WO2019196576A1 - 一种利用植物杂种优势的方法 - Google Patents

一种利用植物杂种优势的方法 Download PDF

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WO2019196576A1
WO2019196576A1 PCT/CN2019/077154 CN2019077154W WO2019196576A1 WO 2019196576 A1 WO2019196576 A1 WO 2019196576A1 CN 2019077154 W CN2019077154 W CN 2019077154W WO 2019196576 A1 WO2019196576 A1 WO 2019196576A1
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protein
sequence
seq
consistent
set forth
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王克剑
王春
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中国水稻研究所
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Priority to EA202092307A priority Critical patent/EA202092307A1/ru
Priority to JP2020556295A priority patent/JP2021520223A/ja
Priority to AU2019250836A priority patent/AU2019250836A1/en
Priority to EP19784291.7A priority patent/EP3777525A4/en
Priority to US17/046,794 priority patent/US20210363537A1/en
Priority to KR1020207032225A priority patent/KR20200140367A/ko
Application filed by 中国水稻研究所 filed Critical 中国水稻研究所
Priority to BR112020020900-7A priority patent/BR112020020900A2/pt
Priority to CA3096898A priority patent/CA3096898A1/en
Publication of WO2019196576A1 publication Critical patent/WO2019196576A1/zh
Priority to PH12020551675A priority patent/PH12020551675A1/en
Priority to ZA2020/06748A priority patent/ZA202006748B/en
Priority to AU2023200393A priority patent/AU2023200393A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
    • A01H1/026—Methods or apparatus for hybridisation; Artificial pollination ; Fertility by treatment with chemicals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A—HUMAN NECESSITIES
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10—Seeds
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
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    • 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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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • 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)
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]

Definitions

  • the present invention relates to the field of biotechnology, and in particular to a method for utilizing plant heterosis.
  • Heterosis refers to the hybridization between two genetically diverse varieties or similar species in the biological world, and the hybrid generation is superior to the parents in terms of growth potential, viability, adaptability and yield. Heterosis is a common phenomenon in the biological world and is widely used in the cultivation and production of crop varieties.
  • the male and female flower crops (such as rice, wheat, etc.) cannot achieve large-scale preparation of hybrid seeds by removing the female pollen pathway.
  • rice Take rice as an example:
  • the way to solve this problem in rice is to use the plant with pollen infertility as the female parent and the pollen hybrid with the other breed as the male parent, that is, the heterosis with male sterility as the core of technology.
  • the utilization of rice heterosis can be divided into two technical approaches, one is the “three-line method” hybridization technology with nuclear-plasma interaction pollen as the core technology, and the other is the light temperature regulated by natural photoperiod and temperature.
  • the nucleus sterility is the "two-line method” hybrid technology at the core of technology.
  • the "three-line method" hybridization technique using the cytoplasmic interaction male sterile line as the female parent, and using the maintainer line as the male parent to breed and still maintain the characteristics of infertility; using the sterile line as the mother
  • This is a large-scale hybrid seed that restores pollen fertility and has heterosis by using the restorer as a male parent, and the hybrid seed is used for producing hybrid rice.
  • the "two-line method" hybridization technique the same rice line, under certain conditions, pollen fertile, using its fertility to propagate sterile seed; under another specific condition, pollen is sterile, using it The sterility is crossed with the male parent to prepare hybrid seeds.
  • hybrid rice utilizes the advantage of hybrid generation, the traits or fertility separation will occur in many generations. Therefore, it must be planted every year and consume a lot of manpower, material resources and land resources.
  • the “three-line method” is restricted by the restoration relationship and has low utilization rate of germplasm resources; the “two-line method” is affected by natural warm light, the reproductive yield of the sterile line is unstable, and there is low temperature induction during hybrid seed production. The self-crossing of the breeding line leads to the risk that the purity of the hybrid seeds is not up to standard.
  • the present invention aims to provide a method for utilizing plant heterosis to enable a hybrid to produce cloned seeds or plants, thereby improving seed production efficiency.
  • a method of utilizing plant heterosis comprises the steps of: S1, using genetic mutation or genetic engineering technology to convert the meiosis of the germ cells of the hybrid into mitosis to obtain a gamete that is consistent with the genotype and chromosome ploidy of the hybrid; and S2, using the gene mutation And genetic engineering techniques affect the involvement of plant gametes or embryo development processes, and the proteins involved are MTL proteins.
  • gene mutations include random mutagenesis and site-directed mutagenesis; among them, random mutagenesis includes chemical mutagenesis, physical mutagenesis and biological mutagenesis; targeted mutagenesis includes gene editing technology, and gene editing technology includes CRISPR/Cas gene editing technology. , CRISPR/Cpf1 gene editing technology, TALEN gene editing technology, homing endonuclease gene editing technology and ZFN gene editing technology; genetic engineering technology includes transgenic technology to induce gene specific expression, translocation expression or gene silencing.
  • S1 comprises taking hybrids and transforming the meiosis of the germ cells into mitosis by genetic mutation or genetic engineering techniques to obtain gametes that are consistent with the hybrid genotype and chromosome ploidy.
  • S1 includes editing the parents of the hybrids by using gene mutation or genetic engineering techniques, and then obtaining hybrids by crossing between the parents, thereby obtaining meiosis of the germ cells and converting them into mitotic-like hybrid gametes.
  • S1 includes using a gene mutation or a genetic engineering technique to edit a protein involved in meiosis in a plant to transform meiosis of a germ cell into a mitosis-like protein; wherein the protein includes a first protein, a second protein, and a third protein, wherein ,
  • the first protein is a protein involved in the formation of a DNA double-strand break, and the first protein is selected from the following proteins:
  • the PAIR1 protein as shown in SEQ ID NO: 13 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and PAIR1 protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with PAIR1 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the PAIR2 protein as shown in SEQ ID NO: 14 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and PAIR2 protein, 85%, 90%, 95% or 98% of sequence-equalized proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with PAIR2 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the PAIR3 protein as shown in SEQ ID NO: 15 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and PAIR3 protein, 85%, 90%, 95% or 98% of sequence-equalized proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with PAIR3 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • PRD1 protein as shown in SEQ ID NO: 16 having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and PRD1 protein, 85%, 90%, 95% or 98% of the sequence-consistent protein, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with the PRD1 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • a PRD2 protein as set forth in SEQ ID NO: 17 having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and PRD2 protein, 85%, 90%, 95% or 98% of the sequence-consistent protein, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with the PRD2 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the SPO11-1 protein as shown in SEQ ID NO: 18 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% with the SPO11-1 protein. 80%, 85%, 90%, 95% or 98% of the sequence-consistent protein, or with the SPO11-1 protein, 45%, 50%, 55%, 60%, 65%, 70%, 75% a protein of sequence similarity of 80%, 85%, 90%, 95% or 98%;
  • the SPO11-2 protein as shown in SEQ ID NO: 19 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% with the SPO11-2 protein. 80%, 85%, 90%, 95% or 98% of the sequence-consistent protein, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70% with the SPO11-2 protein a 75%, 80%, 85%, 90%, 95% or 98% sequence similarity protein;
  • SDS protein as set forth in SEQ ID NO: 20, having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and SDS protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with SDS protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the CRC1 protein as shown in SEQ ID NO: 21 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and CRC1 protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with CRC1 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the P31 comet protein as shown in SEQ ID NO: 22 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80 with the P31 comet protein. %, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% with P31 comet protein a protein of sequence similarity of 80%, 85%, 90%, 95% or 98%;
  • the MTOPVIB protein as set forth in SEQ ID NO: 23 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and MTOPVIB protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with MTOPVIB protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • DFO protein as set forth in SEQ ID NO: 24, having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and DFO protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with DFO protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the second protein is involved in controlling the adhesion between sister chromosomes in the meiotic phase, and the second protein is selected from the following proteins:
  • the REC8 protein as shown in SEQ ID NO: 25 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and REC8 protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with REC8 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the third protein is involved in the second division of meiosis, and the third protein is selected from the following proteins:
  • the OSD1 protein as shown in SEQ ID NO: 26 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and the OSD1 protein, 85%, 90%, 95% or 98% of sequence-equalized proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with OSD1 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the TAM protein as shown in SEQ ID NO: 27 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and TAM protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with TAM protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • TDM1 protein as set forth in SEQ ID NO: 28, having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and TDM1 protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with TDM1 protein , 85%, 90%, 95% or 98% of sequence similarity proteins.
  • S2 includes using genetic mutations and genetic engineering techniques to influence the development of plant gametes or embryos, and to induce gametes to develop into seeds or plants.
  • S2 includes inducing pollen from other plants to induce gamete development into seeds or plants.
  • S2 includes inducing the development of gametes into seeds or plants by physical stimulation, biotic stress or chemical treatment.
  • S2 comprises inducing the development of gametes into seeds or plants by anther culture or pollen culture.
  • the MTL protein is an MTL protein as shown in SEQ ID NO: 29, and has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, and MTL protein, 75%, 80%, 85%, 90%, 95% or 98% of the sequence-consistent protein, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70% with the MTL protein , 75%, 80%, 85%, 90%, 95% or 98% of sequence similarity proteins.
  • plants include monocots and dicots.
  • the plant includes rice, corn, sorghum, millet, barley, wheat, rye, oats, buckwheat, coix seed, sugar cane, asparagus, bamboo shoots, leeks, yam, soybeans, potatoes, peas, mung beans, adzuki beans, broad beans, cowpeas, Beans, lentils, vines, chickpeas, cassava, sweet potatoes, rapeseed, cotton, beets, eggplant, peanuts, tea, mint, coffee, sesame, sunflower, nettle, succulent, safflower, tomato, pepper, cucumber , green vegetables, lettuce, spinach, garlic, kale, mustard greens, scallions, green onions, melon, zucchini, loofah, cabbage, radish, onions, watermelon, grapes, carrots, cauliflower, pumpkin, tobacco, pasture, grassy, pennisetum, Sudanese grass, orchids, lilies, tulips and dragonflies.
  • a plant or seed that retains heterosis is provided.
  • the plant or seed is prepared by any of the above methods.
  • kits for maintaining a plant heterosis includes vectors and/or reagents capable of converting meiosis of plant germ cells into mitotic-like vectors, and vectors and/or reagents for developing gametes into seeds or plants.
  • the vector and/or reagent capable of transforming meiosis of plant germ cells into a mitotic-like vector and/or reagent is to convert the meiosis of the germ cells of the hybrid into a mitotic-like vector and/or reagent by genetic mutation or genetic engineering techniques.
  • random mutagenesis includes chemical mutagenesis, physical mutagenesis and biological mutagenesis
  • targeted mutagenesis includes CRISPR/Cas gene editing technology, CRISPR/Cpf1 gene editing technology, TALEN gene editing technology, homing endonuclease gene editing Technology, ZFN gene editing technology
  • genetic engineering technology includes transgenic technology to induce gene specific expression, translocation expression or gene silencing.
