CN116555472A - Corn pollen quantity related protein RPN1 and application thereof - Google Patents
Corn pollen quantity related protein RPN1 and application thereof Download PDFInfo
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- CN116555472A CN116555472A CN202310446021.5A CN202310446021A CN116555472A CN 116555472 A CN116555472 A CN 116555472A CN 202310446021 A CN202310446021 A CN 202310446021A CN 116555472 A CN116555472 A CN 116555472A
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
- C12N15/827—Flower development or morphology, e.g. flowering promoting factor [FPF]
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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- C12Q2600/00—Oligonucleotides characterized by their use
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
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Abstract
The invention discloses a corn pollen quantity related protein RPN1 and application thereof. The technical problem to be solved is how to regulate and control the quantity of plant pollen. Specifically discloses application of protein, a substance for regulating and controlling expression of a coding gene of the protein or a substance for regulating and controlling activity or content of the protein in regulating and/or controlling the quantity of plant pollen and/or preparing a product for regulating and controlling the quantity of plant pollen and/or preparing a plant breeding product, wherein the protein is shown in a sequence 2; and molecular markers found in nature thereof. The method can realize directional regulation and control of the quantity of plant pollen, and can utilize related molecular markers existing in nature to carry out plant breeding.
Description
Technical Field
The invention particularly relates to a corn pollen quantity related protein RPN1 and application thereof.
Background
Corn is a unique crop with strong male inflorescences in main cultivated crops, and the corn has pollen quantity far exceeding that required by normal pollination due to the characteristics of hermaphrodite plants, air-borne pollination and the like and natural selection of long-term survival competition. Pollen formed by male germ cells of maize is a "pool" formed by the transformation of photosynthetic primary products, requiring substantial photosynthetic energy consumption during production, which affects later grain photosynthetic products. At present, the research on the generation of male germ cells from vegetative growth to germ growth is relatively clear in the time dimension, but in the number dimension, there is no report on how many somatic cells enter the germ cells, so we expect to analyze the quantitative determinants in the process of switching from somatic cells to germ cells by utilizing the characteristic of the number of male germ cells.
During the generation reproduction and genetic recombination of organisms, the development of the reproductive system plays a vital role, while the formation and development mechanisms of the reproductive organs of animals and plants are very different. The formation of the reproductive organs of multicellular animals originates from embryonic stages, differentiating from stem cells into primordial germ cells. The higher plant only has root, stem, leaf and other nutritive organs in early stage, and then the first flower flowers are used as critical stage plant to convert from vegetative growth to reproductive growth, and the sub-epidermal layer begins to differentiate into flower primordium and other reproductive organs. Most of the plant's reproductive organs remain somatic, and only a few differentiate into germ cells. Researchers have early discovered that an SPL gene (also known as NZZ) in arabidopsis plays an important role in the regulation of plant male germ cell differentiation and development to initiate the generation of male germ cells. Several genes have also been found in recent studies that regulate the number of male germ cells in crops during early anther development: the maize mutant anther of the MAC1 gene produces a greater than normal level of sporogenic cells; the msp1 gene mutant in rice produced more microsporocytes than the wild type. The genetic regulation of the plant male gametophyte has a very complex mechanism network, and the effective gene location of the quantitative trait of the plant male germ cells by utilizing the molecular genetic technology plays an important role in deeply knowing the genetic regulation mechanism of the plant male gametophyte.
Corn, one of the most important crops in the world, has a common cross pollination habit. In seed production, one line of male parent is required to produce pollen for multiple lines of female parent, so a sufficient amount of pollen has an important role in ensuring seed production yield of corn. Early researchers have developed a number of genes that regulate maize anther and pollen development based on mutant studies. However, the regulatory mechanism of pollen quantity variation in natural populations is not yet clear.
Disclosure of Invention
The technical problem to be solved by the application is how to regulate and control the pollen quantity of corn, in particular to improve the pollen quantity.
In order to solve the above-described problems, the present application provides the following applications.
Use of a substance that detects a polymorphism or genotype of a SNP to identify or assist in the quantity of maize pollen, or to prepare a product that identifies or assists in the identification of the quantity of maize pollen, or to maize breeding or to prepare a maize breeding product;
the SNP comprises SNP1 and/or SNP2;
SNP1 is one SNP of corn genome, is 4872 nucleotide of SEQ ID No.3 in the sequence table, it is C or T;
SNP2 is one SNP of the corn genome, is the 3476 th nucleotide of SEQ ID No.3 in the sequence table, and is G or A.
In this application, the corn may be wild type W22, wild type B73, mutant UFmu-09290 or EMS4-0bc5bd.
The substance may be a product. The detection substance may include reagents, kits and apparatuses for detecting the above-mentioned single nucleotide polymorphism. Specifically, primers and other reagents and instruments required for in vitro amplification of nucleic acids for detecting the above single nucleotide polymorphisms.
In order to solve the above problems, the present application also provides methods for identifying or aiding in the identification of maize germplasm.
