A maize pollen fertility regulation gene ZmPUB33 and the encoded protein thereof

Abstract

The application belongs to the field of genetic engineering, and discloses a maize pollen fertility regulation gene ZmPUB33 and an encoded protein. The gene regulates maize pollen aperture development and further mediates pollen fertility ability. After mutation, the gene cannot normally blossom, anther husks cannot basically crack, anthers are smaller than wild types, and almost not exposed, pollen grains are not active, and can cause complete male sterility of plants. The sterile line can be used for sterile seed production and production of hybrid crops, and has great application and economic value.

Description

TECHNICAL FIELD

[0001] The present application relates to a maize pollen development regulatory gene ZmPUB33 and its encoded protein, and belongs to the field of genetic engineering. BACKGROUND

[0002] Maize is an important food crop and its heterosis is obvious. Male sterile material is used for hybrid seed production, which can save the cost of artificial or mechanical detasseling and ensure seed purity.

[0003] Maize pollen grain has only one pollen pore, which is a channel for controlling water in and out, ensuring the integrity of pollen grain during the process of dehydration and rehydration from maturity to fertilization. The pollen pore of maize is also a necessary structure for pollen tube germination. Maize pollen grains with defective pollen pores cannot germinate, thereby leading to male sterility.

[0004] The present application provides a DNA sequence of a maize pollen development control gene and its encoded protein. The loss of function of the gene will cause the development defect of maize pollen pore, and the pollen cannot germinate, thereby a new male sterile material can be produced, which has important application value in scientific research and agricultural production. SUMMARY

[0005] The present application provides a new DNA sequence of a maize pollen development control gene ZmPUB33 , a cDNA sequence and an amino acid sequence of the functional protein encoded by the gene. The loss of function of the gene will specifically cause maize pollen sterility.

[0006] The first object of the present application is to provide a new gene for regulating maize pollen development ZmPUB33 , characterized in that it is a DNA molecule selected from 1), 2), 3), 4) or 5) as follows:

[0007] 1) the DNA molecule shown in SEQ ID NO. 1 (cloned from the genomic DNA of maize inbred line Mo17);

[0008] 2) the DNA molecule shown in SEQ ID NO. 2 (cloned from the cDNA of maize inbred line Mo17);

[0009] 3) the DNA molecule formed by one to several base substitutions and / or one to several base insertions and / or deletions and large fragment nucleotide sequence insertions / deletions / transpositions / inversions on the basis of SEQ ID NO. 1, which can affect the fertility of plants;

[0010] 4) DNA molecules capable of hybridizing to the DNA molecule of SEQ ID NO. 2 and encoding a plant pollen development related protein, hybridized and membrane washed in a solution of 0.1 x SSPE (or 0.1 x SSC), 0.1% (w / v) SDS at 65°C;

[0011] 5) DNA molecules having more than 85% homology to the DNA molecule of SEQ ID NO. 2 and encoding a plant pollen development related protein.

[0012] The second object of the present application is to provide a plant pollen development related protein encoded by the above-mentioned gene.

[0013] In one embodiment, the gene encodes a protein as described in 1) or 2) below:

[0014] 1) a protein consisting of the amino acid sequence shown in SEQ ID NO. 3 of the Sequence Listing;

[0015] 2) a protein having one or several amino acid residue substitutions, and / or deletions, and / or additions to SEQ ID NO. 3 of the Sequence Listing and having a plant male fertility related activity.

[0016] The third object of the present application is to provide a recombinant expression vector, an expression cassette, a transgenic cell line or a recombinant bacteria containing the gene and / or the promoter.

[0017] The fourth object of the present application is to provide the use of the above-mentioned gene for transgenic improvement of crops.

[0018] In one embodiment, the gene is used to induce male sterility in crop plants, so as to introduce foreign genes to obtain high-quality transgenic crops.

[0019] In another specific embodiment, the crop is a self-pollinated or cross-pollinated crop.

[0020] In a more specific embodiment, the crop includes but is not limited to corn, wheat, sorghum, rice.

[0021] Compared with the prior art, the present application has the following beneficial effects: the plant floral organ development regulatory gene ZmPUB33 directly involved in the development of the pollen aperture, and when the expression of the gene is inhibited, the pollen aperture development is defective, which ultimately leads to the breaking of pollen homeostasis and the inability to fill starch, or even the pollen grains filled with starch cannot germinate due to the defect of the pollen aperture, resulting in male sterility. Through plant biotechnology, the present application will play a significant role in the utilization of hybrid vigor and the production of hybrid seed in crop breeding.