  • the meiosis of the plant germ cells can be converted into a mitotic-like vector and/or reagent for the use of gene mutation or genetic engineering to edit the meiosis protein in the plant to transform the meiosis of the germ cell into a mitosis-like And a carrier, wherein the protein comprises a first protein, a second protein, and a third protein, wherein
  • the first protein is involved in the formation of a DNA double-strand break, and the first protein is selected from the following proteins:
  • the PAIR1 protein as shown in SEQ ID NO: 13 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and PAIR1 protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with PAIR1 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the PAIR2 protein as shown in SEQ ID NO: 14 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and PAIR2 protein, 85%, 90%, 95% or 98% of sequence-equalized proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with PAIR2 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the PAIR3 protein as shown in SEQ ID NO: 15 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and PAIR3 protein, 85%, 90%, 95% or 98% of sequence-equalized proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with PAIR3 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • PRD1 protein as shown in SEQ ID NO: 16 having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and PRD1 protein, 85%, 90%, 95% or 98% of the sequence-consistent protein, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with the PRD1 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • a PRD2 protein as set forth in SEQ ID NO: 17 having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and PRD2 protein, 85%, 90%, 95% or 98% of the sequence-consistent protein, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with the PRD2 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the SPO11-1 protein as shown in SEQ ID NO: 18 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% with the SPO11-1 protein. 80%, 85%, 90%, 95% or 98% of the sequence-consistent protein, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70% with the SPO11-1 protein a 75%, 80%, 85%, 90%, 95% or 98% sequence similarity protein;
  • the SPO11-2 protein as shown in SEQ ID NO: 19 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% with the SPO11-2 protein. 80%, 85%, 90%, 95% or 98% of the sequence-consistent protein, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70% with the SPO11-2 protein a 75%, 80%, 85%, 90%, 95% or 98% sequence similarity protein;
  • SDS protein as set forth in SEQ ID NO: 20, having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and SDS protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with SDS protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the CRC1 protein as shown in SEQ ID NO: 21 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and CRC1 protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with CRC1 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the P31 comet protein as shown in SEQ ID NO: 22 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80 with the P31 comet protein. %, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% with P31 comet protein a protein of sequence similarity of 80%, 85%, 90%, 95% or 98%;
  • the MTOPVIB protein as set forth in SEQ ID NO: 23 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and MTOPVIB protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with MTOPVIB protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • DFO protein as set forth in SEQ ID NO: 24, having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and DFO protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with DFO protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the second protein is involved in controlling the adhesion between sister chromosomes in the meiotic phase, and the second protein is selected from the following proteins:
  • the REC8 protein as shown in SEQ ID NO: 25 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and REC8 protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with REC8 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the third protein is involved in the second division of meiosis, and the third protein is selected from the following proteins:
  • the OSD1 protein as shown in SEQ ID NO: 26 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and the OSD1 protein, 85%, 90%, 95% or 98% of sequence-equalized proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with OSD1 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the TAM protein as shown in SEQ ID NO: 27 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and TAM protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with TAM protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • TDM1 protein as set forth in SEQ ID NO: 28, having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and TDM1 protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with TDM1 protein , 85%, 90%, 95% or 98% of sequence similarity proteins.
  • the MTL protein is an MTL protein as set forth in SEQ ID NO: 29, and has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, and MTL protein, 75%, 80%, 85%, 90%, 95% or 98% of the sequence-consistent protein, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70% with the MTL protein , 75%, 80%, 85%, 90%, 95% or 98% of sequence similarity proteins.
  • a plant produced using the above kit is provided.
  • the meiosis of the germ cells of the plant is transformed into a mitosis-like trait that produces a gamete that is consistent with the hybrid genotype and chromosome ploidy.
  • gametes of the plant can be induced to develop into plants or seeds.
  • the plant is a genetically modified or genetically engineered plant, and the plant is modulated by a gene mutation or a genetic engineering technique to regulate meiosis in a plant to convert meiosis of the germ cell into a mitosis-like; use of a gene mutation or gene Engineering technology affects the fourth protein involved in gametogenesis or embryo development in a plant to induce gamete development into seeds or plants; wherein the protein includes a first protein, a second protein, and a third protein, wherein
  • the first protein is involved in the formation of a DNA double-strand break, and the first protein is selected from the following proteins:
  • the PAIR1 protein as shown in SEQ ID NO: 13 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and PAIR1 protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with PAIR1 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the PAIR2 protein as shown in SEQ ID NO: 14 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and PAIR2 protein, 85%, 90%, 95% or 98% of sequence-equalized proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with PAIR2 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the PAIR3 protein as shown in SEQ ID NO: 15 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and PAIR3 protein, 85%, 90%, 95% or 98% of sequence-equalized proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with PAIR3 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • PRD1 protein as shown in SEQ ID NO: 16 having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and PRD1 protein, 85%, 90%, 95% or 98% of the sequence-consistent protein, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with the PRD1 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • a PRD2 protein as set forth in SEQ ID NO: 17 having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and PRD2 protein, 85%, 90%, 95% or 98% of the sequence-consistent protein, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with the PRD2 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the SPO11-1 protein as shown in SEQ ID NO: 18 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% with the SPO11-1 protein. 80%, 85%, 90%, 95% or 98% of the sequence-consistent protein, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70% with the SPO11-1 protein a 75%, 80%, 85%, 90%, 95% or 98% sequence similarity protein;
  • the SPO11-2 protein as shown in SEQ ID NO: 19 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% with the SPO11-2 protein. 80%, 85%, 90%, 95% or 98% of the sequence-consistent protein, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70% with the SPO11-2 protein a 75%, 80%, 85%, 90%, 95% or 98% sequence similarity protein;
  • SDS protein as set forth in SEQ ID NO: 20, having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and SDS protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with SDS protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • CRC1 protein as set forth in SEQ ID NO: 21, having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and CRC1 protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with CRC1 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the P31 comet protein as shown in SEQ ID NO: 22 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80 with the P31 comet protein. %, 85%, 90%, 95% or 98% of the sequence-consistent protein, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, with the P31comet protein, 80%, 85%, 90%, 95% or 98% sequence similarity protein;
  • the MTOPVIB protein as set forth in SEQ ID NO: 23 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and MTOPVIB protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with MTOPVIB protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • DFO protein as set forth in SEQ ID NO: 24, having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and DFO protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with DFO protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the second protein is involved in controlling the adhesion between sister chromosomes in the meiotic phase, and the second protein is selected from the following proteins:
  • the REC8 protein as shown in SEQ ID NO: 25 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and REC8 protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with REC8 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the third protein is involved in the second division of meiosis, and the third protein is selected from the following proteins:
  • the OSD1 protein as shown in SEQ ID NO: 26 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and the OSD1 protein, 85%, 90%, 95% or 98% of sequence-equalized proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with OSD1 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the TAM protein as shown in SEQ ID NO: 27 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and TAM protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with TAM protein a protein of sequence similarity of 85%, 90%, 95% or 98%; a TDM1 protein as set forth in SEQ ID NO: 28, having at least 30%, 35%, 40%, 45%, 50% with the TDM1 protein , 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence-equalized protein, or at least 40%, 45%, 50 with TDM1 protein %, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence similarity protein;
  • TDM1 protein as set forth in SEQ ID NO: 28, having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and TDM1 protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with TDM1 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the fourth protein is selected from the following proteins:
  • the MTL protein as shown in SEQ ID NO: 29 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and MTL protein, 85%, 90%, 95%, or 98% of the sequence-consistent protein, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with the MTL protein , 85%, 90%, 95% or 98% of sequence similarity proteins.
  • a method of maintaining plant heterosis comprises the following steps: S1, using gene editing technology to transform the meiosis of the germ cell of the hybrid in the F1 generation into a diploid female gamete similar to mitosis to obtain the F1 generation; and S2, using gene mutation and genetic engineering technology Affecting the development of plant gametes or embryos induces the development of diploid female gametes into seeds, and the proteins involved are MTL proteins.
  • S1 comprises taking hybrid F1 generation seeds and transforming the meiosis of germ cells into mitosis like mitosis to obtain the F1 generation of diploid female gametes.
  • S1 includes editing the parents of the hybrids by using a gene editing technique, obtaining the edited plants whose genes are heterozygously mutated, and then obtaining hybrids by crossing between the parents, and screening the two parents in the hybrids.
  • Many of the genes edited in the Chinese genus are homozygous mutant plants, and the meiosis of germ cells is transformed into a diploid female gamete resembling the mitotic F1 generation.
  • S1 includes knocking out the REC8, OSD1, and PAIR1 genes by gene editing technology to transform meiosis of germ cells into mitosis.
  • S2 comprises administering a diploid female gamete haploid-inducing pollen to induce diploid female gametes to develop into seeds.
  • S2 includes knocking out the MTL gene using a gene editing technique to produce a haploid-inducing pollen.
  • S2 includes the use of haploid-inducing pollen of other plants to induce diploid female gametes to develop into seeds.
  • hybrids simultaneously knocked out the REC8, OSD1, PAIR1 and MTL genes in the F1 generation.
  • the plants include rice, corn, sorghum, millet, barley, and wheat.
  • the hybrid seed can produce cloned seeds which are completely consistent with the genotype of the self, so that the hybrid species can be utilized for a long time, and the hybridization between the parents due to inconsistent flowering period and the like in the utilization of heterosis is solved, and the seed production is difficult. Low, high cost of hybrids and other issues.
  • 1A is a schematic view showing the flow of a three-line cross breeding technique in the prior art
  • 1B is a schematic view showing the flow of a two-line cross breeding technology in the prior art
  • FIGS. 2 and 3 are schematic views showing the maintenance of the F1 generation genotype of the present invention.
  • Fig. 4B shows the results of cell ploidy detection of heterosis-fixed plants in Example 1.
  • Fig. 5 shows the results of whole-genome sequencing of the parental C84, the female parent 16A, the hybrid Chunyou 84 (CY84), and the genotype and ploidy fixed plants in Example 1.
  • Heterosis refers to the phenomenon that the first generation of hybrids is superior to the parents in terms of body shape, growth rate, and behavioral characteristics.
  • Meiosis When the germ cells divide, the chromosomes are only replicated once, and the cells divide twice in succession. This is a special way of dividing the number of chromosomes by half.
  • Mitosis also known as indirect division, is a plant found in E.Strasburger (1880). It is characterized by the presence of spindles and chromosomes in the process of division, so that the sub-chromosomes that have been replicated in S phase are evenly distributed. In the case of daughter cells, this mode of division is commonly found in higher animals and plants (animals and higher plants).
  • Chromosome ploidy refers to the number of genomes or genomes contained in a cell, such as haploid staining and polyploid staining.
  • Diploid female gametes Gametes refer to mature cells produced by the reproductive system when the organism is sexually reproducing, referred to as germ cells. Gametes are divided into male gamete and female gamete; usually germ cell division When the chromosome is only replicated once, the cell divides twice, and the number of chromosomes is halved. However, if the number of chromosomes is not halved when the female gametes are generated, but the number of chromosomes of the somatic cells is the same, it is called double. Female and female gametes.
  • Haploid An individual or cell whose number of somatic cell chromosomes is equal to the number of gametes in this species.
  • Parthenogenesis also known as parthenogenesis, means that the egg can develop into a normal new individual without fertilization.
  • a hybrid refers to a plant or seed heterozygous for genotype, and the offspring of sexual reproduction are genetically separated.
  • a method of utilizing plant heterosis comprises the steps of: S1, using genetic mutation or genetic engineering technology to convert the meiosis of the germ cells of the hybrid into mitosis to obtain a gamete that is consistent with the genotype and chromosome ploidy of the hybrid; and S2, using the gene mutation And genetic engineering techniques affect the involvement of plant gametes or embryo development processes, and the proteins involved are MTL proteins.