Detecting the genotype of the SNP in the corn to be detected, wherein the number of corn pollens with the genotype of SNP1 being CC is higher than or candidate higher than that of corn with the genotype of SNP1 being TT, and the number of corn pollens with the genotype of SNP2 being Aa is higher than or candidate higher than that of corn pollens with the genotypes of SNP2 being Aa and AA;
SNP1 genotype is CC and is homozygous that the 4872 nucleotide of SEQ ID No.3 in the sequence table is C; SNP1 genotype TT is homozygous with the 4872 th nucleotide of SEQ ID No.3 in the sequence table as T;
SNP2 genotype aa is homozygous with the 3476 th nucleotide of SEQ ID No.3 in the sequence table as A; SNP2 genotype AA is the homozygote of the 3476 th nucleotide of SEQ ID No.3 in the sequence table as G; SNP2 genotype Aa is a hybrid of A as 3476 th nucleotide of SEQ ID No.3 in the sequence table and G as 3476 th nucleotide of SEQ ID No.3 in the sequence table.
The sequence 3 is specifically as follows:
GGGTTGTACATGCCCTCGGGCGGTTCCTGAGGGCTCGCGCTCGCTGCATCTGACCGCGCCGGCGGGCGCACGTGGCAACGTCCGCCGGCCGACGAGCTTTAGAATCCACGCGTCTCCTCCCCTCGCAACTCGCGACATTCGCCGATTCGAATCCCACCCACGACCCCCGTCCCGTCCCGTCCCGTCCGCCGCTGCCACCACCACCCCACCCCAGAAACCTGACCTCCGCGCCCCCGCGCGTCATTGAGACTCCTGCGCGCCGCCGAATATTATTATTCTCATCCCGCCGCGGCCGCAGGCAGGGCCAGTTCCGTCCGACGCGTCGCAACAACCCCCAGGGCTCAATTGACGCGCGGGGGCGTTTCGGTCATCCAGTCATCCCCCTCCCCTGCCAAAGGCGAAGAGCAGAAGCAGAAGCAGCAGCAGCGTACGCCCTTTTCCTTTTTCTAGTTCCGTGTGCGCTGCCTGCTACCTTTGCTTCTCGTTCTTCCCCCTCGCCTCTCTCTCCTTCCACCGAGTCACGCCGCTTCCAGAAACAAGGGAGGGAACCGGAGACCCGGGAGCCGAGCAGAGTCGCACCAAATGCTCTGATCCCCGTTCGTGGGCTTTCCTCCCCGTCGATCCCTCCTCCATGGCGCGCGTCCTCCCGCTCGAGACTGCCGAGATGGCCAGGTACGCGCGCGGGGTCAAAAAAAAAAAACTTCTTCATCAACTTCAGATTACCTCTTTCCTTCCTCTCATCCCCTCCCCCCCTGGAAATTTTGCTTCGGCTGCAGGCTTGAGAGACTCTGTTCGGTATTTCTGTTGCGTTAGAGTTCTTCTTAAGGTCGTGAGGTGTGGTGTAGCTGCCGCCCGCAGGCACAGGTGGATTGCGCTTCTTCACGGGGTTTTCATTCGTTCCGGGAGGAAGATGTTCTTCTTGCTCCCTTCCTTCTTTCCGCTTGGTCGGGAAATGTCCCGTGGCCCCCACACCTTTTCCTTGCTCGGTGCGCTTAAGTTTCCAAGAATGGCGGTCGGTGGTGCCTTCCGGAGCAACGGCGGCCGCCGCCGGCCAGCCACCGGAGCCGCCGAGCGTCCATGCCACGCCACGCGTCGCCGTGTGGTCACCCCTGGTGCCAGTGTCACGCACGCGCCGCGCGTGGATCTTTTTTAAAAACGTAGAGTCCGAAGCTTGGTCGAATTTGGGGAACATAAT
TATTGCTACTAGTTAATGATGTTCCAACTTCCAATCCTATCCGAGTATATTAAAATCTCTCTCGTATGAGTATGAGAGTA
TCTCGATTGGGCCACTGGCCGTTGCACTGTACCGACTGGTGGTGGTGGGTGGTTTCATAGACTACTGTGGTTGTGGATGA
ACTGTACTGATGAACACAAGTGGATGCTTTTGACAATCTTGCTCTTGCACTGATGTAGTTGCAATACTACACCAAACAAA
GAACACATGGCGGACCTTTTTTGTGGGTAAATTCGACTTTGTTTCACTAGGGTCGTCAAATGTGACCTTGAAATAAAGAG
GGCAGGTAATGTTTTACTGCTAATTTTAGCCAGAGAGCACCTGCACTTCAACTTCCAAAGAGACTGCAACCTAGAACCTA
TGTTCCCCTCATTCCGTTGGCTGGGTTTCTATTGTGCATGCGTGAATACCGTGGTATTGTATATATTGCAAAAATGAGAT
CTCATTACGATAGCATGCGAACCATATAAGGTTCCTTAGTTGATTAGTTCGCTTCTTGGATATATGCTACATGGTAGCCT
TCTGGCACAGTTTCATAAGCTGTTCTAGTATACAGCACAAGTATGTCACAGCCTTACATTGATATGTTTTGTTTGGTTGA
TAAACAGAGGCGAAGAGGCAGTGGTAGACATGCCAAGGTCATCAGCCGGGGCGTCATGCACAGCAGTTGACCATTCTGAG
CAGAGCAGAAAGGATGATGAATATGAGAGGCTGGTTACAAGAGCTCAACATGCAACATCTGATGTTGGCACGACAATTTT
ATCTGAGCAACCAAAGTCAAGATCTTTCATTTGGTGGATGAAAGTTCTGCTTGGCTGCTTCCTTCTTATATTAGTGGGCT
ATGTCTTCGTGAAATGGGGGGTCCCCTTTGTCTTTGAGAAGGTGAGCACTGATACTCTTTATCACCACTTCTGCAGGCAT
TCTTTTCTCTAACCTGAAAGGTCTTGTCATCTGCATAGATGAATCAGATCATTAGGATATTGCTAGGACAACAACAATTA
TATTTCAGAGATAAACTGAAATATGCTACTTCAAAAGGACTTAAAGGATAGTTACATTTTTTTGTCTGATGGCTTGCTTG
ATTGATATGGTACATATGCCTCGAACCTTCAAGATATACGTTTCTTATGCTTGCTAATTACTTCAAACTGTCATGATAAA
CAATACTCCCTCCATTCCAAAATATAGTTCTTTCTAGCCCTCTTTTTTCCGACCTCATTCGAATGAATAATAATGAAAAT
AGATATACAAACAAACTACATTCATAGGTTAATTAATGAATGCATGTTTAGTCAAACAAAGTATATTTTGAGACAGAGGG
AGTATAAAAAACTTGACTTACGGGGGTATTGCATTAATAAGAAGACCTCACCTTGGTCGAGAAACCCTTGAACCCCTGCC
TCGTCTATACACAACAACACCGTATACCATGTGAGACTGACTGGGACCATGCCATAGACATCTGCTTTGGCGTGAGACCA
GTGGTGGACCCAGAAAATTTCCAAAGCCTGGGCAAATATTAGAAGATAACATTGCCACAAACATAAAGCTTAATTGTTGC
CCACACTAAAGTATATTTTACTGCTGAGATACATATATTTATACACGTTTGCAGCAGGTGAGCCCGGGCAGCCGCTCGGG
GCAGCCGGGCGGTGACAGGCGAGAGGTTTTTTTAACCTGAAATTTGTGGCCTTAGACATGTGCTTTTGCGCTTCCATGAG
GAGTCGAACTCAGGACCTAAAGAGTGCAACTCAGATCACTTATCGACTCAGCTAGAAGCCCTTTCACCATGATAAAGAAT
ATGCAAGATGTCTTAAATATCTTTTTTATGCTATTTGGAATAAAAAAATAAGTAGGTTTTAGATGTATATATGCCATAAA
GCTTGTAGTTCAGAAATTATCACTCAGTAACAAACACATGGTACCAGGGCCCATGTCTCGTTGTATCCTACAACACTGGT
GATTTTTTTTTCTTAAAAACAGTGAGTATGCTGTACTTCAAGTGGCAAACATCCTAGCACACTCTTATCATTTTGTAACA
AGCAGTTGATAAGTTGATACAATATGTTGTAGTTGAAGTGAAATGTATTTTGTGGTCTATGTCTTTATGAAATTATCGCT
TGTGCCATCCTTCTTCATGCTATTTTGTGTTAGTTACATGCTTTCTCACAGCACTTCTTCAAATTATTGATACATGTACT
TTTCCTGTTTGTTGTAGGTTCTCTTGCCAATCATGCAATGGGAAGCCAGTGCTTTTGGGCGTCCAGTATTAGCTATTGTT
CTCGTTGCATCTTTGGCTCTCTTTCCAGTTATTCTAGTTCCTTCTGGACCTTCTATGTGGTTAGCAGGAATGATCTTTGG
ATATGGTTGGGGTTTCTTGATTATAATGGCCGGGAGTACTATTGGCATGGTGGTTCCATATTGGATTGGTTCATTGTTCC
GTGAACGTCTGCATGTAAAATTTCTAAGCCATATGTTCTGTGATTTGTAATTTAAGAGGTAGATCTTTTCTATGTTAGGA
TTATTCCCTGATTCGTTTTTTTTCTCTACTTCCTTTCTCTGTTTCTTTTTAAAAATACAGGTATGGCTGACAAAGTGGCC
TCAGCAGATAGCACTAATAAAACTCGCGGGTGAAGGAAATTGGTTCCAGCAGTTTCGGGTTGTCGCGCTTTTCAGAATCT
CACCATTCCCATATACAATTTTTAATTATGCCGTGACTGTGACAGAAATTAAGTTCAACCCTTATCTATGTGGTTCTGTT
GCTGGAATGGTACCTGAGGCCTTCATTTATATCTACAGGTTAGACATGATCTCTAATACATTGCAGATTATCGATCCATT
GAGTTTAGCATGTTCTTTTTTTCATCTACCATGTAAATCTGTATCATCTACTTGTGTGCGAATTCTTTTCACATGATTCA
CGTACTGAGAAAGTAGCATGCCGAATTTCGGACCATGAATCTAACCAATCATTGTTTCTTATATAAGTTCACCAGTTGAA
GGATATCTTGCTTCTTGGTGTACTCTTCTTATAAGCTACTCCCTCCGTCCCAAATTAAAATTCGTTTTACCTTTTAAATA
GATTCATAAAATAATTAATGTATGTGTTCTATATACGTGTCTAGATTCTTCATCATCAATTTGAATATAGACATAAAAAT
AAAGAGCTACAATGACTAATATTTCGTGATGGGGTACTATATAATATATCTGACCATGTAGAAGTTTATTTAGTTTATTT
GAAAACTTCGTTGAGTTTTTTTGGTTCCCGGTATATTGGGACAACTCACCCAATGTACTTACTAAGTTATTGAATGCATT
TATATTGGTTCCCTGTGTTTTGCTTATGTTGCTGTGTTTTGCTTATGTTGCCGTGTTTTTTTTTTTGGTGCAGTGGGCGT
TTAATACGTACATTGGCTGACATGAAGTACGGCAACTATAAAATGACACCAGTGGAAATAGCATACAACGCCATTTCCTT
CGTCATCGCTATCGCCCTCACGGTCGCCTTCACGGTGTACGCGAAGAGAGCTCTGGGTGACATAAAAAGTCCGGACGATG
GCATCGGTAAAGACGAAGAAGATCACGGCCCAAATGGCTCAGGGGTGCGTATGAATCGTCGTCAGGAGCGTGCCGACGCA
CGTTACATAGAACTAGATGATATGTGATGGTGTGTTGGCCCGGATCTTGCTTGGAAGGAGGCACCAGTAGGTCATTAGGT
GCACGGCTACGGTAGGTAGCTAGCTATAGTTTACAAGAGGAGGCTAAAATAATCCACACCCAGCTGACGTGGTTTGCGTG
ATTCGTTTCGTGTGCTTTGCCTACTGCACTGCCATACTGTCTGAATCTGAAGGTGCAGGGCTACTCACTACTGAGTTACT
GACCGAGCGATGCTGCTCTCAACGATGTTTCTGTGACCATCAGCGTATCAATCAGTCCTTGATCTGAAGCCTACACTGGA
GCTTCATGTTGAAGCATACATTGGACTTGAGTAGTACATTTGTATATAACGTAACTGCTAATAGAGTTAGCATATGATCT
AGTTTTGAACATGAAGCTCGTGGGCTGTTCAGCCATGTTCGGTCCCATCCTGTCTCTGTCTCAAATTATATGTTATTATGGGTTTTGTTTTTTCTGT。
in the above, the corn variety to be tested is wild type corn B73 or W22.