[0022] Terminology Definition

[0023] 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.

[0024] 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.

[0025] 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).

[0026] 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.

[0027] 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.

[0028] The "gene capable of regulating pollen development in plants" also includes a kind of amino acid sequence capable of translating a function of regulating pollen development in corn, such as the amino acid sequence of SEQ ID NO. 3. The kind of amino acid sequence also includes a variant of SEQ ID NO. 3 having the same function as the natural protein capable of regulating pollen development in corn. The variants include (but are not limited to) deletion, insertion and / or substitution of one or several amino acids, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and / or N-terminal. In the art, substitution with similar or similar amino acids usually does not change the function of the protein; addition of one or several amino acids at the C-terminal and / or N-terminal usually does not change the function of the protein.

[0029] In addition, the nucleotide full-length sequence or fragment of the "gene capable of regulating pollen development in plants" can be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed according to the nucleotide sequence disclosed in the embodiments, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared according to conventional methods known to those skilled in the art is used as a template to amplify the relevant sequence. When the sequence is long, two or more PCR amplifications are often required, and then the fragments amplified in each amplification are spliced together in the correct order. Once the relevant sequence is obtained, recombination can be used to obtain the relevant sequence in large quantities. Usually, it is cloned into a vector, and then the relevant sequence is separated from the proliferating host cells by conventional methods such as cell transformation. In addition, mutations can be introduced into the protein sequence of the embodiments by chemical synthesis. In addition to being produced by recombination, fragments of the embodiments can be produced by directly synthesizing polypeptides using solid-phase technology. In vitro synthesis of proteins can be done manually or automatically, and each fragment of the embodiments can be chemically synthesized, and then linked by chemical methods to produce a full-length protein molecule. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 For normal development of corn wild type Mo17 and ZmPUB33 Gene mutant ms34-6013 Observation of tassel and anther development, pollen KI-I2 staining and pollen germination detection. A, wild type Mo17; B, ZmPUB33 Gene mutant ms34-6013 .

[0031] Figure 2 For ZmPUB33 Fine mapping and map-based cloning results of the gene: A, ZmPUB33 Genetic mapping results of the gene based on the use of 144 sterile single plant mapping population and 5 pairs of molecular markers; B, ZmPUB33The 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 .

[0032] 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: ZmPUB33 Genes 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 results in 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.

[0033] Figure 4 for ZmPUB33 The coding region (CDS) of the gene in wild-type Mo17 and mutant ms34-6013 Nucleotide sequence comparison.

[0034] Figure 5 for ZmPUB33 Genes in wild-type Mo17 and mutant ms34-6013 Comparison of the amino acid sequences translated in the text.

[0035] 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.

[0036] Figure 7PCR 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 ).

[0037] Figure 8 for ZmPUB33 Expression 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.

[0038] 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

[0039] 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.

[0040] 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.

[0041] Therefore, the present application also includes using the sequences described in the sequence listing to regulate the fertility of plants, i.e. using the genetic sequences provided by the present application to affect the function of the same or homologous genes in other plants at the genomic, and / or transcriptomic, and / or proteomic level to achieve the purpose of controlling male fertility. For example, but not limited to the following methods: affecting or changing the function of plant genes by causing gene expression inhibition or loss of protein function through variation of the natural sequence, by introducing an antisense sequence of the gene or introducing a hairpin structure into the plant, or combining the gene with other sequences (DNA or RNA) to produce a new DNA or RNA chain with functional activity, by CRISPR / Cas9 and other similar gene editing methods to inhibit the expression or activity of the gene. Or any one of the technical methods known to those skilled in the art that can be used to affect the male fertility of plants.

[0042] It is worth mentioning that the present application includes corn ZmPUB33 The DNA and coding sequence of the gene are different in different corn inbred genetic backgrounds, such as the ZmPUB33 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 The dominant alleles of the gene have a key role in the male fertility of plants, and the loss-of-function recessive alleles will cause male sterility. The gene is located on chromosome 7 of corn, and the specific location of the gene is shown in Figure 2 .

[0043] The gene sequence and its homologous sequences can be obtained from any flowering plant, including but not limited to corn ( Zea mays ), common wheat ( Triticum aestivum ), sorghum ( Sorghum bicolor ), rice ( Oryza sativa ), brachypodium ( Brachypodium distachyon ), millet ( Setaria italica ), barley ( Hordeum vulgare ), rye ( Secale cereale ), thick goat grass ( Aegilops tauschii ), Arabidopsis ( Arabidopsis thaliana ), cabbage ( Brassica oleracea ), soybean ( Glycine max ), tomato ( Lycopersicon esculintum ), etc. The method of obtaining includes but is not limited to: cloning the gene from corn ZmPUB33Gene 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.