  • gene mutations include random mutagenesis and site-directed mutagenesis; random mutagenesis includes chemical mutagenesis, physical mutagenesis and biological mutagenesis; targeted mutagenesis includes gene editing technology, preferably, gene editing technology including CRISPR/Cas gene editing technology , CRISPR/Cpf1 gene editing technology, TALEN gene editing technology, homing endonuclease gene editing technology and ZFN gene editing technology; genetic engineering technology includes transgenic technology to induce gene specific expression, translocation expression or gene silencing.
  • common methods of physical mutagenesis include: radiation (ultraviolet rays, X-rays, ray rays, neutron rays), laser microbeams, ion beams, microwaves, ultrasonic waves, heat, and the like.
  • Common methods of chemical mutagenesis dipping method, smearing method, dropping method, injection method, application method and fumigation method, chemical mutagens include: alkylating agent, base analog, lithium chloride, nitroso compound, Azide, antibiotic, hydroxylamine, acridine, diethyl sulfate (DFS), 5-bromouracil (5-BU), nitrogen mustard (Nm), N' wide methyl N' nitrosoguanidine (NTG) Wait.
  • Biological mutagenesis methods include: space condition treatment mutagenesis, pathogenic microorganism mutagenesis, tissue culture mutagenesis, transgenic mutagenesis.
  • TILLING Targeting Induced Local Lesions IN Genomes
  • McCallum et al. Plant Physiology, 2000, 123, 439-442
  • Targeted mutagenesis is performed using standard techniques known in the art and utilizes homologous recombination, preferably in combination with nucleases such as TALEN or CRISPR.
  • the method comprises the steps of: S1, using genetic mutation or genetic engineering techniques to transform the meiosis of the germ cells of the hybrid into mitosis-like to obtain genotype and chromosome ploidy with the hybrid Consistent gametes; and S2, induce gametes to develop into seeds or plants.
  • the hybrid seed can produce cloned seeds or plants which are completely consistent with the genotype of the self, so that the hybrid species can be utilized for a long time, and it is difficult to solve the hybridization between the parents due to the inconsistent flowering period in the utilization of the heterosis. Problems such as low yield and high cost of hybrids.
  • S1 comprises taking a hybrid, and using genetic mutation or genetic engineering technology to transform the meiosis of the germ cell into a mitosis-like manner to obtain a gamete that is consistent with the genotype and chromosome ploidy of the hybrid,
  • the specific operation may be:
  • S1 includes taking hybrid F1 generation seeds, and using genetic engineering techniques to transform meiosis of germ cells into mitosis like mitosis to obtain F1 generation diploid gametes.
  • the specific method may be: taking the F1 generation hybrid seed, and editing the key gene involved in meiosis by introducing the gene editing system to obtain the genetically edited F1 generation plant, and the female gamete of the F1 generation plant after editing the gene is doubled.
  • the gamete preferably, the key genes involved in meiosis are the three genes REC8, OSD1, and PAIR1.
  • S1 includes editing a parent of a hybrid using gene mutation or genetic engineering technology, and then obtaining a hybrid by crossing between the parents, thereby obtaining meiosis of the germ cell into mitosis-like Hybrid gametes.
  • the specific operation may be: S1 includes editing a parent of the hybrid using genetic engineering technology, obtaining a hybrid mutant in which the key genes involved in meiosis are heterozygous mutation states, and then obtaining hybridization by crossing between the parents.
  • the key genes involved in meiosis are screened as homozygous mutant plants, and then the meiosis of germ cells is transformed into a diploid female gamete similar to the mitotic F1 generation.
  • the specific method may be: taking the male parent and the female parent of the hybrid respectively, and editing the above three key genes involved in meiosis by introducing a gene editing system, and obtaining the above three genes after the gene editing are all heterozygous.
  • the female gametes of the F1 generation seeds are diploid female gametes.
  • S1 comprises using a gene mutation or a genetic engineering technique to edit a protein involved in meiosis in a plant to transform meiosis of a germ cell into a mitosis-like; the protein includes a first protein, a second Protein and third protein, among them,
  • the first protein is involved in the formation of a DNA double-strand break, and the first protein is selected from the following proteins:
  • the SEQ ID NO: 13 (MKLKMNKACDIASISVLPPRRTGGSSGASASGSVAVAVASQPRSQPLSQSQQSFSQGASASLLHSQSQFSQVSLDDNLLTLLPSPTRDQRFGLHDDSSKRMSSLPASSASCAREESQLQLAKLPSNPVHRWNPSIADTRSGQVTNEDVERKFQHLASSVHKMGMVVDSVQSDVMQLNRAMKEASLDSGSIRQKIAVLESSLQQILKGQDDLKALFGSSTKHNPDQTSVLNSLGSKLNEISSTLATLQTQMQARQLQGDQTTVLNSNASKSNEISSTLATLQTQMQADIRQLRCDVFRVFTKEMEGVVRAIRSVNSRPAAMQMMADQSYQVPVSNGWTQINQTPVAAGRSPMNRAPVAAGRSRMNQLPETKVLSAHLVYPAKVTDLKPKVEQGKVKAAPQKPFASSYY
  • the PAIR2 protein as shown in SEQ ID NO: 14 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80 with the PAIR2 protein. %, 85%, 90%, 95% or 98% of the sequence-consistent protein, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, with the PAIR2 protein, 80%, 85%, 90%, 95% or 98% sequence similarity protein;
  • the SEQ ID NO: 15 (MEVELTNIQKATSSDYWSLASNQYPCGKFPKVSVGVTIPRTSSVSRGRDAASTAAFEKNLSQGTDGRSRPPKMDNASLQVSPEAANHGGSAKEVPKPVPAKVSVSQPDDNAIEQTGTFSFGTRREQDSHLDQLDRPPLVSSQGKRQVESADKNKPNSEMLRMKLWEILGGTSQNKEAVASPNPEDIETPCQPKSQIANGPSSGRQKVFTSPVPYNIKTPAQFNSQTANKPSS) PAIR3 protein shown with pair3 protein having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% 85%, 90%, 95% or 98% of sequence-equalized proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with PAIR3 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the SEQ ID NO: 16 (MEMVLIMSFRVLLYHRLTAQTGPFKLHCLGILLNSTKDAATYIGDKQSLYLNLVNNLRLPSDEIRGEILFVLYKLSLLNATPWDDICDNDNVDLSAIGRSLLQFSLEVLLKTQNDDVRLNCIALLLTLAKKGAFDILLLSDPSLINSAEAEDNVPLNDSLVILFAEAVKGSLLSTNIEVQTGTLELIFHFLSSDANIFVLKTLIDQNVADYVFEVLRLSGMRNHLLQSSNASQFLTKLLYVSGNNDPLVISSIKVLSILANSEERFKEKLAIAVSTLLPVLHYVSEIPFHPVQSQVLRLVCISIINCSGILSLSQEEQIACTLSAILRRHGNGELGMSSETFALVCSMLVEILKLPSADDIQKLPSFIVEASKHAISLTFSHEYDCLFLIPHSLLLLKEALIFCLEGNKDQILRKKSLEDSIIETCETYLLPWLESAIVDGNDEET
  • the SEQ ID NO: 17 (MAPPASRPPTPTPTPTANAAASSSRIESPSLRAALAMALIHYNRLPSRAAAAAAPSPQALLNWKRKAKDRKREILRLREELKLLQDGARGEEMEPPVASCRCHFFDGCGDLPPPTDGDAGEHWVDDVLRRRFVRLVRWKDKRRRLDRSLPTSSLMEYNTEDEVQQLSLSIDFLVELSDGLFAKREAGSSFTTFSHQAVDFILASLKNILSSEREKEIIEEIINGLVARLMKRMCTTPENAGSVDCSDAQFSLQHLFRKLGNEEFVGQRIILAISQKISNVSEKLLLADPFDDGFPE MHSNMFIMIQLIEFLISDSFNNWLCRDHFDRKLFEEWVRSILKARKDLEVLDGRNGLYVVYIERVIGRLAREVAPAAHQGKLDLEVLSKLLY) PRD2 protein shown with PRD2 protein having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%
  • the SEQ ID NO: 18 (MAGREKRRRVAALDGEERRRRQEEAATLLHRIRGLVRWVVAEVAAGRSPTVALHRYQNYCSSASAAAASPCACSYDVPVGTDVLSLLHRGSHASRLNVLLRVLLVVQQLLQNKHCSKRDIYYMYPSIFQEQAVVDRAINDICVLFKCSRHNLNVVPVAKGLVMGWIRFLEGEKEVYCVTNVNAAFSIPVSIEAIKDVVSVADYILIVEKETVFQRLANDKFCERNRCIVITGRGYPDIPTRRFLRYLVEQLHLPVYCLVDADPYGFDILATYKFGSLQLAYDANFLRVPDIRWLGVFTSDFEDYRLPDCCLLHLSSEDRRKAEGILSRCYLHREAPQWRLELEAMLQKGVKFEIEALSACSISFLSEEYIPKKIKQGRHI) SPO11-1 protein shown with Spo1 1-1 protein having at least 30%, 35%, 40%, 45%, 50%, 55%,
  • SEQ ID NO: 19 (MAEAGVAAASLFGADRRLCSADILPPAEVRARIEVAVLNFLAALTDPAAPAISALPLISRGAANRGLRRALLRDDVSSVYLSYASCKRSLTRANDAKAFVRVWKVMEMCYKILGEGKLVTLRELFYTLLSESPTYFTCQRHVNQTVQDVVSLLRCTRQSLGIMASSRGALIGRLVVQGPEEEHVDCSILGPSGHAITGDLNVLSKLIFSSDARYIIVVEKDAIFQRLAEDRIYSHLPCILITAKGYPDLATRFILHRLSQTYPNMPIFALVDWNPAGLAILCTYKYGSISMGLESYRYACNVKWLGLRGDDLQLIPQSAYQELKPRDLQIAKSLLSSKFLQDKHRAELTLMLETGKRAEIEALYSHGFDFLGKYVARKIVQGDYI) SPO11-2 protein shown with SPO11-2 protein having at least 30%, 35%, 40%, 45%, 50%, 55%
  • the SEQ ID NO: 21 (MSAPMEVSFSAPPPPDAASAAAAAPSLVPAVSAAAVAATTVSCSPQPPTGSPSADDRILVSVEVLLHATSTARAEDVCAAVERMLEARSLSYVDGPVPIPNDDPFLLANVKRIQICDTDEWTENHKVLLFWQVRPVVHVFQLSEDGPGEEPGEDDTLSSFNEWALPAK EFDGLWESLLYEVGLKQRLLRYAASALLFTEKGVDPCLVSWNRIVLLHGPPGTGKTSLCKALAQKLSIRFKSRYSMCQLIEVNAHSLFSKWFSESGKLVAKLFQKIQEMVEEESNLVFVLIDEVESLAAARQAAISGSEPSDSIRVVNALLTQMDKLKSWPNVIILTTSNITTAIDIAFVDRADIKAYVGPPTLQARYEILRSCLQELLRVGILTHTQGGNSLCLLSYFSLMENQHCPEVADPHGSVHLSGLLHKAAEICEGLSGRTLRKLPFLAHASVANPSC
  • the SEQ ID NO: 23 (MASSPPPSTASPTSSSPYRKLLHSLIYWAVQRCRMSESPCRLTVSVKRSPEPAGSSPLRISVSDTGVGSKLEEFLELDALARETPVEKWDGTLLITTTGIDDKAIYRYQFNLQEDTSSSTRFTKLATMYKSRAIFSGTEVCLCLPTEADVDDLILWLVGFVRKIFVLRASNLACELFVAQTDSAGSGDVCLSQDSDDVHISITTSSIDRLVSGLKDYALSHANTSDRCEACYMNRDRLKIGTGTAKYVDKRKAKGQLVEVVIMIAPTSSDLSCWMTNCSSTQVLHFVEFIPCPISQSSLSALMSIDWQSYGFKFKGGFIDDDGNAELQWDNMAFSHVDIAIHTYHEGAVDEWKSSQPERHLLRKALKSALFGLKADHAEDFLSCHGQKVREYVPDLAESIAGLILSSNDQEFQDECIALLGLGSDQDLTEGAVR
  • the SEQ ID NO: 24 (MRHNIKFKSKGTLKIRNTAQISLWKKCSDSMIADQTYLFINRVQDRRFDEESLRILELSLVAMNVKSFLEVRSRLRDFMRSESVVIFGELTGESMVAKLSVLEFFARAFALLGDMESCLAMRYEALNLRQLKSPSCLWLGVSHSEWTKFAVQSMENGFPSIAGKASENALLSLKKDSLIEPKSEDNSDILDAAEKVRRLRDSAASLTSSHSGIFIYIVSSLKFAVCNRLLTTF) DFO protein illustrated with DFO protein having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence-consistent protein, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% with DFO protein a protein of sequence similarity of 80%, 85%, 90%, 95% or 98%;