The maize variety to be tested is the offspring of the wild type maize B73 and W22 crosses.
The application of the method in corn breeding.
To solve the above problems, the present application provides a product.
The product contains the substance for detecting the SNP polymorphism or genotype of the corn genome, and is any one of the following G1) to G3):
g1 A product for detecting a single nucleotide polymorphism or genotype associated with the quantity of maize pollen;
g2 Identifying or aiding in identifying a product of maize pollen quantity;
g3 A product for corn breeding.
In the above, the product is applied to corn pollen quantitative breeding or improvement.
In the above, the purpose of the breeding in the above applications, methods or products includes maize variety breeding. The corn variety breeding may be to obtain corn with more pollen count values or corn with less pollen count values.
The above application, the above method, and the above product, wherein the product of the substance for detecting a SNP polymorphism or genotype of a corn genome is D1), D2), D3), or D4):
d1 An in vitro nucleic acid amplification primer that specifically amplifies said SNP 1;
D2 An in vitro nucleic acid amplification reagent comprising D1) the in vitro nucleic acid amplification primer;
d3 A kit comprising D1) the in vitro nucleic acid amplification primer or D2) the in vitro nucleic acid amplification reagent;
d4 A detection instrument comprising D1) said in vitro nucleic acid amplification primer, D2) said in vitro nucleic acid amplification reagent, or D3) said kit.
The in vitro nucleic acid amplification technique may be Polymerase Chain Reaction (PCR), strand Displacement Amplification (SDA), ligase Chain Reaction (LCR) and nucleic acid sequence dependent amplification (NASBA), rolling circle nucleic acid amplification (RCA), loop-mediated isothermal amplification (lamp), helicase dependent isothermal amplification technique (HDA) or qβ replication technique.
The present application uses Polymerase Chain Reaction (PCR) as an amplification means to detect polymorphisms.
D1 The specific amplification can be performed by detecting the nucleotide sequence of the SNP1-SNP40 polymorphic site by the presence or absence of the amplification product or by the presence or absence of the amplification product in combination with an auxiliary reagent such as a probe.
In the above applications, methods and products, the in vitro nucleic acid amplification primers may or may not be labeled with a label. The label refers to any atom or molecule that can be used to provide a detectable effect and that can be attached to a nucleic acid. Markers include, but are not limited to, dyes; radiolabels, such as 32P; binding moieties such as biotin (biotin); hapten such as Digoxin (DIG); a luminescent, phosphorescent or fluorescent moiety; and fluorescent dyes alone or in combination with a portion of the emission spectrum that can be suppressed or shifted by Fluorescence Resonance Energy Transfer (FRET). The label may provide a signal detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, or the like. The label may be a charged moiety (positive or negative) or alternatively may be charge neutral. The label may comprise or be a combination of nucleic acid or protein sequences, provided that the sequence comprising the label is detectable. In some embodiments, the nucleic acid is detected directly without a label.
In the present application, the primer of D1) may be as follows: RPN1-EMS-F TGGCAAACATCCTAGCACAC;
RPN1-EMS-R:TCTGTCACAGTCACGGCATA。
the use of the above method in maize breeding.
The number of the corn pollens with the SNP2 genotype Aa is higher than or is higher than the number of the corn pollens with the SNP2 genotypes Aa and AA;
SNP2 genotype aa is homozygous with the 3476 th nucleotide of SEQ ID No.3 in the sequence table as A; SNP2 genotype AA is the homozygote of the 3476 th nucleotide of SEQ ID No.3 in the sequence table as G; SNP2 genotype Aa is a hybrid of A as 3476 th nucleotide of SEQ ID No.3 in the sequence table and G as 3476 th nucleotide of SEQ ID No.3 in the sequence table.
The corn may be a filial generation of a corn carrying the SNP2 and having nucleotide a at position 3476 of SEQ ID No.3 and a corn carrying the SNP2 and having nucleotide G at position 3476 of SEQ ID No. 3.
The 3476 th nucleotide carrying SEQ ID No.3 is homozygous for A.
The A maize can be maize EMS mutant EMS4-0bc5bd and the B maize can be B73.
In the above-mentioned application, the purpose of the breeding is to breed high pollen corn.
In the above, the pollen quantity of the corn with high pollen quantity is higher than that of the parent.
In order to solve the above-described problems, the present application provides the following applications.
Use of a protein, a substance regulating expression of a gene encoding said protein or a substance regulating activity or content of said protein for regulating the amount of maize pollen and/or for the preparation of a product regulating the amount of maize pollen and/or for maize breeding and/or for the preparation of a maize breeding product; the protein is any one of the following:
a1 Amino acid sequence is a protein shown in sequence 2;
a2 Protein which is obtained by substituting and/or deleting and/or adding amino acid residues of the protein A1), has more than 80 percent of identity with the protein A1) and has the function of regulating and controlling the quantity of corn pollen;
a3 Fusion proteins obtained by ligating the N-terminal or/and C-terminal of A1) or A2) with a protein tag.
Among the above proteins, the protein tag (protein-tag) refers to a polypeptide or protein that is fusion expressed together with a target protein by using a DNA in vitro recombination technique, so as to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag may be a Flag tag, his tag, MBP tag, HA tag, myc tag, GST tag, and/or SUMO tag, etc.
In the above proteins, the identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, the identity of a pair of amino acid sequences can be searched for by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, per residue gap cost and Lambda ratio to 11,1 and 0.85 (default values), respectively, and calculating, and then obtaining the value (%) of the identity.
In the above protein, the 80% or more identity may be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.
In the above protein, sequence 2 (SEQ ID No. 2) consists of 347 amino acid residues. This was designated as RPN1 protein. The coding gene is RPN1 gene.
The sequence 2 is specifically as follows:
MARVLPLETAEMARGEEAVVDMPRSSAGASCTAVDHSEQSRKDDEYERLVTRAQHATSDVGTTILSEQPKSRSFIWWMKVLLGCFLLILVGYVFVKWGVPFVFEKVLLPIMQWEASAFGRPVLAIVLVASLALFPVILVPSGPSMWLAGMIFGYGWGFLIIMAGSTIGMVVPYWIGSLFRERLHVWLTKWPQQIALIKLAGEGNWFQQFRVVALFRISPFPYTIFNYAVTVTEIKFNPYLCGSVAGMVPEAFIYIYSGRLIRTLADMKYGNYKMTPVEIAYNAISFVIAIALTVAFTVYAKRALGDIKSPDDGIGKDEEDHGPNGSGVRMNRRQERADARYIELDDM。
in this application, the modulation may be up-regulation or enhancement or elevation. The modulation may also be down-regulation or attenuation or reduction.
In the above, the up-regulation or enhancement or increase of the expression of the gene encoding the above protein or the activity or content of the above protein can reduce the number of plant pollens. The regulation can also be down regulation or weakening or reducing the expression of the coding gene of the protein or the activity or the content of the protein can increase the quantity of the corn pollen.
In the above uses, the protein is derived from corn.
Above, the corn may be wild type B73, wild type W22, mutant UFmu-09290 or EMS4-0bc5bd.
In the above, the substance that regulates gene expression may be a substance that performs at least one of the following 6 regulation: 1) Regulation at the level of transcription of said gene; 2) Regulation after transcription of the gene (i.e., regulation of splicing or processing of the primary transcript of the gene); 3) Regulation of RNA transport of the gene (i.e., regulation of nuclear to cytoplasmic transport of mRNA of the gene); 4) Regulation of translation of the gene; 5) Regulation of mRNA degradation of the gene; 6) Post-translational regulation of the gene (i.e., regulation of the activity of the protein translated by the gene).