[0044] “ 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 / .

[0045] The following description provides a more detailed account, but it is not intended to limit the scope of the invention.

[0046] Example 1. ms34-6013 Phenotypic analysis of sterile lines

[0047] 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...ms34-6013 Pollen 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.

[0048] Example 2. ZmPUB33 Identification of gene mutants and fine mapping of genes

[0049] 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.

[0050] 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. ms34-6013The gene was located between two markers 6_24 and 8_40, about 0.78 Mb (Table 2) Figure 2 A).

[0051] In summer 2019, the mapping population was expanded to 730 sterile single plants for fine mapping. InDel markers were designed in the linkage interval between the two markers that had been determined (Table 1), and ms34-6013 The candidate gene was located in the interval of 190 Kb between molecular markers 7088R1 and 24_33, in which marker 12_19 was co-segregated with the sterile phenotype (Table 2) Figure 2 B).

[0052]

[0053] The genomic sequence of Mo17 was retrieved from Maize GDB, and after removing pseudogene and transposon sequences, there were 3 functional genes in the 190 Kb interval (Table 3) Figure 2 C), from ms34-6013 After cloning the coding sequences of the 3 genes from the cDNA of sterile plants, it was found that only Zm00014a007089 there was a base deletion mutation, resulting in premature termination of the reading frame, and the coding sequences of the other two genes had no mutations. Zm00014a007089 predicted as a U-box family gene, the corresponding gene in the maize B73 reference genome (version 5) was Zm00001eb312220 , annotated as pub33 in maize GDB. Therefore, the gene was named ZmPUB33 .

[0054] Based on the sequence information of the maize B73 genome, ZmPUB33 The full-length of the gene is 18422 bp, and the structural characteristics of the genomic sequence of the gene are as follows (Table 4) Figure 3A): contains 12 exons and 11 introns, with the first base of the cDNA (transcription start site) as +1 nucleotide, +1 to +210 nucleotides as the first exon, +211 to +706 nucleotides as the first intron, +707 to +947 nucleotides as the second exon, +948 to +1034 nucleotides as the second intron, +1035 to +1153 nucleotides as the third exon, +1154 to +1462 nucleotides as the third intron, +1463 to +1584 nucleotides as the fourth exon, +1585 to +1661 nucleotides as the fourth intron, +1662 to +1855 nucleotides as the fifth exon, +1856 to +2165 nucleotides as the fifth intron, +2166 to +2407 nucleotides as the sixth exon, +2408 to +8620 nucleotides as the sixth intron, +8621 to +8734 nucleotides as the seventh exon, +8735 to +9001 nucleotides as the seventh intron, +9002 to +9115 nucleotides as the eighth exon, +9116 to +15514 nucleotides as the eighth intron, +15515 to +15616 nucleotides as the ninth exon, +15617 to +15702 nucleotides as the ninth intron, +15703 to +16131 nucleotides as the tenth exon, +16132 to +16347 nucleotides as the tenth intron, +16348 to +17103 nucleotides as the eleventh exon, +17104 to +17539 nucleotides as the eleventh intron, +17540 to +18422 nucleotides as the twelfth exon, +749 to +751 nucleotides as the translation start codon, +17741 to +18422 nucleotides as the 3' untranslated region (3'-UTR).

[0055] Example 3. ZmPUB33 Cloning of the gene in the mutant

[0056] Based on ZmPUB33 According to the annotation information of the gene in the B73 genome, the full length of the gene is 18422 bp, containing 11 introns. The primers listed in Table 2 were used to specifically clone the gene in Mo17 and ms34-6013 Specific cloning in the mutant 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.

[0057] 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.

[0058]

[0059] Using the same primer combination from allelic mutants ms34-6004 Cloned Zm00001eb312220 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... ms34- 6004It carries an unknown fragment of about 5Kb. Figure 3 C), further sequencing revealed that, ms34-6004 mutant ZmPUB33 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.

[0060] 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 ).

[0061] Example 4. Identification of homozygous recessive sterile plants at the seedling stage using diagnostic markers.

[0062] 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.

[0063] 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.