  • the second protein is involved in controlling the adhesion between sister chromosomes in the meiotic phase, and the second protein is selected from the following proteins:
  • the REC8 protein as shown in SEQ ID NO: 25 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80 with the REC8 protein. %, 85%, 90%, 95% or 98% of the sequence-consistent protein, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, with the REC8 protein, 80%, 85%, 90%, 95% or 98% sequence similarity protein;
  • the third protein is involved in the second division of meiosis, and the third protein is selected from the following proteins:
  • the SEQ ID NO: 27 (MSSSSRNLSQENPIPRPNLAKTRTSLRDVGNRRAPLGDITNQKNGSRNPSPSSTLVNCSNKIGQSKKAPKPALSRNWNLGILDSGLPPKPNAKSNIIVPYEDTELLQSDDSLLCSSPALSLDASPTQSDPSISTHDSLTNHVVDYMVESTTDDGNDDDDDEIVNIDSDLMDPQLCASFACDIYEHLRVSEVNKRPALDYMERTQSSINASMRSILIDWLVEVAEEYRLSPETLYLAVNYVDRYLTGNAINKQNLQLLGVTCMMIAAKYEEVCVPQVEDFCYITDNTYLRNELLEMESSVLNYLKFELTTPTAKCFLRRFLRAAQGRKEVPSLLSECLACYLTELSLLDYAMLRYAPSLVAASAVFLAQYTLHPSRKPWNATLEHYTSYRAKHMEACVKNLLQLCNEKLSSDVVAIRKKYSQHKYKFAAKKLCPTSLPQELFL
  • the SEQ ID NO: 28 (MCPCVERRAPPGVYYTPPPARTSDHVAAMPMTERRRPPYSCSSSSERRDPFHIVHKVPSGDSPYVRAKHAQLIDKDPNRAISLFWTAINAGDRVDSALKDMAVVMKQLGRSDEGIEAIKSFRYLCSFESQDSIDNLLLELYKKSGRIEEEAVLLEHKLQTLEQGMGFGGRVSRAKRVQGKHVIMTIEQEKARILGNLGWVHLQLHNYGIAEQHYRFGFVTKIPNIDYCLVMRALGLERDKNKLCNLAICLMRMSRIPEAKSLLDDVRDSPAESECGDEPFAKSYDRAVEMLA EIESKKPEADLSEKFYAGCSFVNRMKENIAPGTANKNYSDVSSSPASVRPNSAGLYTQPRRCRLFEEETRGAARKLLFGKPQPFGSEQMKILERGEEEPMKRKKLDQNMIQYLHEFVKDTADGPKSESKKSWADIAEEEEEEEEEEEERL
  • PAIR1 protein is involved in the initiation of meiotic recombination, catalyzes the formation of DNA double-strand breaks, and the deletion of PAIR1 gene leads to the loss of recombination process;
  • REC8 protein is responsible for the close association of newly replicated sister chromosomes, which is to ensure sisters ( Or homologous) a key regulatory factor that is correctly isolated and assigned to daughter cells, and its loss of function results in the separation of sister chromatids at the end of the first division of the meiosis, shifting to the cell poles; loss of OSD1 gene function , will lead to the formation of gametes directly skip the second division process of the subtraction.
  • Knocking out the above genes is a simple and effective method to transform meiosis of germ cells into mitosis-like.
  • the inhibitory protein described in the present invention refers to a gene encoding a protein or a promoter thereof by mutagenesis, and selects a part or all of the activity of the lost protein, including, by expressing a silencing RNA in a plant, to obtain inhibition of the related protein. .
  • S2 includes utilizing genetic mutation and genetic engineering techniques to influence the development of plant gametes or embryos, and to induce gamete development into seeds or plants.
  • S2 may include inducing pollen from other plants to induce gamete development into seeds or plants, for example, S2 includes administering diploid female gamete haploid-inducing pollen to induce diploid female gametes to develop into seeds; S2 includes inducing gametes to develop into seeds or plants by physical stimulation, biotic stress or chemical treatment; for example, S2 includes inducing gametes to develop into seeds or plants by anther culture or pollen culture.
  • MTL protein is SEQ ID NO: 29 (MAASYSCRRTCEACSTRAMAGCVVGEPASAPGQRVTLLAIDGGGIRGLIPGTILAFLEARLQELDGPDARLADYFDCIAGTSTGGLITAMLAAPGDHGRPLFAASDINRFYLDNGPLIFPQKRCGMAAAMAALTRPRYNGKYLQGKIRKMLGETRVRDTLTNVVIPTFDVRLLQPTIFSTYDAKSMPLKNALLSDICISTSAAPTYLPAHCFQTTDDATGKVREFDLIDGGVAANNPTMVAMTQITKKIMVKDKEELYPVKPSDCGKFLVLSVGTGSTSDQGMYTARQCSRWGIVRWLRNKGMAPIIDIFMAASSDLVDIHAAVMFQSLHSDGDYLRIQDNTLHGDAATVDAATRDNMRALVGIGERMLAQRVSRVNVETGRYVEVPGAGSNADALRGFARQLSEERRARLGRRNACGGGGEGEPSGVACKR) protein shown MTL, the
  • the inducing pollen may be from a plant that produces a genotype and a ploidy homozygous game with the hybrid, or may be derived from other plants.
  • the inducing pollen is derived from the genotype and the ploidy and the hybrid. The plants were achieved by simultaneously knocking out the REC8, OSD1, PAIR1 and MTL genes in the hybrid.
  • the plant comprises monocotyledonous and dicotyledonous plants; preferably, the plant comprises rice, corn, sorghum, millet, barley, wheat, rye, oats, buckwheat, coix seed, sugar cane, asparagus, Bamboo shoots, leeks, yam, soybeans, potatoes, peas, mung beans, adzuki beans, broad beans, cowpeas, kidney beans, lentils, peas, chickpeas, cassava, sweet potatoes, rapeseed, cotton, beets, eggplant, peanuts, tea, mint, Coffee, sesame, sunflower, ramie, sorghum, safflower, tomato, pepper, cucumber, greens, lettuce, spinach, garlic, kale, mustard, scallions, green onions, melon, zucchini, loofah, cabbage, radish, onion, watermelon , grapes, carrots, cauliflower, pumpkin, tobacco, pasture, grassy
  • the principle of apomixis in this application is to directly form embryos and produce seeds by bypassing meiosis and fertilization processes, which are mainly divided into two major steps:
  • meiosis is a special process of cell division during the reproductive period of animals and plants. At the time of meiosis, the genetic information from the parents will be recombined, producing a gamete with a halved number of chromosomes.
  • the number and genotype of chromosomes in the female and male gametophytes produced by MiMe plants are completely consistent with those of somatic cells, and the self-crossing progeny are genotypes heterozygous tetraploids, which proves that hybrid plants can be bypassed by simultaneously mutating three genes.
  • the meiotic process produces cloned gametes that are consistent with somatic genotypes.
  • Step 2 The pollen-specific phospholipase gene (MATRILINEAL, MTL) mainly acts on plant male gametes and is a gene that controls haploid induction and is first cloned in maize.
  • the haploid inducing material mtl can be obtained by knocking out the MTL gene.
  • the genome of the mtl male gamete in the zygote was degraded, that is, the paternal sperm nucleus did not form a zygote with the recipient nucleus, and induced nucleus haploid fruiting.
  • the following steps are included: 1) converting meiosis during gametogenesis into mitosis.
  • This material is named MiMe, Mitosis instead of Meiosis
  • the chromosomes are replicated once, and the germ cells are changed from the original division to the division.
  • the number of chromosomes formed in the gametes thus formed was not halved, which was consistent with somatic cells.
  • the meiosis is shifted to a similar mitosis to achieve the purpose of chromosome doubling; 2) the female gametes produced are stimulated by pollen that can induce the development of female gametes, that is, the female gametes do not fuse with the sperm cell chromosomes to develop embryos, forming and Seeds with completely identical somatic genotypes. Pollen that induces haploid production can be obtained by knocking out the MTL gene. Using hybrid seeds as the transgenic background, using gene mutation or genetic engineering technology to simultaneously knock out four genes of REC8, OSD1, PAIR1 and MTL, the female gametes produced by this plant are the same as the somatic cells, and the MTL gene is destroyed.
  • the pollen produced can induce the development of female gametes into seeds or plants, so that the obtained seeds or plants do not have gene isolation (separation of traits or fertility), and genotypes and mother cells (for genetically modified background material hybrids) are The same, ultimately to achieve the purpose of fixed heterosis.
  • FIGS. 2 and 3 clearly show that the F1 progeny genotype and ploidy of the F1 of the present invention are consistent with the hybrid mother cells.
  • a plant or seed that maintains a heterosis is provided.
  • the plant or seed is prepared by any of the above methods, and the seed is capable of fixing the hybridization advantage well.
  • a kit for maintaining a plant heterosis includes vectors and/or reagents capable of converting meiosis of plant germ cells into mitotic-like vectors, and vectors and/or reagents for developing gametes into seeds or plants.
  • the vector and/or reagent capable of transforming meiosis of plant germ cells into a mitotic-like vector and/or reagent and a vector and/or reagent for inducing parthenogenesis of the plant gamete is random mutagenesis or directed mutagenesis.
  • random mutagenesis includes chemical mutagenesis, physical mutagenesis and biological mutagenesis
  • targeted mutagenesis includes CRISPR/Cas gene editing technology, CRISPR/Cpf1 gene editing technology, TALEN gene editing technology, homing endonuclease gene editing technology.
  • ZFN gene editing technology genetic engineering technology includes transgenic technology to induce gene specific expression, translocation expression or gene silencing.
  • the meiosis of plant germ cells can be converted into a mitotic-like vector and/or reagent for meiosis of germ cells by using genetic engineering techniques to inhibit proteins involved in meiotic recombination in plants.