In the above-mentioned applications, the gene encoding the protein may be any of the following:
c1 A DNA molecule having a nucleotide sequence of the coding strand of sequence 1;
c2 Nucleic acid molecules which are obtained by substituting and/or deleting and/or adding nucleotides of the nucleic acid molecules of the C1), have more than 80 percent of identity with the nucleic acid molecules shown in the C1) and have the function of regulating the quantity of corn pollen.
The sequence 1 is specifically as follows:
ATGGCGCGCGTCCTCCCGCTCGAGACTGCCGAGATGGCCAGAGGCGAAGAGGCAGTGGTAGACATGCCAAGGTCATCAGC
CGGGGCGTCATGCACAGCAGTTGACCATTCTGAGCAGAGCAGAAAGGATGATGAATATGAGAGGCTGGTTACAAGAGCTC
AACATGCAACATCTGATGTTGGCACGACAATTTTATCTGAGCAACCAAAGTCAAGATCTTTCATTTGGTGGATGAAAGTT
CTGCTTGGCTGCTTCCTTCTTATATTAGTGGGCTATGTCTTCGTGAAATGGGGGGTCCCCTTTGTCTTTGAGAAGGTTCT
CTTGCCAATCATGCAATGGGAAGCCAGTGCTTTTGGGCGTCCAGTATTAGCTATTGTTCTCGTTGCATCTTTGGCTCTCT
TTCCAGTTATTCTAGTTCCTTCTGGACCTTCTATGTGGTTAGCAGGAATGATCTTTGGATATGGTTGGGGTTTCTTGATT
ATAATGGCCGGGAGTACTATTGGCATGGTGGTTCCATATTGGATTGGTTCATTGTTCCGTGAACGTCTGCATGTATGGCT
GACAAAGTGGCCTCAGCAGATAGCACTAATAAAACTCGCGGGTGAAGGAAATTGGTTCCAGCAGTTTCGGGTTGTCGCGC
TTTTCAGAATCTCACCATTCCCATATACAATTTTTAATTATGCCGTGACTGTGACAGAAATTAAGTTCAACCCTTATCTA
TGTGGTTCTGTTGCTGGAATGGTACCTGAGGCCTTCATTTATATCTACAGTGGGCGTTTAATACGTACATTGGCTGACAT
GAAGTACGGCAACTATAAAATGACACCAGTGGAAATAGCATACAACGCCATTTCCTTCGTCATCGCTATCGCCCTCACGG
TCGCCTTCACGGTGTACGCGAAGAGAGCTCTGGGTGACATAAAAAGTCCGGACGATGGCATCGGTAAAGACGAAGAAGAT
CACGGCCCAAATGGCTCAGGGGTGCGTATGAATCGTCGTCAGGAGCGTGCCGACGCACGTTACATAGAACTAGATGATATGTGA。
advantageous effects
In the invention, the natural variation of the pollen quantity of the corn is utilized, and the pollen quantity is measured in the corn association population, so that the corn is found to have wide natural variation in the pollen quantity. The MLM model is utilized to analyze the fertility character of pollen, and we excavate a candidate gene which is obviously related to the quantity of corn pollen, namely the protein RPN1 involved in regulating and controlling the quantity of corn pollen is obtained, and the gene verification and the function analysis are carried out on the protein RPN 1. Amino acid sequence comparison analysis shows that the gene belongs to TVP38 family and contains a highly conserved SNARE-associated structural domain. The mutant of the gene backcross 3 generations and EMS mutant are utilized to verify that the mutant of the RPN1 can obviously increase the quantity of the corn pollen, which proves that the RPN1 plays an important role in regulating the quantity of the corn pollen.
The application discloses a corn pollen quantity related protein RPN1 and application thereof. The application finds out an SNP related to the pollen quantity by analyzing 391 corn inbred line with wide variation and locates the SNP on a gene GRMZM 5G806784 (V4 version gene number is Zm00001d 047403), which is also called RPN1 gene. The gene sequence has the following polymorphism: the 4872 nucleotide of SEQ ID No.3 in the sequence Listing is C or T, and is designated as SNP1. The corn plant with SNP1 locus genotype of CC has two chromosomes in the genome as follows: the 4872 base of sequence 3 (SEQ ID No. 3) is C. The corn plant with SNP1 locus genotype TT has two chromosomes in the genome as follows: the 4872 base of sequence 3 (SEQ ID No. 3) is T. The pollen quantity of the corn with the genotype of the SNP1 locus CC is obviously higher than that of the corn with the genotype of the SNP1 locus TT.
Further, a maize mutant UFU-09290 with the function of RPN1 deleted by T-DNA insertion is found, after the mutant UFU-09290 is backcrossed with W22 and the mutant EMS4-0bc5bd is backcrossed with B73 for 3 generations, the selfing is carried out again, the consistency is found, and the quantity of maize pollen is increased due to the function of RPN1 deleted.
Drawings
FIG. 1 is a schematic representation of a protein conserved domain.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The following examples use EXCEL statistical software to process data, with experimental results expressed as averages, using the T-test. P < 0.05 (x) indicates a significant difference, P < 0.01 (x) indicates a significant difference, and P < 0.001 (x) indicates a significant difference.
The W22 mutants referred to in the present invention are available in the maize genome public database (www.maizeGDB.org).
Maize W22 is described in the following documents: huang C, chen Q, xu G, xu D, tian J, tian F.identification and fine mapping of quantitative trait loci for the number of vascular bundle in maize stem.J Integr Plant biol.2016Jan;58 (1) 81-90.Doi:10.1111/j ipb.12358.Epub 2015Jul 16.PMID:25845500; PMCID PMC5034846.
Maize material 177, maize material 238, maize material 5237, maize material 7327, maize material 526018, maize material 04K5686, maize material 04K5702, maize material 07KS4, maize material 3H-2, maize material 835A, maize material A619, maize material B110, maize material B111, maize material B114, maize material B151, maize material BS16, maize material BY4944, maize material BY4960, maize material BY813, maize material BY815, maize material BY855, maize material CF3, maize material CHANG7-2, maize material CIMBL1, maize material CIMBL10, maize material CIMBL101, maize material CIMBL102, maize material CIMBL105, maize material CIMBL109, maize material CIMBL112, maize material CIMBL113, maize material CIMBL115, maize material CIMBL120, maize material CIMBL122, maize material CIMBL127, maize material CIMBL129, maize material CIMBL138, CIMBL142, CIMBL material CIMBL141, and maize material CIMBL. Corn material CIMBL145, corn material CIMBL70, corn material CIMBL73, corn material CIMBL74, corn material CIMBL75, corn material CIMBL78, corn material CIMBL79, corn material CIMBL8, corn material CIMBL80, corn material CIMBL81, corn material CIMBL83, corn material CIMBL84, corn material CIMBL85, corn material CIMBL87, corn material CIMBL88, corn material CIMBL89, corn material CIMBL9, corn material CIMBL92, corn material CIMBL93, corn material CIMBL94, corn material CIMBL95, corn material CIMBL corn material CIMBL97, corn material CIMBL98, corn material CML114, corn material CML116, corn material CML118, corn material CML121, corn material CML122, corn material CML130, corn material CML139, corn material CML168, corn material CML169, corn material CML170, corn material CML171, corn material CML172, corn material CML191, corn material CML223, corn material CML225, corn material CML228, corn material CML304, corn material CML32, corn material CML323, corn material CML324, corn material CML325, corn material CML326, corn material CML327, corn material GEMS65, corn material GY1007, corn material GY1032, corn material GY220, corn material GY386B, corn material GY923, corn material HTH-17, corn material HUA83-2, corn material HUANGC, corn material HYS, corn material HZS, corn material JH59, corn material JH96C, corn material JI53, corn material JI63, corn material JI846, corn material JI853, corn material JIAO51, corn material JY01, corn material K10, corn material K12, corn material K14, corn material LG001, corn material LIAO5262, corn material LIAO5263, corn material LK11, corn material LV28, corn material LX9801, corn material 042, RY material M97, corn material MO17, corn material Q1261, corn material R15, X691, X694, corn material Y713, corn material S22, corn material 1148 and corn material L713; maize material SHEN5003, maize material SI444, maize material SI446, maize material SW92E114, maize material SY1032, maize material SY1035, maize material 05WN230, maize material 4F1, maize material 7884-4HT, maize material 975-12, maize material B113, maize material B73, maize material B77, maize material BY4839, maize material BY804, maize material BY807, maize material BY809, maize material C8605, maize material CHUAN48-2, maize material CI7, maize material CIMBL106, maize material CIMBL108, maize material CIMBL111, maize material CIMBL114, maize material CIMBL116, maize material CIMBL123, maize material CIMBL125, maize material CIMBL143, maize material CIMBL144, maize material CIMBL146, maize material CIMBL148, maize material CIMBL157, maize material CIMBL17, maize material CIMBL2, maize material CIMBL42, maize