5. The method of claim 1 ZmPUB33 mutant gene of a gene ms34-6004, characterized in that the mutant gene ms34-6004 In claim 1 ZmPUB33 a sequence of 5334 bp is inserted at +488 of the gene, the inserted nucleotide sequence is shown as SEQ ID NO.

32.

6. The mutant gene of claim 4 ms34-6013 diagnostic marker for identifying the gene of claim 1 ZmPUB33 gene and the mutant gene of claim 4 ms34-6013 characterized in that Using the primer combination of SEQ ID NO. 32 and SEQ ID NO. 33 a 98 bp band can be specifically amplified in the seedling stage ZmPUB33 homozygous recessive ms34-6013 / ms34-6013 sterile plants and ZmPUB33 / ms34-6013 ms34-6013 heterozygous fertile plants, wherein homozygous recessive genotype ms34-6013 / ms34-6013 of the single plants can be specifically amplified a 98 bp band, and the heterozygous fertile plant ZmPUB33 / ms34-6013 can be detected a 98 bp and 96 bp bands simultaneously.

7. The mutant gene of claim 5 ms34-6004 diagnostic marker for identifying the gene of claim 1 ZmPUB33 gene and the mutant gene of claim 5 ms34-6004 characterized in that Using the primer combination of SEQ ID NO. 30 with SEQ ID NO. 31 and SEQ ID NO. 35 with SEQ ID NO. 31 at the seedling stage ZmPUB33 homozygous recessive ms34-6004 / ms34-6004 sterile plants and ZmPUB33 / ms34-6004 ms34-6004 heterozygous fertile plants, wherein homozygous recessive genotype ms34-6004 / ms34-6004 ms34-6004 of the single plants can be specifically amplified a band of 558 bp, and the heterozygous fertile plant ZmPUB33 / ms34-6004 can be detected simultaneously two bands of 558 bp and 669 bp in molecular weight.

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