  • the first protein is involved in the formation of a DNA double-strand break, and the first protein is selected from the following proteins:
  • the PAIR1 protein as shown in SEQ ID NO: 13 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and PAIR1 protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with PAIR1 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the PAIR2 protein as shown in SEQ ID NO: 14 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and PAIR2 protein, 85%, 90%, 95% or 98% of sequence-equalized proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with PAIR2 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the PAIR3 protein as shown in SEQ ID NO: 15 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and PAIR3 protein, 85%, 90%, 95% or 98% of sequence-equalized proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with PAIR3 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • PRD1 protein as shown in SEQ ID NO: 16 having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and PRD1 protein, 85%, 90%, 95% or 98% of the sequence-consistent protein, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with the PRD1 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • a PRD2 protein as set forth in SEQ ID NO: 17 having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and PRD2 protein, 85%, 90%, 95% or 98% of the sequence-consistent protein, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with the PRD2 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the SPO11-1 protein as shown in SEQ ID NO: 18 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% with the SPO11-1 protein. 80%, 85%, 90%, 95% or 98% of the sequence-consistent protein, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70% with the SPO11-1 protein a 75%, 80%, 85%, 90%, 95% or 98% sequence similarity protein;
  • the SPO11-2 protein as shown in SEQ ID NO: 19 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% with the SPO11-2 protein. 80%, 85%, 90%, 95% or 98% of the sequence-consistent protein, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70% with the SPO11-2 protein a 75%, 80%, 85%, 90%, 95% or 98% sequence similarity protein;
  • SDS protein as set forth in SEQ ID NO: 20, having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and SDS protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with SDS protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the CRC1 protein as shown in SEQ ID NO: 21 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and CRC1 protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with CRC1 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the P31 comet protein as shown in SEQ ID NO: 22 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80 with the P31 comet protein. %, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% with P31 comet protein a protein of sequence similarity of 80%, 85%, 90%, 95% or 98%;
  • the MTOPVIB protein as set forth in SEQ ID NO: 23 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and MTOPVIB protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with MTOPVIB protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • DFO protein as set forth in SEQ ID NO: 24, having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and DFO protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with DFO protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the second protein is involved in controlling the adhesion between sister chromosomes in the meiotic phase, and the second protein is selected from the following proteins:
  • the REC8 protein as shown in SEQ ID NO: 25 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and REC8 protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with REC8 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the third protein is involved in the second division of meiosis, and the third protein is selected from the following proteins:
  • the OSD1 protein as shown in SEQ ID NO: 26 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and the OSD1 protein, 85%, 90%, 95% or 98% of sequence-equalized proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with OSD1 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the TAM protein as shown in SEQ ID NO: 27 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and TAM protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with TAM protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • TDM1 protein as set forth in SEQ ID NO: 28, having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and TDM1 protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with TDM1 protein , 85%, 90%, 95% or 98% of sequence similarity proteins.
  • the MTL protein is an MTL protein as set forth in SEQ ID NO: 29, and has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, and MTL protein, 75%, 80%, 85%, 90%, 95% or 98% of the sequence-consistent protein, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70% with the MTL protein , 75%, 80%, 85%, 90%, 95% or 98% of sequence similarity proteins.
  • kits comprise vectors and/or reagents for simultaneously knocking out the REC8, OSD1, PAIR1 and MTL genes in the hybrid.
  • a plant is provided.
  • the meiosis of the germ cell of the plant is converted to mitosis to produce a gamete that is consistent with the genotype and chromosome ploidy of the hybrid; for example, the meiosis of the germ cell of the plant is transformed into a mitosis-like transformation to produce a hybrid Gametes with ploidy and genotypes.
  • the plant is capable of inducing gamete development into plants or seeds.
  • the plant is a genetically modified or genetically engineered plant, and the plant utilizes gene mutation or genetic engineering technology to regulate the protein involved in meiosis in the plant to transform the meiosis of the germ cell into a mitosis-like; Mutation or genetic engineering techniques affect the MTL protein involved in gametogenesis development in plants and induce gametes to develop into seeds or plants; wherein the protein includes a first protein, a second protein, and a third protein, wherein
  • the first protein is involved in the formation of a DNA double-strand break, and the first protein is selected from the following proteins:
  • the PAIR1 protein as shown in SEQ ID NO: 13 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and PAIR1 protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with PAIR1 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the PAIR2 protein as shown in SEQ ID NO: 14 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and PAIR2 protein, 85%, 90%, 95% or 98% of sequence-equalized proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with PAIR2 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the PAIR3 protein as shown in SEQ ID NO: 15 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and PAIR3 protein, 85%, 90%, 95% or 98% of sequence-equalized proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with PAIR3 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • PRD1 protein as shown in SEQ ID NO: 16 having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and PRD1 protein, 85%, 90%, 95% or 98% of the sequence-consistent protein, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with the PRD1 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • a PRD2 protein as set forth in SEQ ID NO: 17 having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and PRD2 protein, 85%, 90%, 95% or 98% of the sequence-consistent protein, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with the PRD2 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the SPO11-1 protein as shown in SEQ ID NO: 18 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% with the SPO11-1 protein. 80%, 85%, 90%, 95% or 98% of the sequence-consistent protein, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70% with the SPO11-1 protein a 75%, 80%, 85%, 90%, 95% or 98% sequence similarity protein;
  • the SPO11-2 protein as shown in SEQ ID NO: 19 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% with the SPO11-2 protein. 80%, 85%, 90%, 95% or 98% of the sequence-consistent protein, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70% with the SPO11-2 protein a 75%, 80%, 85%, 90%, 95% or 98% sequence similarity protein;
  • SDS protein as set forth in SEQ ID NO: 20, having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and SDS protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with SDS protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the CRC1 protein as shown in SEQ ID NO: 21 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and CRC1 protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with CRC1 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the P31 comet protein as shown in SEQ ID NO: 22 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80 with the P31 comet protein. %, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% with P31 comet protein a protein of sequence similarity of 80%, 85%, 90%, 95% or 98%;
  • the MTOPVIB protein as set forth in SEQ ID NO: 23 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and MTOPVIB protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with MTOPVIB protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • DFO protein as set forth in SEQ ID NO: 24, having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and DFO protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with DFO protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the second protein is involved in controlling the adhesion between sister chromosomes in the meiotic phase, and the second protein is selected from the following proteins:
  • the REC8 protein as shown in SEQ ID NO: 25 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and REC8 protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with REC8 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the third protein is involved in the second division of meiosis, and the third protein is selected from the following proteins:
  • the OSD1 protein as shown in SEQ ID NO: 26 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and the OSD1 protein, 85%, 90%, 95% or 98% of sequence-equalized proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with OSD1 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the TAM protein as shown in SEQ ID NO: 27 has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and TAM protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with TAM protein a protein of sequence similarity of 85%, 90%, 95% or 98%; a TDM1 protein as set forth in SEQ ID NO: 28, having at least 30%, 35%, 40%, 45%, 50% with the TDM1 protein , 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence-equalized protein, or at least 40%, 45%, 50 with TDM1 protein %, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence similarity protein;
  • TDM1 protein as set forth in SEQ ID NO: 28, having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, and TDM1 protein, 85%, 90%, 95% or 98% of sequence-consistent proteins, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% with TDM1 protein a protein of sequence similarity of 85%, 90%, 95% or 98%;
  • the MTL protein is an MTL protein as set forth in SEQ ID NO: 29, and has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, and MTL protein, 80%, 85%, 90%, 95% or 98% sequence-equalized protein, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% with the MTL protein , 80%, 85%, 90%, 95% or 98% of sequence similarity proteins.
  • the F 1 hybrid used was the approved commercial hybrid rice variety Chunyou 84.
  • Chunyou 84 is a new hybrid rice combination of the subspecies of the cultivar, which is bred by the combination of Chunjiang 16A and the middle-type broad-affinity restorer line C84 in early flowering.
  • the hybrid rice has the advantages of large yield potential, high seed production, excellent agronomic traits, good resistance to mites and wide adaptability.
  • the genetically transformed background material used in the present example is a callus obtained by inducing hybrid rice F 1 seeds, and has not undergone a sexual reproduction stage. Therefore, the transgenic T 0 generation material obtained after transgenic is genetically related to hybrid rice. F 1 plants are consistent.
  • OSD1 gene knockout site (SEQ ID NO: 1): CTGCCGCCGACGAGCAACA AGG
  • PAIR1 gene knockout site (SEQ ID NO: 2): AAGCAACCCAGTGCACCGC TGG
  • MTL gene knockout site (SEQ ID NO: 4): GGTCACGCGCGAGACCGGC AGG
  • SK-gRNA There are two AarI cleavage sites on SK-gRNA, which are digested with AarI to form a vector with sticky ends; after denaturing annealing of the designed target sequence forward and reverse primers, T4 ligase is ligated before construction.
  • Intermediate vector SK-gRNA forming a single gRNA of interest;
  • NheI and XbaI, SalI and XhoI are the properties of homologous enzymes for the polymerization of gRNA: SK-gRNA OSD1 was digested with KpnI and XhoI as a vector; SK-gRNA PAIR1 was digested with SalI and XbaI.
  • the PAIR1sgRNA fragment, SK-gRNA REC8 is digested with NheI and BamHI to provide the REC8sgRNA fragment, and the SK-gRNA MTL is digested with BglII and KpnI to provide the MTL sgRNA fragment, and the one-step rapid polymerization of gRNA within four is performed; gRNA OSD1-gRNA REC8-gRNA PAIR1-gRNA MTL fragment was digested with KpnI and BglII and the fragment was recovered and ligated into the binary vector pC1300-Cas9 expressing the Cas9 protein (between KpnI and BamHI sites).
  • a multi-knocker vector pC1300-Cas9-gRNA OSD1-gRNA REC8-gRNA PAIR1-gRNA MTL which is a gene for REC8, OSD1, PAIR1 and MTL, was used for transgenic rice multi-mutant.
  • the multi-gene knockout binary expression vector pC1300-Cas9-gRNA OSD1-gRNA REC8-gRNA PAIR1-gRNA MTL was transferred into AgroBacterium tumefaciens strain EHA105 by electroporation, and this pair was amplified by Agrobacterium-mediated method.
  • the meta-expression vector was transferred into the callus of rice Chunyou 84.
  • the specific method of transformation is to sterilize the embryo of hybrid rice Chunyou 84 seed and inoculate it into the medium for inducing callus. After 1 week of culture, a vigorously growing, light-colored, relatively loose embryogenic callus was selected and used as a receptor for transformation.
  • the rice calli were infested with EHA105 strain containing pC1300-Cas9-gRNA OSD1-gRNA REC8-gRNA PAIR1-gRNA MTL plasmid, and cultured in the dark at 25 ° C for 3 days, on a selection medium containing 50 mg / l hygromycin. Screening for resistant calli and transgenic plants. Transgenic plants that grew normally on hygromycin selection medium were selected.
  • OSD1-F (SEQ ID NO: 5): atctccaggatgcctgaagtgag
  • OSD1-R (SEQ ID NO: 6): cctagactgctactcttgctagtgat
  • PAIR1-F (SEQ ID NO: 7): ctgtacctgtgcatctaattacag
  • PAIR1-R (SEQ ID NO: 8): ccccatcttatgtactgagcttgccag
  • MTL-F (SEQ ID NO: 11): acagtgactagtgacaaacgatcg
  • MTL-R (SEQ ID NO: 12): gatcgcgtcagcatgatgcgtgtac
  • the obtained PCR product was sent to a sequencing company and sequenced using OSD1-F, PAIR1-F, REC8-F, and MTL-F as sequencing primers, respectively.