material CIMBL46, CIMBL54, CIMBL71, CIMBL and CIMBL71 Corn material CIMBL86, corn material CIMBL90, corn material CIMBL96, corn material CIMBL99, corn material CML115, corn material CML162, corn material CML163, corn material CML189, corn material CML290, corn material CML31, corn material CML338, corn material CML361, corn material K22, corn material L3180, corn material LIAO159, corn material LIAO5114, corn material LXN, corn material LY, corn material M153, corn material M165, corn material P138, corn material QI319, corn material R08, corn material RY729, corn material RY737, corn material SI273, corn material SY3073, corn material TIE7922, corn material CIMBL147, corn material CIMBL149, corn material CIMBL15, corn material CIMBL150, corn material MBL151, corn material CIMBL153, corn material CIMBL154, corn material CIMBL155, corn material CIMBL156, corn material CIMBL21, CIMBL23, CIMBL24, CIMBL30, CIMBL23, CIMBL30, and corn material CIMBL 23. Corn material CIMBL32, corn material CIMBL34, corn material CIMBL38, corn material CIMBL39, corn material CIMBL4, corn material CIMBL40, corn material CIMBL43, corn material CIMBL44, corn material CIMBL45, corn material CIMBL47, corn material CIMBL48, corn material CIMBL49, corn material CIMBL5, corn material CIMBL50, corn material CIMBL51, corn material CIMBL52, corn material CIMBL56, corn material CIMBL58, corn material CIMBL59, corn material CIMBL6, corn material CIMBL60, corn material CIMBL corn material CIMBL61, corn material CIMBL62, corn material CIMBL63, corn material CIMBL65, corn material CIMBL66, corn material CIMBL67, corn material CIMBL68, corn material CIMBL69, corn material CML411, corn material CML412, corn material CML415, corn material CML422, corn material CML430, corn material CML431, corn material CML432, corn material CML433, corn material CML451, corn material CML454, corn material CML470, corn material CML479, corn material CML480, corn material CML, corn material CML486, corn material CML496, corn material CML51, corn material DAN340, corn material DAN4245, corn material DAN598, corn material DH29, corn material ES40, corn material GEMS1, corn material GEMS12, corn material GEMS2, corn material GEMS27, corn material GEMS28, corn material GEMS3, corn material GEMS30, corn material GEMS32, corn material GEMS33, corn material GEMS35, corn material GEMS37, corn material GEMS39, corn material GEMS40, corn material GEMS41, corn material GEMS42, corn material GEMS43, corn material GEMS45, corn material GEMS47, corn material GEMS48, corn material GEMS50, corn material GEMS57, corn material GEMS58, corn material GEMS59, corn material GEMS60, corn material SY1039, corn material SY1052, corn material SY1128, corn material SY998, corn material SY999, corn material N77, corn material SY 5, corn material TY11, corn material TY2, corn material TY2, corn material GEMS corn material TY4, corn material TY5, corn material TY6, corn material TY7, corn material TY8, corn material TY9, corn material W138, corn material WH413, corn material XI502, corn material YAN414, corn material YE52106, corn material YU374, corn material YU87-1, corn material Z2018F, corn material ZB648, corn material ZHENG22, corn material ZHENG28, corn material ZHENG30, corn material ZHENG653, corn material ZHI41, corn material ZI330, corn material ZONG3, corn material ZZ01, corn material ZZ03, corn material 150, corn material 268, corn material 501, corn material 647, corn material 812, corn material 1462, corn material 3411, corn material 4019, corn material 5213, corn material 9782, corn material 81162, corn material 05W002, corn material CML423, corn material L, corn material 493, corn material CML50, corn material CML 313, DAD 863, DAN 30, and so on the like Maize material DH3732, maize material EN25, maize material FCD0602, maize material GEMS11, maize material GEMS13, maize material GEMS14, maize material GEMS15, maize material GEMS16, maize material GEMS17, maize material GEMS18, maize material GEMS19, maize material GEMS20, maize material GEMS21, maize material GEMS23, maize material GEMS25, maize material GEMS29, maize material GEMS4, maize material GEMS44, maize material GEMS46, maize material GEMS49, maize material GEMS5, maize material GEMS51, maize material GEMS52, maize material GEMS53, maize material GEMS54, maize material GEMS55, maize material GEMS56, maize material GEMS6, maize material GEMS61, maize material GEMS62, maize material GEMS63, maize material GEMS64, maize material GEMS66, maize material GEMS9, maize material GY237, maize material IRF291, maize material IRF314, maize material J4112, maize TT16, maize material U62, HER 109, HEU 976, HEYE material 978, YE material YE 32, ZYE 32 and maize material ZYE 32 in the following documents: yang X, gao S, xu S, zhang Z, prasanna BM, lin L, li J, yan J (2011) Characterization of a global germplasm collection and its potential utilization for analysis of complex quantitative traits in mail.molecular Breeding 28:511-526.
EXAMPLE 1 discovery of molecular markers
(1) Prophase utilization 391 (177, 238, 5237, 7327, 526018, 04K5686, 04K5702, 07KS4, 3H-2, 835A, A619, B110, B111, B114, B151, BS16, BY4944, BY4960, BY813, BY815, BY855, CF3, CHANG7-2, CIMBL1, CIMBL10, CIMBL101, CIMBL102, CIMBL105, CIMBL109, CIMBL112, CIMBL113, CIMBL115, CIMBL120, CIMBL122, CIMBL127, CIMBL128, CIMBL129, CIMBL133, CIMBL138, CIMBL139, CIMBL140, CIMBL141, CIMBL142, CIMBL145, CIMBL70, CIMBL73, CIMBL74, CIMBL75, CIMBL78, CIMBL79, CIMBL8, CIMBL80, CIMBL83, CIMBL81, CIMBL85, CIMBL. CIMBL87, CIMBL88, CIMBL89, CIMBL9, CIMBL92, CIMBL93, CIMBL94, CIMBL95, CIMBL97, CIMBL98, CML114, CML116, CML118, CML121, CML122, CML130, CML139, CML168, CML169, CML170, CML171, CML172, CML191, CML223, CML225, CML228, CML304, CML32, CML323, CML324, CML325, CML326, CML327, GEMS65, GY1007, GY1032, GY220, GY386, GY386, HTH-17, HUA83-2, HUANGC, HYS, HZS, JH59, JH96C, JI, JI63, JI846, JI853, JI 51, JY01, K10, K12, K14, LG001, LIAO5262, LILV 11, LX 63, LX 01, LX 97, L982, L98, L97, L98, L01, L98, L35; MO17, Q1261, R15X1141, RY684, RY697, RY713, S22, SHEN5003, SI444, SI446, SW92E114, SY1032, SY1035, 05WN230, 4F1, 7884-4HT, 975-12, B113, B73, B77, BY4839, BY804, BY807, BY809, C8605, CHUAN48-2, CI7, CIMBL106, CIMBL108, CIMBL111, CIMBL114, CIMBL116, CIMBL123, CIMBL125, CIMBL143, CIMBL144, CIMBL146, CIMBL148, CIMBL157, CIMBL17, CIMBL2, CIMBL42, CIMBL46, CIMBL54, CIMBL55, CIMBL71, CIMBL77, CIMBL86, CIMBL90, CIMBL96, CIMBL99, CML115, CML162, CML189, CML and CML 162. CML338, CML361, K22, L3180, LIAO159, LIAO5114, LXN, LY, M153, M165, P138, QI319, R08, RY729, RY737, SI273, SY3073, TIE7922, CIMBL147, CIMBL149, CIMBL15, CIMBL150, CIMBL151, CIMBL153, CIMBL154, CIMBL155, CIMBL156, CIMBL16, CIMBL21, CIMBL23, CIMBL24, CIMBL29, CIMBL3, CIMBL30, CIMBL32, CIMBL34, CIMBL38, CIMBL39, CIMBL4, CIMBL40, CIMBL43, CIMBL44, CIMBL45, CIMBL47, CIMBL48, CIMBL49, CIMBL5, CIMBL50, CIMBL51, CIMBL52, CIMBL58, CIMBL62, CIMBL60, CIMBL61 ZHENG28, zhen 30, zhen 653, ZHI41, ZI330, ZONG3, ZZ01, ZZ03, 150, 268, 501, 647, 812, 1462, 3411, 4019, 5213, 9782, 81162, 05W002, CML423, CML471, CML493, CML50, D863F, DAN3130, DAN360, DH3732, EN25, FCD0602, GEMS11, GEMS13, GEMS14, GEMS15, GEMS16, GEMS17, GEMS18, GEMS19, GEMS20, GEMS21, GEMS23, GEMS1, GEMS2, and GEMS2 ZHENG28, ZHENG30, ZHENG653, ZHI41, ZI330, ZONG3, ZZ01, ZZ03, 150, 268, 501, 647, 812, 1462, 3411, 4019, 5213, 9782, 81162, 05W002, CML423, CML471, CML493, CML50, D863F, DAN3130, DAN360, DH3732, EN25, FCD0602, GEMS11, GEMS13, GEMS14, GEMS15, GEMS16, GEMS17, GEMS18, GEMS19, GEMS20, GEMS21, GEMS23 GEMS25, 29, 4, 44, 46, 49, 5, 51, 52, 53, 54, 55, 56, 6, 61, 62, 63, 64, 66, 9, 237, 291, 314, J4112, TT16, WMR, WU109, XUN971, 107, 478, 515, 8001, ZAC546, ZH68, ZHENG29, ZHENG32, ZHENG35 and ZHENG58, each line is 3) of related populations composed of maize inbred lines with wide variation, pollen quantity phenotypes are counted, a Mixed Linear Model (MLM) is utilized to carry out GWAS analysis on the BLUP value of 391 phenotypes, the 4872 th nucleotide of SEQ ID No.3 in a SNP site sequence table showing extremely obvious correlation with the maize pollen quantity is C or T, the SNP is positioned on a gene GRMZM5G806784 (V4 version gene number is Zm00001D 047403), the gene is also called RPN1 gene, and the SNP is called SNP1.