[0064] Example 5. ZmPUB33 Transcript level analysis of genes in B73

[0065] Roots, stems, leaves, ears, filaments, mature pollen and anthers at different developmental stages were collected from tasseling stage plants of maize inbred line B73. Anther samples of different lengths were collected according to the length of the anthers; 20 fresh anthers of similar length were collected for each sample, 3 of which were fixed in FAA solution (Coolaber, China) to determine the specific developmental stage by resin semi-thin sectioning experiment, and the remaining 17 anthers were immediately frozen in liquid nitrogen for RNA extraction.

[0066] Fixed anthers for resin sectioning were dehydrated using gradient ethanol (50%, 70%, 90%, 100%) for 15-30 minutes at each step. Anthers can be stored in 70% ethanol during dehydration; to facilitate later embedding, materials can be stained by adding 0.1% eosin to 90% ethanol; to ensure complete dehydration, materials must be dehydrated in anhydrous ethanol for 2-3 times. Then resin replacement was performed, placing the anthers in liquid with a volume ratio of ethanol to Spurr resin of 3:1, 1:1, 1:3 for 2-4 hours, and finally placing them in pure resin overnight. After resin replacement was completed, 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 polymerization overnight. Then the block was trimmed, and sections were cut using a Leica microtome at a thickness of 2 μm; the cut sections were picked up with tweezers and placed in sterile water in the center of a glass slide, and the sections were spread overnight at 42°C. The glass slide with the sample fixed on it was immersed in 0.1% toluidine blue staining solution for 1 minute, then rinsed with deionized water, and placed on a spreading table to dry before being used for microscopic observation; it can also be mounted for long-term storage. The results of the resin sections were analyzed, and the specific developmental stage of each sample was determined according to the cytological characteristics of the 14 different developmental stages of maize (Stage 1-Stage 14: S1-S14).

[0067] Total RNA of the above root, stem, leaf, ear axis, filament, mature pollen samples and identified anthers at different developmental stages (S5-S12, ref. 2) were extracted with Trizol reagent (Invitrogen, USA); then cDNA was synthesized using 5X All-in-One RT Master Mix (ABM, Canada); quantitative reverse transcription polymerase chain reaction was detected on QuantStudio5 QuantStudio 5 Real-Time PCR System (ABI, USA) using TB Green™ PreMix Ex Taq™ (TaKaRa, Japan), and 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 the reference gene, and the amplification primers were: UBI2-F (SEQ ID NO. 38: 5'-CGACAACGTGAAGGCGAAGA-3') and UBI2-R (SEQ ID NO. 39: 5'-ACGCAGATACCCAGGTACAGC-3'); each developmental stage included three biological replicates, and each sample had three technical replicates; the data were given in the form of means ± standard deviation (Means ± SD) after analysis. -ΔΔCt Methods were analyzed, and quantitative results were given in the form of means ± standard deviation (Means ± SD).

[0068] ZmPUB33 The gene was highly expressed in the early stage of ear axis and anther development (S6), and maintained a medium level of expression in the middle and late stages of anther development Figure 8 .

[0069] Example 6. ZmPUB33 Effect of gene mutation on the development of pollen pores

[0070] Pollen pores are the location of corn pollen germination. In order to analyze ZmPUB33 the effect of gene mutation on pollen fertility, 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).

[0071] 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 ms34-6004Transmission electron microscopy (TEM) analysis of mutant sterile plant pollen aperture structure. The specific method is as follows: the anthers of wild type and mutant at the mature stage (S13) are peeled and placed in a freshly prepared 3% glutaraldehyde solution fixing solution. The glutaraldehyde is absorbed, and the anthers are washed with 0.1 mol / L PBS for 5 times, 30 min each time. The washed anthers are fixed with 2% osmium acid fixing solution for 2-3 h. The anthers fixed by osmium acid are washed with 0.1 mol / L PBS for 3 times, 15 min each time. The anthers are dehydrated with 50% ethanol, 60% ethanol, 70% ethanol, 85% ethanol, 95% ethanol, and 100% ethanol in sequence. Shake in 100% ethanol at 4°C overnight. The dehydrated anthers are replaced with anhydrous acetone for 1-2 h, and then replaced with resin in multiple times, each time after replacement, shake at low speed for 2-3 h, until the anthers are completely immersed in 100% resin, and then placed in a 30°C oven overnight. The resin-permeated anthers are embedded in a silica gel mold, and the embedded plate is placed in a 37°C oven for 2 h, a 45°C oven for 2 h, and a 65°C oven overnight for resin polymerization. The anther samples are cut into 70 nm ultrathin sections using an ultramicrotome (Leica EM UC6), and then placed in a water tank. The ultrathin sections are then fished out with a copper mesh, and then dried on filter paper to fix the ultrathin sections on the copper mesh. First, the ultrathin sections on the copper mesh are stained with 2% (m / v) uranyl acetate for 15 min, and then washed with double-distilled water for 5 times, 5 min each time. Then, the copper mesh is placed in 2.6% (m / v) lead citrate for 15 min, and then washed with double-distilled water for 5 times, 5 min each time. After drying, the copper mesh is placed in a transmission electron microscope (Hitachi H-7500) for observation and photography. It is found that the wild type pollen aperture has a complete "Z" structure under the cap, while the mutant sterile plant pollen aperture of the pollen grain without starch filling lacks the "Z" structure, and the pollen grain with partially filled starch also has a burst pollen aperture due to the lack of "Z" structure ms34-6013 and ms34-6004 the mutant sterile plant pollen aperture of the pollen grain without starch filling lacks the "Z" structure, and the pollen grain with partially filled starch also has a burst pollen aperture due to the lack of "Z" structure Figure 9 lower row). ms34-6013 and ms34-6004 The defect in the development of the pollen aperture of the mutant sterile plant is the reason for the failure of pollen germination and complete sterility.