  • the results obtained were aligned with wild type sequences.
  • the sequencing results were bimodal, and the mutation information was directly obtained by degenerate codon strategy analysis (http://dsdecode.scgene.com/ for peak map analysis). Four mutants in which all four genes are biallelic mutations are screened.
  • Fig. 4A shows the results of cell ploidy detection of F1 generation plant Chunyou 84; and Fig. 4B shows the results of cell ploidy detection of heterosis fixed plants.
  • the maintenance system Chunjiang 16B and the ⁇ intermediate type broad affinity recovery system C84 were used.
  • the genetically transformed background material used in this example is a callus obtained by induction with a parental seed.
  • OSD1 gene knockout site (SEQ ID NO: 1): CTGCCGCCGACGAGCAACA AGG
  • PAIR1 gene knockout site (SEQ ID NO: 2): AAGCAACCCAGTGCACCGC TGG
  • MTL gene knockout site (SEQ ID NO: 4): GGTCACGCGCGAGACCGGC AGG
  • SK-gRNA There are two AarI cleavage sites on SK-gRNA, which are digested with AarI to form a vector with sticky ends; after denaturing annealing of the designed target sequence forward and reverse primers, T4 ligase is ligated before construction.
  • Intermediate vector SK-gRNA forming a single gRNA of interest;
  • NheI and XbaI, SalI and XhoI are the properties of homologous enzymes for the polymerization of gRNA: SK-gRNA OSD1 was digested with KpnI and XhoI as a vector; SK-gRNA PAIR1 was digested with SalI and XbaI The PAIR1sgRNA fragment, SK-gRNA REC8 is digested with NheI and BamHI to provide the REC8sgRNA fragment, and the SK-gRNA MTL is digested with BglII and KpnI to provide the MTL sgRNA fragment, and the one-step rapid polymerization of gRNA within four is performed; gRNA OSD1-gRNA REC8-gRNA PAIR1-gRNA MTL fragment was digested with KpnI and BglII and the fragment was recovered and ligated into the binary vector pC1300-Cas9 expressing the
  • a multi-knocker vector pC1300-Cas9-gRNA OSD1-gRNA REC8-gRNA PAIR1-gRNA MTL which is a gene for the four genes of REC8, OSD1, PAIR1 and MTL, was used for transgenic rice multi-mutant.
  • the multi-gene knockout binary expression vector pC1300-Cas9-gRNA OSD1-gRNA REC8-gRNA PAIR1-gRNA MTL was transferred into AgroBacterium tumefaciens strain EHA105 by electroporation, and this pair was amplified by Agrobacterium-mediated method.
  • the meta-expression vector was transferred into the callus of Chunjiang 16B and C84.
  • the specific method of transformation is to sterilize the embryo of the seed and inoculate it into the medium for inducing the callus. After 1 week of culture, a vigorously growing, light-colored, relatively loose embryogenic callus was selected and used as a receptor for transformation.
  • the rice calli were infested with EHA105 strain containing pC1300-Cas9-gRNA OSD1-gRNA REC8-gRNA PAIR1-gRNA MTL plasmid, and cultured in the dark at 25 ° C for 3 days, on a selection medium containing 50 mg / l hygromycin. Screening for resistant calli and transgenic plants. Transgenic plants that grew normally on hygromycin selection medium were selected.
  • OSD1-F (SEQ ID NO: 5): atctccaggatgcctgaagtgag
  • OSD1-R (SEQ ID NO: 6): cctagactgctactcttgctagtgat
  • PAIR1-F (SEQ ID NO: 7): ctgtacctgtgcatctaattacag
  • PAIR1-R (SEQ ID NO: 8): ccccatcttatgtactgagcttgccag
  • MTL-F (SEQ ID NO: 11): acagtgactagtgacaaacgatcg
  • MTL-R (SEQ ID NO: 12): gatcgcgtcagcatgatgcgtgtac
  • the obtained PCR product was sent to a sequencing company and sequenced using OSD1-F, PAIR1-F, REC8-F, and MTL-F as sequencing primers, respectively.
  • the obtained results are compared with the wild type sequence, and the mutation information can be directly obtained.
  • cell ploidy was screened by flow cytometry, and the plants having the cell ploidy and the mother plants were selected.
  • the leaves of two parents, Chunjiang 16B and C84, Chunyou 84 and ploidy fixed generation (two randomly selected plants) were selected and DNA was extracted for whole genome sequencing. According to the results of genome-wide sequencing, there are many different homozygous genotypes between Chunjiang 16B and C84.
  • the genotypes of hybrids Chunyou 84 at these loci are heterozygous for both Chunjiang 16B and C84 genotypes.
  • the genotypes of the two plants tested were consistent with Chunyou 84, and they were all heterozygous. From the molecular biological point of view, the genotype was completely consistent with the hybrid mother cells.
  • the F 1 hybrid used was the approved commercial hybrid rice variety Chunyou 84.
  • Chunyou 84 is a new hybrid rice combination of the subspecies of the cultivar, which is bred by the combination of the sterile line Chunjiang 16A and the middle-type broad-affinity restorer line C84.
  • the hybrid rice has the advantages of large yield potential, high seed production, excellent agronomic traits, good resistance to mites and wide adaptability.
  • the genetically transformed background material used in the present example is a callus obtained by inducing hybrid rice F 1 seeds, and has not undergone a sexual reproduction stage. Therefore, the transgenic T 0 generation material obtained after transgenic is genetically related to hybrid rice. F 1 plants are consistent.
  • OSD1 gene knockout site (SEQ ID NO: 1): CTGCCGCCGACGAGCAACA AGG
  • PAIR1 gene knockout site (SEQ ID NO: 2): AAGCAACCCAGTGCACCGC TGG
  • SK-gRNA There are two AarI cleavage sites on SK-gRNA, which are digested with AarI to form a vector with sticky ends; after denaturing annealing of the designed target sequence forward and reverse primers, T4 ligase is ligated before construction.
  • Intermediate vector SK-gRNA forming a single gRNA of interest;
  • the multi-gene knockout binary expression vector pC1300-Cas9-gRNA OSD1-gRNA REC8-gRNA PAIR1-gRNA was transferred into AgroBacterium tumefaciens strain EHA105 by electroporation, and the dual element was amplified by Agrobacterium-mediated method.
  • the expression vector was transferred into the callus of rice Chunyou 84.
  • the specific method of transformation is to sterilize the embryo of hybrid rice Chunyou 84 seed and inoculate it into the medium for inducing callus. After 1 week of culture, a vigorously growing, light-colored, relatively loose embryogenic callus was selected and used as a receptor for transformation.
  • Rice calli were infested with EHA105 strain containing pC1300-Cas9-gRNA OSD1-gRNA REC8-gRNA PAIR1 plasmid, and cultured for 3 days at 25 ° C in the dark, and screened for resistance on selection medium containing 50 mg/l hygromycin. Callus and transgenic plants. Transgenic plants that grew normally on hygromycin selection medium were selected.
  • OSD1-F (SEQ ID NO: 5): atctccaggatgcctgaagtgag
  • OSD1-R (SEQ ID NO: 6): cctagactgctactcttgctagtgat
  • PAIR1-F (SEQ ID NO: 7): ctgtacctgtgcatctaattacag
  • PAIR1-R (SEQ ID NO: 8): ccccatcttatgtactgagcttgccag
  • the obtained PCR product was sent to a sequencing company and sequenced using OSD1-F, PAIR1-F, and REC8-F as sequencing primers, respectively.
  • the results obtained were aligned with wild type sequences.
  • the sequencing results were bimodal, and the mutation information was directly obtained by degenerate codon strategy analysis (http://dsdecode.scgene.com/ for peak map analysis).
  • loci are mutants of biallelic mutations, which are plants that can produce gametes that are consistent with somatic genotypes and ploidy.
  • the genotypes of the four plants tested were consistent with Chunyou 84, and they were all heterozygous. From the molecular biology point of view, the genotype was completely consistent with the hybrid mother cells.
  • the F 1 hybrid used was the approved commercial hybrid rice variety Chunyou 84.
  • Chunyou 84 is a new hybrid rice combination of the subspecies of the cultivar, which is bred by the combination of the sterile line Chunjiang 16A and the middle-type broad-affinity restorer line C84.
  • the hybrid rice has the advantages of large yield potential, high seed production, excellent agronomic traits, good resistance to mites and wide adaptability.
  • the genetically transformed background material used in the present example is a callus obtained by inducing hybrid rice F 1 seeds, and has not undergone a sexual reproduction stage. Therefore, the transgenic T 0 generation material obtained after transgenic is genetically related to hybrid rice. F 1 plants are consistent.
  • OSD1 gene knockout site (SEQ ID NO: 1): CTGCCGCCGACGAGCAACA AGG
  • PAIR1 gene knockout site (SEQ ID NO: 2): AAGCAACCCAGTGCACCGC TGG
  • SK-gRNA There are two AarI cleavage sites on SK-gRNA, which are digested with AarI to form a vector with sticky ends; after denaturing annealing of the designed target sequence forward and reverse primers, T4 ligase is ligated before construction.
  • Intermediate vector SK-gRNA forming a single gRNA of interest;
  • the multi-gene knockout binary expression vector pC1300-Cas9-gRNA OSD1-gRNA REC8-gRNA PAIR1-gRNA MTL was transferred into AgroBacterium tumefaciens strain EHA105 by electroporation, and this pair was amplified by Agrobacterium-mediated method.
  • the meta-expression vector was transferred into the callus of rice Chunyou 84.
  • the specific method of transformation is to sterilize the embryo of hybrid rice Chunyou 84 seed and inoculate it into the medium for inducing callus. After 1 week of culture, a vigorously growing, light-colored, relatively loose embryogenic callus was selected and used as a receptor for transformation.
  • Rice calli were infested with EHA105 strain containing pC1300-Cas9-gRNA OSD1-gRNA REC8-gRNA PAIR1 plasmid, and cultured for 3 days at 25 ° C in the dark, and screened for resistance on selection medium containing 50 mg/l hygromycin. Callus and transgenic plants. Transgenic plants that grew normally on hygromycin selection medium were selected.
  • OSD1-F (SEQ ID NO: 5): atctccaggatgcctgaagtgag
  • OSD1-R (SEQ ID NO: 6): cctagactgctactcttgctagtgat
  • PAIR1-F (SEQ ID NO: 7): ctgtacctgtgcatctaattacag
  • PAIR1-R (SEQ ID NO: 8): ccccatcttatgtactgagcttgccag
  • the obtained PCR product was sent to a sequencing company and sequenced using OSD1-F, PAIR1-F, and REC8-F as sequencing primers, respectively.
  • the results obtained were aligned with wild type sequences.
  • the sequencing results were bimodal, and the mutation information was directly obtained by degenerate codon strategy analysis (http://dsdecode.scgene.com/ for peak map analysis).
  • loci are mutants of biallelic mutations, which are plants that can produce gametes that are consistent with somatic genotypes and ploidy.
  • the anthers or pollen are inoculated into the artificially arranged anther culture medium by aseptic operation, and the callus is induced to form, and then the plants are obtained through tissue culture.
  • cell ploidy was screened by flow cytometry, and the plants having the cell ploidy and the mother plants were selected.