The pollen quantity counting method comprises the following steps:
by utilizing 391 parts of related groups formed by maize inbred lines with wide variation, 10 grains are sowed in each row, three single plants with relatively uniform growth vigor are selected from a single row of the same family from the time of emasculation of maize tassel to the time of powder scattering, 2 pairs of small flowers are taken from one half of main shaft tassel of each single plant, 6 pairs of small flowers are taken from each family, and the small flowers are stored in a 2ml centrifuge tube filled with FAA fixing liquid.
Each corn tassel is provided with a main shaft spike and a plurality of side branch spikes, hundreds of flowers are attached, and each flower is provided with 3 clustered upper anthers and 3 clustered lower anthers. Selecting small flowers in the middle of a main shaft of a tassel, sampling, mixing 9 anthers from three small flowers, putting the anthers into a 2ml centrifuge tube containing 0.9ml of pure water and one small glass bead, performing shaking breaking (1200 rpm,2 minutes) of a proofing machine, breaking anther walls, fully entering pollen grains into the solution, adding preheated 0.9ml of 0.2% agarose solution, and fully shaking uniformly. The optimized result shows that pollen grains can be uniformly distributed in 0.1% agarose solution after shaking. 20ul of solution was randomly pipetted onto slides for titration, observation and counting under a stereoscope. Each observation included at least three technical replicates and three biological replicates. The results are shown in Table 3. The pollen quantity of the corn with the genotype of the SNP1 locus CC is obviously higher than that of the corn with the genotype of the SNP1 locus TT.
The corn plant with SNP1 locus genotype of CC has two chromosomes in the genome as follows: the 4872 base of sequence 3 (SEQ ID No. 3) is C.
The corn plant with SNP1 locus genotype TT has two chromosomes in the genome as follows: the 4872 base of sequence 3 (SEQ ID No. 3) is T.
(2) The GRMZM5G806784 gene is located on chromosome 9, has the total length of 5372bp, and has 5 exons and codes 347 amino acids. Searching was performed using multiple protein domain prediction software (NCBI, CDD, PFAM), and the results indicated that the gene encoded protein contained a TVP38 domain. TVP38 is a family of membrane proteins TVP38/TMEM64, the Golgi protein domain associated with the SNARE protein, indicating that this gene encodes a typical membrane protein (FIG. 1).
TABLE 3 Table 3
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Example 2 molecular marker functional validation
(1) From the maize genome public database (www.maizeGDB.org), 1 Unifromu insertion mutant UFmu-09290 (also known as UFmu-09290 or mutant UFmu-09290) was obtained at the first exon of the RPN1 gene (GRMZM 5G806784 or Zm00001d 047403). Both chromosomes of the insertion mutant UFMu-09290 were mutated as follows: the fragment between the GTCCTCCCGCTCGAGACTGCC and AGGTACGCGCGCGGGGTCAAA sequences of sequence 3 (SEQ ID No. 3) was replaced with a transposon sequence, resulting in premature termination and mutation of the encoded protein sequence to MARVLPLETA.
The transposon sequence is specifically as follows:
TCGGCAGGCGAGATAATTGTCATTATAGACGAAGAGCGGACGGGATTCGACGAAATGGAGGCGATGGCGTTGGCTTCTCT
GTTCTGGAAACGCAGACGACAGCCAAACGCCAAAACGGAAAGGAGACAGCGCTTGGAGCTGTGTAAACAGGTATTAGTCT
CCTGTCCCCGTTTACCGTTCGCCTGCGCAGACGCCGTCTGGCATACTCCTCTCTACGCCGTCTCTTCTTGCGGCTGCTCT
CGGGGTCGGCCTGCTCGCAATACCTGTTTTGACACAAGCAGCGCCGGAGCGGGCCGCGCAGGTTGGCCTCGAACAGCCCC
GTGATCTTGCGCTCGCGTTGAACGCGCCACGGTGCGCAGGTTGGCCACGGCTCGCCCGCGATCTGCGTGGCCCTGGCGTG
CGCGGCCTCCAGGTCCCCCGAGAAGCCCTTCATGAACATCTTCTGCAGCACGGTGGCGCCCACGACGAGCGGGAACACGG
CGAGGAGCACGAGCGCGAGGCGCACTGGAGGACGAACCCCGCGGTGTCAGGCCACCAGCATCAGCGCCGAGTTCTGGACG
ATGACGGAGATGCGGTCCCCGATGGCGGACGGCACGTTCTGGGCGTCCAGCGCGAGCCTGGCGCCACGCGCGCGCTGGCG
TTCTCGTCCGTGTCGAACCAGGCGATCTCGTTGCGGAGCACGGCGTCGAACATCTTCTCGCGCACCCGCTTGGTCAGGTT
CTCGCCCACCGTGTCCCAGMCACTGCTGCACCGTGTTGAACAGCAGCGCGCGAGGACATGCCGATGAGCAGGTAGCAGTA
TTTGGCGATCTCGCGCTTCATGTACCGCGGGTCCGGCGCGTAGTACACGCTGAGCACGGCGCTGAGGATGTAGGCGAAGA
TGGCGCTGAAGGAGCCGCGGACCATGGAGCCGGCGAGCGCGTAGGCCCACTCGGGCGAGTTCATCCTGGCGAGGCGCAGG
AAGGAGCTGGCGCCGGCGCGGAACGCCAGCTGCTTGTCGGCCATGGTCCGGTGGTGGTGGTGCGGGTCGTGGATGGAGAG
GGTGAAGTCGGAGGTGGAGAAGTCGGAGAGGCGGCGGGAGTAGGGGGAGCGGCCGTAGAGGAGTTGCGCGTCATGATGGG
CGAGCTGACGGAGTTGCGGGCGCTGGAGGGCCTGGCGCTGCGCCGTGCTGGTGCAATGTCGACCCCGAGAGCATGAACAC
GAGAGCATGAACACGAAACGGCGGCTAGGGCAGCGTCTGCGCAGACGAACGGTAAACGGGGACAGGAGACTAATACCTGT
TTACGCAGCTCCAAGCGCTGTCTTCTTTCCGTTTTGGCGTTTGGCTGTCGCCTGCGTCTCCAGAACAGAGAAGCCAACGCCATCGCCTCCATTTCGTCGAATCCCGTCCGCTCTTCGTCTATAATGGCAATTATCTCTCGGCAGGC。
taking a wild type W22 material as a recurrent parent, taking a UFmu-09290 homozygous mutant obtained from a corn genome public database as a donor parent, taking W22 tassel pollen in a pollination period, and manually pollinating female ears of the obtained UFmu-09290 homozygous mutant to obtain a BC1F1 material; sowing the BC1F1 material, taking the tassel pollen of W22 in the pollination period, and manually pollinating female ears of the BC1F1 material to obtain a BC2F1 material; sowing the BC2F1 material, taking the tassel pollen of W22 in the pollination period, manually pollinating female ears of the BC2F1 material to obtain the BC3F1 material, taking the BC3F1 material obtained by taking the UFU-09290 homozygous mutant as a donor parent as BC3F1-RPN1, sowing the BC3F1-RPN1 material, manually bagging and self-pollinating in the pollination period, and finally obtaining the RPN1-BC3F2 mutant material. Mutant materials with homozygous mutant genotypes were selected for phenotypic analysis by the following genotyping methods.