[0072] REFERENCES

[0073] 1. Trimnell, MR et al. (1999) New chromosome 7L male-sterile mutant ms34. Maize Genet. Coop. Newsletter 73:49;

[0074] 2. Tian Y, Wan X. Research methods of cell biology and molecular genetics of maize anther development. Chinese Journal of Biological Engineering, 2018, 38: 88-99.

Claims

1. Corn ZmPUB33 The application of genes in regulating maize pollen development is characterized by, Suppress the ZmPUB33 Gene expression and / or activity to induce male sterility in maize plants; SEQ ID NO.2 is the one described. ZmPUB33 The coding region sequence of the gene, SEQ ID NO.3 is described. ZmPUB33 The amino acid sequence encoded by the gene.

2. The application according to claim 1, characterized in that, Suppress the ZmPUB33 Methods for gene expression and / or activation, including gene editing, RNA interference, and T-DNA insertion.

3. The corn as described in claim 1 ZmPUB33 The application of genes in maize improvement is characterized by, Suppress the ZmPUB33 Gene expression and / or activity, acquired Zmpub33 Male-sterile maize lines will be obtained Zmpub33 Maize male-sterile lines are hybridized and backcrossed with other target materials to obtain the desired male-sterile line. Zmpub33 Male sterility genes and male sterility traits will include Zmpub33 Male-sterile maize lines with male-sterile genes are used for hybrid maize breeding, seed production, and maize production.

4. Identification of maize at the seedling stage using primer combinations SEQ ID NO.33 and SEQ ID NO.34 ZmPUB33 homozygous recessive at loci ms34-6013 / ms34-6013 sterile single plants and ZmPUB33 / ms34-6013 Its application in heterozygous fertile plants is characterized by... Identification of maize at the seedling stage using primer combinations of SEQ ID NO.33 and SEQ ID NO.34 ZmPUB33 homozygous recessive at loci ms34- 6013 / ms34-6013 sterile single plants and ZmPUB33 / ms34-6013 Heterozygous fertile plants, among which homozygous recessive genotype ms34- 6013 / ms34-6013 A single plant can specifically amplify a 98bp band, and heterozygous fertile plants ZmPUB33 / ms34-6013 It can simultaneously detect bands of two molecular weights, 98bp and 96bp.

5. Primer combinations SEQ ID NO.30 and SEQ ID NO.31 and SEQ ID NO.35 and SEQ ID NO.31 were used to identify maize at the seedling stage. ZmPUB33 homozygous recessive at loci ms34-6004 / ms34-6004 sterile single plants and ZmPUB33 / ms34- 6004 Its application in heterozygous fertile plants is characterized by... The primer combinations SEQ ID NO. 30 and SEQ ID NO. 31 and SEQ ID NO. 35 and SEQ ID NO. 31 were used to identify maize at the seedling stage. ZmPUB33 homozygous recessive at loci ms34-6004 / ms34-6004 sterile single plants and ZmPUB33 / ms34-6004 Heterozygous fertile plants, among which homozygous recessive genotype ms34-6004 / ms34-6004 A single plant can specifically amplify a 558 bp band, and heterozygous fertile plants ZmPUB33 / ms34-6004 It can simultaneously detect bands of two molecular weights: 558bp and 669bp.

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