  • the genotypes of the four plants tested were consistent with Chunyou 84, and they were all heterozygous. From the molecular biology point of view, the genotype was completely consistent with the hybrid mother cells.
  • the F 1 hybrid used was the approved commercial hybrid rice variety Chunyou 84.
  • Chunyou 84 is a new hybrid rice combination of the subspecies of the cultivar, which is bred by the combination of the sterile line Chunjiang 16A and the middle-type broad-affinity restorer line C84.
  • the hybrid rice has the advantages of large yield potential, high seed production, excellent agronomic traits, good resistance to mites and wide adaptability.
  • the genetically transformed background material used in the present example is a callus obtained by inducing hybrid rice F 1 seeds, and has not undergone a sexual reproduction stage. Therefore, the transgenic T 0 generation material obtained after transgenic is genetically related to hybrid rice. F 1 plants are consistent.
  • OSD1 gene knockout site (SEQ ID NO: 1): CTGCCGCCGACGAGCAACA AGG
  • PAIR1 gene knockout site (SEQ ID NO: 2): AAGCAACCCAGTGCACCGC TGG
  • SK-gRNA There are two AarI cleavage sites on SK-gRNA, which are digested with AarI to form a vector with sticky ends; after denaturing annealing of the designed target sequence forward and reverse primers, T4 ligase is ligated before construction.
  • Intermediate vector SK-gRNA forming a single gRNA of interest;
  • the multi-gene knockout binary expression vector pC1300-Cas9-gRNA OSD1-gRNA REC8-gRNA PAIR1-gRNA MTL was transferred into AgroBacterium tumefaciens strain EHA105 by electroporation, and this pair was amplified by Agrobacterium-mediated method.
  • the meta-expression vector was transferred into the callus of rice Chunyou 84.
  • the specific method of transformation is to sterilize the embryo of hybrid rice Chunyou 84 seed and inoculate it into the medium for inducing callus. After 1 week of culture, a vigorously growing, light-colored, relatively loose embryogenic callus was selected and used as a receptor for transformation.
  • Rice calli were infested with EHA105 strain containing pC1300-Cas9-gRNA OSD1-gRNA REC8-gRNA PAIR1 plasmid, and cultured for 3 days at 25 ° C in the dark, and screened for resistance on selection medium containing 50 mg/l hygromycin. Callus and transgenic plants. Transgenic plants that grew normally on hygromycin selection medium were selected.
  • OSD1-F (SEQ ID NO: 5): atctccaggatgcctgaagtgag
  • OSD1-R (SEQ ID NO: 6): cctagactgctactcttgctagtgat
  • PAIR1-F (SEQ ID NO: 7): ctgtacctgtgcatctaattacag
  • PAIR1-R (SEQ ID NO: 8): ccccatcttatgtactgagcttgccag
  • the obtained PCR product was sent to a sequencing company and sequenced using OSD1-F, PAIR1-F, and REC8-F as sequencing primers, respectively.
  • the results obtained were aligned with wild type sequences.
  • the sequencing results were bimodal, and the mutation information was directly obtained by degenerate codon strategy analysis (http://dsdecode.scgene.com/ for peak map analysis).
  • loci are mutants of biallelic mutations, which are plants that can produce gametes that are consistent with somatic genotypes and ploidy.
  • the genotypes of the four plants tested were consistent with Chunyou 84, and they were all heterozygous. From the molecular biology point of view, the genotype was completely consistent with the hybrid mother cells.
  • the plants with heterozygous mutations of REC8 and OSD1 were screened by high-throughput sequencing technology in the offspring, and the hybrids and offspring were screened by hybrid plants to obtain both REC8 and OSD1.
  • the plants were heterozygous mutants; in the soybean variety Qihuang 34, the plants with heterozygous mutations of SPO11-1 and CENH3 genes were screened by high-throughput sequencing in the offspring by EMS mutagenesis.
  • a binary vector with an EC1.2 gene promoter specifically expressed by egg cells was driven to drive wild-type CENH3 expression, and the vector was transformed into four plants with heterozygous mutations; identified and screened in self-crossing progeny of plants.
  • the REC8, OSD1, SPO11-1 and CENH3 genes are homozygous mutants and have a single plant of the EC1.2::CENH3 transgenic component, and the selfed seeds on the plants are harvested.
  • the primary fixed sub-generation (four randomly selected strains) and the leaves of the previous generation plants were selected to extract DNA; 16 hybridized sites were randomly selected from the previous generation of hybrid materials, and the detection primers were designed. Genotype detection of the first generation plants showed that the genotypes of the four plants at 16 loci were identical to those of the previous generation, that is, they were all heterozygous, and the hybrid genotype was not recombined from the molecular biological point of view. With separation.
  • the F 1 hybrid used was a corn hybrid Jiahe 158, which was made up of LD140 ⁇ LD975.
  • ZmOSD1 gene knockout site (SEQ ID NO: 30): TCTGCCTGTACTGGAGTTAT TGG
  • ZmPAIR1 gene knockout site (SEQ ID NO: 31): GGATTGCTGCGACAGCGGCT GGG
  • ZmREC8 gene knockout site (SEQ ID NO: 32): GGAAGTCCCACGAGTAATTA TGG
  • ZmMTL gene knockout site (SEQ ID NO: 33): GGAAGGGGAGGATGGTTCCC GGG
  • NheI and XbaI, SalI and XhoI are the properties of the homologous enzyme, and the gRNA is polymerized: SK-gRNA ZmOSD1 is digested with KpnI and XhoI as a vector; SK-gRNA ZmPAIR1 is digested with SalI and XbaI.
  • ZmPAIR1sgRNA fragment SK-gRNA ZmREC8 is digested with NheI and BamHI to provide ZmREC8sgRNA fragment, SK-gRNA ZmMTL is digested with BglII and KpnI to provide ZmMTL sgRNA fragment, and one-step rapid polymerization of gRNA within 4; gRNA ZmOSD1-gRNA ZmREC8-gRNA ZmPAIR1-gRNA ZmMTL fragment was digested with KpnI and BglII and the fragment was recovered and ligated into the binary vector pC1300-Cas9 expressing the Cas9 protein (between KpnI and BamHI sites).
  • the polygenic knockout vector pC1300-Cas9-gRNA ZmOSD1-gRNA ZmREC8-gRNA ZmPAIR1-gRNA ZmMTL which is a gene for maize REC8, OSD1, PAIR1 and MTL, was used for transgenic preparation of maize multi-mutant.
  • the maize multi-knockout vector obtained in the previous step was transferred into Agrobacterium tumefaciens strain LBA4404 by electroporation, and the binary expression vector was transferred into the maize hybrid Jiahe 158 callus by Agrobacterium-mediated method.
  • the bag After corn pollination, the bag is artificially bagged for 9-12 days. The ears are stripped of the leaves, and each layer of eucalyptus leaves is stripped and sprayed with 75% alcohol for surface disinfection. The blade is used to pick 1.0-1.2mm under the ultra-clean workbench. The young embryos of the size are placed in a high permeate for use and placed in the permeate for no more than one hour.
  • Agrobacterium When Agrobacterium was cultured to an OD600 value of 0.8, the cells were collected by centrifugation, suspended in a 1 mol/L suspension, and then added with acetosyringone to a final concentration of 200 ⁇ mol/L. The inoculum was inoculated with the bacterial solution for 5 min, and then transferred. In the co-culture medium, dark culture was carried out for 7 days at 25 ° C, and the immature embryos were transferred to a selection medium containing 15 mg/l hygromycin and a later regeneration medium to screen resistant calli and transgenic plants.
  • the CTAB method was used to extract the genomic DNA of transgenic maize, and Hi-Tom was used to identify the mutation of the target gene (see CN201710504178.3 for details).
  • the leaves of two parental LD140 and LD975, Jiahe 158 and ploidy fixed generations of maize plants were selected and DNA was extracted for whole genome sequencing.
  • the genotypes of the tested generations of maize plants were consistent with those of Jiahe 158, and they were all heterozygous. From the molecular biological point of view, the genotypes were completely consistent with the hybrid mother cells.
  • the F 1 hybrid used is the tomato hybrid Eliza
  • the female parent is a low temperature resistant inbred line "S 2-4”
  • the male parent is a high quality disease resistant inbred line "S28”.
  • loci were selected as the CRISPR-Cas9 gene editing system to knock out the sites of tomato REC8, OSD1, SPO11 and MTL (PAM sequences underlined):
  • SlOSD1 gene knockout site (SEQ ID NO: 34): CAGAAGCAGGGAGAATGGC AGG
  • SlSPO11 gene knockout site (SEQ ID NO: 35): TGAGGATCTCGCTCGAGGT AGG
  • SlREC8 gene knockout site (SEQ ID NO: 36): GCACAGGAGGAACCTGCTA AGG
  • SlMTL gene knockout site (SEQ ID NO: 37): TGATTGCCGGAACGAGCAC CGG
  • NheI and XbaI, SalI and XhoI are the properties of the homologous enzyme, and the gRNA is polymerized: SK-gRNA SlOSD1 is digested with KpnI and XhoI as a vector; SK-gRNA SlSPO11 is digested with SalI and XbaI.
  • SK-gRNA SlREC8 was digested with NheI and BamHI to provide SlREC8sgRNA fragment
  • SK-gRNA SlMTL was digested with BglII and KpnI to provide SlMTL sgRNA fragment, and one-step rapid polymerization of gRNA within 4
  • gRNA SlOSD1-gRNA SlREC8-gRNA SlPAIR1-gRNA SlMTL fragment was digested with KpnI and BglII and the fragment was recovered and ligated into the binary vector pC1300-Cas9 expressing the Cas9 protein (between KpnI and BamHI sites), which was finally knockable.
  • the polygenic knockout vector pC1300-Cas9-gRNA SlOSD1-gRNA SlREC8-gRNA SlSPO11-gRNA SlMTL of the four genes of tomato REC8, OSD1, SPO11 and MTL was used for transgenic preparation of tomato multi-mutant.
  • the tomato multi-knockout vector obtained in the previous step was transferred into Agrobacterium strain EHA105 by electroporation by leaf disc method, and the binary expression vector was transferred into tomato hybrid Eliza callus by Agrobacterium-mediated method. in.
  • Tomato seeds were aseptically sown on 1/2MS medium, incubated for 2-3 days in dark culture, and cultured in light. After 10-12 days, when the cotyledons of the seedlings were fully expanded, but no true leaves were formed, cotyledons were selected. For explants, the cotyledons are cut at both ends, and the middle portion is divided into two horizontally, and the cut small pieces are leaf discs. The leaf discs were inoculated in pre-culture medium with the leaves facing up and pre-incubated for 2 days.
  • the pre-cultured cotyledon leaf discs were soaked in the prepared Agrobacterium liquid, fully infiltrated for 5 minutes, the leaf discs were appropriately blotted by sterile filter paper, the back side of the leaves was upward, and the dark culture was carried out for 48-72 hours, and the culture temperature was 28 °C.
  • the leaf discs co-cultured with Agrobacterium were transferred to a de-bacterifying medium and cultured by light. After 5 days, the leaf discs were transferred to the screening medium and transferred once every 14 days. When the resistant buds grow to about 2 cm, they are cut from the explants and transferred to the rooting medium. After the roots are developed, they are transplanted into the soil.
  • the CTAB method was used to extract transgenic tomato genomic DNA, and Hi-Tom was used to identify the mutation of the target gene (see CN201710504178.3 for details).