After backcrossing the mutant UFMu-09290 with maize W22 (as male parent) for three generations (as described above), the presence of the above-described insertional mutation and homozygosity of the gene GRMZM5G806784 was maintained; then selfing (according to the method), planting 100 selfing offspring seeds (the RPN1-BC3F2 mutant materials) respectively, carrying out genotype identification (the specific method is as follows) and carrying out pollen quantity representative analysis on the selfing offspring by using the pollen quantity statistical method in the example 1, wherein the offspring generate 3 genotypes, namely genotype RPN1-AA (genotype is AA), genotype RPN1-AA (genotype is AA) and genotype RPN1-AA (genotype is AA).
The genome of the genotype RPN1-AA is as follows: there was no transposon insertion between the GTCCTCCCGCTCGAGACTGCC sequence and AGGTACGCGCGCGGGGTCAAA sequence of sequence 3 (SEQ ID No. 3).
The genome of the genotype RPN1-a1a1 has two chromosomes as follows: the fragment between the GTCCTCCCGCTCGAGACTGCC sequence and AGGTACGCGCGCGGGGTCAAA sequence of sequence 3 (SEQ ID No. 3) was replaced with a transposon sequence.
The two chromosomes in the genome of the genotype RPN1-Aa1 are as follows: there was no transposon insertion between the GTCCTCCCGCTCGAGACTGCC and AGGTACGCGCGCGGGGTCAAA sequences of one chromosome 3 (SEQ ID No. 3) and the fragment between the GTCCTCCCGCTCGAGACTGCC and AGGTACGCGCGCGGGGTCAAA sequences of the other chromosome 3 (SEQ ID No. 3) was replaced with a transposon sequence.
As a result, as shown in Table 1, the insertion mutation of the RPN1 gene (genotype RPN1-a1a 1) resulted in about 16% increase in pollen number, and no significant change in other traits of the plants was observed.
TABLE 1
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When the genotype is identified, a PCR amplification and agarose gel electrophoresis combined method is adopted, if the left primer and the right primer are simultaneously amplified with the MuTIR primer to form a band, and the left primer and the right primer are not amplified with the band, the genotype is a mutant, namely the genotype RPN1-a1a1; if the left primer and the right primer and the MuTIR primer do not amplify a band, and the left primer and the right primer amplify 747bp, the wild type is genotype RPN1-AA; if the bands are amplified, the bands are heterozygotes, namely genotype RPN1-Aa1. The mutant genotype identification primers were:
Left primer rpn-UFmu-09290-F: TTTCTAGTTCCGTGTGCGCT
Right primer rpn-UFMu-09290-R: GTTCCCCAAATTCGACCAAGC
MuTIR:AGAGAAGCCAACGCCAWCGCCTCYATTTCGTC。
The wild type gene of the corn W22 RPN1 is simply called A, and the sequence of the RPN1 gene is shown as a sequence 3. The RPN1 mutant gene in UFU-09290 is named as a1, namely, the RPN1 mutant gene is inserted into a transposon (the sequence is shown as SEQ I) at the base of ' GAGATGGCC ' of the 1 st exon of the RPN1 wild type gene (namely, after 662 of the sequence shown as SEQ ID No. 3), and the mutation leads to the transcription of the mRNA of the RPN1 gene to be stopped from the 31 st position corresponding to the 5' end of SEQ ID No.1, thereby leading to premature termination and the loss of the function of the RPN1 protein. The transposon sequences are specifically as indicated above.
The primer combinations MuTIR, RPN1-UFmu-09290-F and RPN1-UFmu-09290-R amplify bands, the primer combinations RPN-UFmu-09290-F and RPN-UFmu-09290-R do not amplify bands, and the genotype of the offspring plant is a1a1, and the genotype indicates that the RPN1 genes in 2 homologous chromosomes in the genome of the plant are all a1 genes. This progeny plant was designated as RPN1-a1a1 mutant.
No band is amplified by the primer combination MuTIR, RPN1-UFmu-09290-F and RPN1-UFmu-09290-R, 747bp bands are amplified by the primer combination RPN-UFmu-09290-F and RPN-UFmu-09290-R, the genotype of the offspring plant is AA, and the genotype indicates that the RPN1 genes in 2 homologous chromosomes in the genome of the plant are all A genes. This progeny plant was designated RPN1-AA.
The primer combination MuTIR, RPN1-UFmu-09290-F and RPN1-UFmu-09290-R amplify bands, the primer combination RPN-UFmu-09290-F and RPN-UFmu-09290-R amplify 747bp bands, the genotype of the offspring plant is Aa1, the genotype indicates that the RPN1 gene in 1 homologous chromosome in the genome of the plant is A gene, and the RPN1 genes in 1 homologous chromosome are a1 gene. This progeny plant was designated RPN1-Aa1.
Example 3 molecular marker functional validation
From the maize EMS-induced mutant library (MEMD: https:// elabacas. Cn/MEMD/public/index. Html#), 1 independent B73-based GRMZM5G806784 (v 4: zm00001d 047403) gene EMS mutant EMS4-0bc5bd was obtained, which was sold by mutant MEMD and publicly available. From MEMD website, it is known that the following mutations occur on both chromosomes of EMS mutant EMS4-0bc5 bd: the 3476 th base G of the sequence 3 (SEQ ID No. 3) is mutated into A, so that the coded protein stops translation in advance, the function of the gene is deleted, the sequencing result is consistent with the description, namely SNP2, and the SNP2 is the 3476 th nucleotide of the SEQ ID No.3 in the sequence table, and is G or A.
3 genotypes can be generated: genotype RPN1-EMS-AA (SNP 2 genotype is AA), genotype RPN1-EMS-Aa2 (SNP 2 genotype is AA 2) and genotype RPN1-EMS-a2a2 (SNP 2 genotype is a2a 2), SNP2 genotype is a2a2 is the homozygous type with nucleotide 3476 of SEQ ID No.3 in the sequence table as A; SNP2 genotype AA is the homozygote of the 3476 th nucleotide of SEQ ID No.3 in the sequence table as G; SNP2 genotype Aa2 is the heterozygous type with G and A at 3476 th nucleotide of SE Q ID No.3 in the sequence table.
Plants of genotype RPN1-EMS-a2a2, both chromosomes in their genome were mutated as follows: base G at position 3476 of sequence 3 (SE Q ID No. 3) was mutated to A.
Plants of genotype RPN1-EMS-Aa2, whose genomic chromosomes were mutated as follows: a mutation of base G at position 3476 of chromosome sequence 3 (S EQ ID No. 3) to A; no mutation occurred at base 3476 of the other chromosome sequence 3.
The plant of the genotype RPN1-EMS-AA has two chromosomes in the genome as follows: no mutation occurs at the 3476 th base of the sequence 3.
Taking a wild type B73 material as a recurrent parent, taking EMS4-0BC5bd obtained from a corn EMS induced mutant library as a donor parent, taking tassel pollen of B73 in a pollination period, and manually pollinating the obtained EMS4-0BC5bd tassel respectively to obtain a BC1F1 material; sowing the BC1F1 material, taking the tassel pollen of B73 in the pollination period, and manually pollinating female ears of the BC1F1 material to obtain a BC2F1 material; and sowing the BC2F1 material, and carrying out artificial bagging self-pollination in the pollination period to obtain the BC2F2 mutant material. 100 BC2F2 mutant materials (seeds) were planted, respectively identified by genotype and analyzed for the representative number of selfed pollen by the pollen number statistical method of example 1, as follows:
Identification method of 100-grain RPN1 mutant co-sown in beijing city in summer in 2020 (specifically as follows)
Genotype detection is carried out on the BC2F1 material (the method is as follows), and the BC2F1 material is detected to be heterozygote, namely the chromosome in the genome is mutated as follows: the 3476 th base G of sequence 3 (SEQ ID No. 3) was mutated to A; the other chromosome sequence 3 was not mutated at the 3476 th base and was called an RPN1 heterozygous mutant. Seeds obtained by selfing the RPN1 heterozygous mutant are planted, 10 seeds are sown in each row, 10 rows are added, seedling leaves are taken after emergence of seedlings, genotype identification is carried out according to a method for identifying the R PN1 mutant, 28 homozygous wild type AA single plants (RPN 1 wild type plants, genotypes are AA, and are concretely described above) are obtained, homozygous mutant a2a2 (RPN 1 homozygous mutant plants, genotypes are a2a2, and are concretely described above) single 25 plants, and heterozygous genotype AA2 (genotypes are Aa2, and are concretely described above) single 47 plants. The obtained homozygous wild type AA single plant, homozygous mutant a2a2 single plant and heterozygous genotype AA2 single plant were each selected from 20 single plants with good growth vigor, and the pollen quantity was measured, in the same manner as in example 1.
The genotype a2a2 is homozygote with the 3476 th nucleotide of SEQ ID No.3 in the sequence table as A; the genotype A A is the homozygote of the 3476 th nucleotide of SEQ ID No.3 in the sequence table; genotype Aa2 is a hybrid of A as the 3476 th nucleotide of SEQ ID No.3 in the sequence table and G as the 3476 th nucleotide of SEQ ID No.3 in the sequence table.
Identification of RPN1 mutants:
the RPN1 mutant genotype is identified by adopting a left primer RPN1-EMS-F and a right primer RPN 1-EMS-R. Amplifying a band with RPN1-EMS-F and RPN1-EMS-R, carrying out sanger sequencing with 724bp length, and if the sequencing result is still G after no change of a base at a mutation position (3476 base of a sequence 3), obtaining a single plant as a homozygous wild type material AA (an RPN1 wild type plant, the genotype of which is AA, and the specific is as above); if the sequencing result of the RPN1-EMS-F and the RPN1-EMS-R amplified bands shows that the mutation site base is A, the single plant is a homozygous mutant material a2a2 (an RPN1 homozygous mutant plant, the genotype is a2a2, and the specific is as above); if the sequencing result of the strips amplified by the RPN1-EMS-F and the RPN1-EMS-R is that the mutation sites exist G and A simultaneously, the single plant is a heterozygous mutant material Aa2 (an RPN1 heterozygous mutant plant, the genotype of which is Aa2, and the specific is as above).
Mutant genotyping primers:
RPN1-EMS-F:TGGCAAACATCCTAGCACAC
RPN1-EMS-R:TCTGTCACAGTCACGGCATA
TABLE 2
As a result, as shown in Table 2, the deletion mutation of the RPN1 gene (genotype RPN1-a2a 2) resulted in about 10% increase in pollen number, and no significant change in other traits of the plants was observed.
The data were processed using EXCEL statistical software, and experimental results were expressed as average values using T-test. P < 0.05 (x) indicates significant differences, P < 0.01 (x) indicates significant differences, and P < 0.001 (x) indicates significant differences
There was a significant difference between the AA genotype and the a2a2 genotype, but there was no significant difference between the AA genotype and the AA2 genotype.
The EMS4-0bc5bd mutant resulted in an increase in pollen numbers of about 10% compared to wild type. Mutant phenotype statistics indicate that GRMZM5G806784 plays an important role in controlling maize pollen numbers. This gene is designated herein as RP N1 (REGULATOR OF POLLEN NUMBER 1).
The number of the corn pollens with the SNP2 genotype AA is higher than or is higher than the number of the corn pollens with the SNP2 genotype AA;
SNP2 genotype aa (i.e., SNP2 genotype a2a 2) is homozygous for nucleotide No. 3476 of SEQ ID No.3 in the sequence Listing as A; SNP2 genotype is AA (namely SNP2 genotype is AA) and is homozygous type that the 3476 th nucleotide of SEQ ID No.3 in the sequence table is G; SNP2 genotype Aa (i.e., SNP2 genotype Aa 2) is a hybrid of nucleotide A at position 3476 of SEQ ID No.3 in the sequence Listing and nucleotide G at position 3476 of SEQ ID No.3 in the sequence Listing.
The results show that: the number of pollen per anther of homozygous wild type AA was 3144 on average and the number of pollen per anther of homozygous mutant AA was 3448 on average. The pollen number of homozygous mutant AA increased by about 10% compared to homozygous wild type AA, and no significant change in other traits of the plant was observed. The phenotype statistics of the transgenic knockout and EMS mutant show that: RPN1 plays an important role in regulating pollen quantity, and RPN1 has the function of regulating corn pollen quantity.
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Claims (10)
1. Use of a substance that detects a polymorphism or genotype of a SNP to identify or assist in the quantity of maize pollen, or to prepare a product that identifies or assists in the identification of the quantity of maize pollen, or to maize breeding or to prepare a maize breeding product;
the SNP comprises SNP1 and/or SNP2;
SNP1 is one SNP of corn genome, is 4872 nucleotide of SEQ ID No.3 in the sequence table, it is C or T;
SNP2 is one SNP of the corn genome, is the 3476 th nucleotide of SEQ ID No.3 in the sequence table, and is G or A.
2. A method for identifying or aiding in the identification of maize germplasm, said method comprising detecting in a test maize the genotype of the SNP of claim 1, the number of maize pollen with the SNP1 genotype of CC being higher or candidate higher than that of maize with the SNP1 genotype of TT, the number of maize pollen with the SNP2 genotype of Aa being higher or candidate higher than that of maize pollen with the SNP2 genotypes of Aa and Aa;
SNP1 genotype is CC and is homozygous that the 4872 nucleotide of SEQ ID No.3 in the sequence table is C; SNP1 genotype TT is homozygous with the 4872 th nucleotide of SEQ ID No.3 in the sequence table as T;
SNP2 genotype aa is homozygous with the 3476 th nucleotide of SEQ ID No.3 in the sequence table as A; SNP2 genotype AA is the homozygote of the 3476 th nucleotide of SEQ ID No.3 in the sequence table as G; SNP2 genotype Aa is a hybrid of A as 3476 th nucleotide of SEQ ID No.3 in the sequence table and G as 3476 th nucleotide of SEQ ID No.3 in the sequence table.
3. Use of the method of claim 2 in maize breeding.
4. A product comprising the substance for detecting a corn genomic SNP polymorphism or genotype according to claim 1, and being any one of G1) to G3) below:
G1 A product for detecting a single nucleotide polymorphism or genotype associated with the quantity of maize pollen;
g2 Identifying or aiding in identifying a product of maize pollen quantity;
g3 A product for corn breeding.
5. The use according to claim 1 or 3, the method according to claim 2, the product according to claim 4, characterized in that the product of the substance for detecting a maize genomic SNP polymorphism or genotype is D1), D2), D3) or D4) as follows:
d1 An in vitro nucleic acid amplification primer that specifically amplifies said SNP 1;
d2 An in vitro nucleic acid amplification reagent comprising D1) the in vitro nucleic acid amplification primer;
d3 A kit comprising D1) the in vitro nucleic acid amplification primer or D2) the in vitro nucleic acid amplification reagent;
d4 A detection instrument comprising D1) said in vitro nucleic acid amplification primer, D2) said in vitro nucleic acid amplification reagent, or D3) said kit.
6. Use of the method of claim 2 in maize breeding.
7. The use according to claim 6, wherein the purpose of said breeding is to breed high pollen corn.
8. Use of a protein, a substance regulating expression of a gene encoding said protein or a substance regulating activity or content of said protein for regulating the amount of maize pollen and/or for the preparation of a product regulating the amount of maize pollen and/or for maize breeding and/or for the preparation of a maize breeding product; the protein is any one of the following:
A1 Amino acid sequence is a protein shown in sequence 2;
a2 Protein which is obtained by substituting and/or deleting and/or adding amino acid residues of the protein A1), has more than 80 percent of identity with the protein A1) and has the function of regulating and controlling the quantity of corn pollen;
a3 Fusion proteins obtained by ligating the N-terminal or/and C-terminal of A1) or A2) with a protein tag.
9. The use according to claim 8, wherein the protein is derived from corn.
10. The use according to claim 8 or 9, wherein the coding gene of the protein is any one of the following:
c1 A DNA molecule having a nucleotide sequence of the coding strand of sequence 1;
c2 Nucleic acid molecules which are obtained by substituting and/or deleting and/or adding nucleotides of the nucleic acid molecules of the C1), have more than 80 percent of identity with the nucleic acid molecules shown in the C1) and have the function of regulating the quantity of corn pollen.
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