  • the two parents "S 2-4" and “S28”, tomato hybrid Eliza and the leaves of the ploided sub-generation tomato plants were selected and the DNA was extracted for genome-wide sequencing.
  • the genotypes of the detected sub-generation tomato plants were consistent with Eliza, and they were all heterozygous. From the molecular biological point of view, the genotypes were completely consistent with the hybrid mother cells.

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Abstract

提供了一种利用植物杂种优势的方法，该方法包括以下步骤：S1，利用基因突变或基因工程技术将杂交种的生殖细胞的减数分裂转变为类似有丝分裂从而得到与杂交种基因型和染色体倍性一致的配子；以及S2，利用基因突变和基因工程技术影响参与植物配子或胚发育过程，其中涉及的蛋白为MTL蛋白。

Description

一种利用植物杂种优势的方法 技术领域
本发明涉及生物技术领域，具体而言，涉及一种利用植物杂种优势的方法。
背景技术
杂种优势是指在生物界中，两个遗传基础不同的品种间或相近物种间进行杂交，其杂交子一代在生长势、生活力、适应性和产量等性状上优于双亲的现象。杂种优势是生物界一种普遍的现象，被广泛地运用到农作物的品种培育和生产实践中。
对于杂种优势在农业生产中的应用，最重要的环节之一是进行杂交种的高效制备。在玉米等雌雄异花的作物中，可以通过人工(或利用机械)去除母本自交系的雄花，以另一自交系(父本)的花粉进行授粉获得杂交种子，其操作相对简单易行，故玉米的杂种优势利用得早，且体系成熟，应用广泛。但也存在有些亲本开花时期不一致，导致在田间无法进行大规模杂交制种的问题。
而雌雄同花作物(如水稻、小麦等)无法通过去除母本的花粉途径实现大规模制备杂交种子。以水稻为例：目前，在水稻中解决这个问题的途径是利用具有花粉不育特性的植株作为母本，以另一品种为父本提供花粉杂交，即以雄性不育为技术核心的杂种优势利用体系。其中，水稻杂种优势的利用又可分为两个技术途径，一是以核质互作花粉不育为技术核心的“三系法”杂交技术，二是以受自然光周期、温度调控的光温敏核不育为技术核心的“两系法”杂交技术。
如图1A所示，“三系法”杂交技术：利用核质互作雄性不育系为母本、以保持系为父本批量化繁殖仍保持不育特性的种子；用不育系为母本、以恢复系为父本大规模生产恢复花粉可育性且具杂种优势的杂交种子，该杂交种子用于生产杂交稻。
如图1B所示，“两系法”杂交技术：同一水稻株系，在一定条件下花粉可育，利用其可育性繁殖不育系种子；在另一特定条件下花粉不育，利用其不育性与父本杂交，制备杂交种子。
由于杂交水稻利用的是杂种一代优势，以后多代都会发生性状或育性的分离，因此必须年年制种，耗费大量的人力、物力和土地资源。另外，“三系法”受恢保关系的制约而对种质资源利用率低；“两系法”受自然温光影响，不育系的繁殖产量不稳定、杂交制种期间存在低温诱导不育系自交结实导致杂交种子纯度不达标的风险。
另外，还有一些相关文献报道了杂种优势的利用及与植物生殖有关的基因，例如：Turning rice meiosis into mitosis，(Cell Research(2016)26:1242-1254)公开了可以通过无融合生殖的种子使F1杂种自我繁殖保持优良性状，其中通过杂交引入了外源修饰表达的CENH3基因。 US 2014/0298507 A1公开了通过将无融合配子转化为克隆胚或种子。四川大学学报(自然科学版)，Vol.29No.2 1992公开了无融合生殖在植物育种中的应用及细胞胚胎学研究方法。
发明内容
本发明旨在提供一种利用植物杂种优势的方法，以使杂交种产生克隆种子或植株，从而提高制种效率。
为了实现上述目的，根据本发明的一个方面，提供了利用植物杂种优势的方法。该方法包括以下步骤：S1，利用基因突变或基因工程技术将杂交种的生殖细胞的减数分裂转变为类似有丝分裂从而得到与杂交种基因型和染色体倍性一致的配子；以及S2，利用基因突变和基因工程技术影响参与植物配子或胚发育过程，其中涉及的蛋白为MTL蛋白。
进一步地，基因突变包括随机诱变和定向诱变；其中，随机诱变包括化学诱变、物理诱变和生物诱变；定向诱变包括基因编辑技术，基因编辑技术包括CRISPR/Cas基因编辑技术、CRISPR/Cpf1基因编辑技术、TALEN基因编辑技术、归巢核酸内切酶基因编辑技术和ZFN基因编辑技术；基因工程技术包括转基因技术诱导基因的特异表达、易位表达或基因沉默。
进一步地，S1包括取杂交种，利用基因突变或基因工程技术将其生殖细胞的减数分裂转变为类似有丝分裂从而得到与杂交种基因型和染色体倍性一致的配子。
进一步地，S1包括利用基因突变或基因工程技术将杂交种的亲本进行编辑，然后通过亲本之间杂交获得杂交种，进而获得生殖细胞的减数分裂转变为类似有丝分裂的杂交种配子。
进一步地，S1包括利用基因突变或基因工程技术编辑参与植物中减数分裂的蛋白实现将生殖细胞的减数分裂转变为类似有丝分裂；其中蛋白包括第一蛋白、第二蛋白和第三蛋白，其中，
第一蛋白为参与DNA双链断裂形成的蛋白，第一蛋白选自以下蛋白：
如SEQ ID NO:13所示的PAIR1蛋白，与PAIR1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与PAIR1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:14所示的PAIR2蛋白，与PAIR2蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与PAIR2蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:15所示的PAIR3蛋白，与PAIR3蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与PAIR3蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、 90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:16所示的PRD1蛋白，与PRD1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与PRD1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:17所示的PRD2蛋白，与PRD2蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与PRD2蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:18所示的SPO11-1蛋白，与SPO11-1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与SPO11-1蛋白具、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:19所示的SPO11-2蛋白，与SPO11-2蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与SPO11-2蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:20所示的SDS蛋白，与SDS蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与SDS蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:21所示的CRC1蛋白，与CRC1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与CRC1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:22所示的P31 comet蛋白，与P31 comet蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与P31 comet蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:23所示的MTOPVIB蛋白，与MTOPVIB蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与MTOPVIB蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:24所示的DFO蛋白，与DFO蛋白具有至少30％、35％、40％、45％、50％、 55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与DFO蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
第二蛋白参与控制减数分裂期姐妹染色体间的黏连，第二蛋白选自以下蛋白：
如SEQ ID NO:25所示的REC8蛋白，与REC8蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与REC8蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
第三蛋白参与减数分裂的第二次分裂，第三蛋白选自以下蛋白：
如SEQ ID NO:26所示的OSD1蛋白，与OSD1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与OSD1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:27所示的TAM蛋白，与TAM蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与TAM蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:28所示的TDM1蛋白，与TDM1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与TDM1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白。
进一步地，S2包括利用基因突变和基因工程技术影响参与植物配子或胚发育过程，诱导配子发育成种子或植株。
进一步地，S2包括授以其它植株的诱导花粉，诱导配子发育成种子或植株。
进一步地，S2包括通过物理刺激、生物胁迫或化学药剂处理，诱导配子发育成种子或植株。
进一步地，S2包括通过花药培养或花粉培养诱导配子发育成种子或植株。
进一步地，MTL蛋白为如SEQ ID NO:29所示的MTL蛋白，与MTL蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与MTL蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白。
进一步地，植物包括单子叶植物和双子叶植物。
进一步地，植物包括水稻、玉米、高粱、谷子、大麦、小麦、黑麦、燕麦、荞麦、薏仁、甘蔗、芦笋、竹笋、韭菜、山药、大豆、土豆、豌豆、绿豆、小豆、蚕豆、豇豆、菜豆、小扁豆、蔓豆、鹰嘴豆、木薯、甘薯、油菜、棉花、甜菜、茄子、花生、茶叶、薄荷、咖啡、芝麻、向日葵、蓖麻、苏子、红花、番茄、辣椒、黄瓜、青菜、生菜、菠菜、大蒜、甘蓝、芥菜、茭白、大葱、冬瓜、西葫芦、丝瓜、白菜、萝卜、洋葱、西瓜、葡萄、胡萝卜、花菜、南瓜、烟草、牧草、象草、狼尾草、苏丹草、兰花、百合、郁金香和苜蓿。
根据本发明的另一个方面，提供一种保持有杂种优势的植物或种子。该植物或种子通过上述任一种方法制备得到。
根据本发明的再一个方面，提供一种用于使植物保持杂种优势的试剂盒。该试剂盒包括能够使植物生殖细胞的减数分裂转变为类似有丝分裂的载体和/或试剂，和使配子发育成种子或植株的载体和/或试剂。
进一步地，能够使植物生殖细胞的减数分裂转变为类似有丝分裂的载体和/或试剂为通过基因突变或基因工程技术将杂交种的生殖细胞的减数分裂转变为类似有丝分裂的载体和/或试剂，优选为随机诱变或定向诱变的载体和/或试剂。
进一步地，随机诱变包括化学诱变、物理诱变和生物诱变；定向诱变包括CRISPR/Cas基因编辑技术、CRISPR/Cpf1基因编辑技术、TALEN基因编辑技术、归巢核酸内切酶基因编辑技术、ZFN基因编辑技术；基因工程技术包括转基因技术诱导基因的特异表达、易位表达或基因沉默。
进一步地，能够使植物生殖细胞的减数分裂转变为类似有丝分裂的载体和/或试剂为利用基因突变或基因工程技术编辑参与植物中减数分裂蛋白实现将生殖细胞的减数分裂转变为类似有丝分裂的载体和/或试剂，其中蛋白包括第一蛋白、第二蛋白和第三蛋白，其中，
第一蛋白参与DNA双链断裂形成的蛋白，第一蛋白选自以下蛋白：
如SEQ ID NO:13所示的PAIR1蛋白，与PAIR1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与PAIR1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:14所示的PAIR2蛋白，与PAIR2蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与PAIR2蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:15所示的PAIR3蛋白，与PAIR3蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与PAIR3蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:16所示的PRD1蛋白，与PRD1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与PRD1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:17所示的PRD2蛋白，与PRD2蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与PRD2蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:18所示的SPO11-1蛋白，与SPO11-1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与SPO11-1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:19所示的SPO11-2蛋白，与SPO11-2蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与SPO11-2蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:20所示的SDS蛋白，与SDS蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与SDS蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:21所示的CRC1蛋白，与CRC1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与CRC1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:22所示的P31 comet蛋白，与P31 comet蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与P31 comet蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:23所示的MTOPVIB蛋白，与MTOPVIB蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与MTOPVIB蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:24所示的DFO蛋白，与DFO蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与 DFO蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
第二蛋白参与控制减数分裂期姐妹染色体间的黏连，第二蛋白选自以下蛋白：
如SEQ ID NO:25所示的REC8蛋白，与REC8蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与REC8蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
第三蛋白参与减数分裂的第二次分裂，第三蛋白选自以下蛋白：
如SEQ ID NO:26所示的OSD1蛋白，与OSD1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与OSD1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:27所示的TAM蛋白，与TAM蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与TAM蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:28所示的TDM1蛋白，与TDM1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与TDM1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白。
进一步地，使配子发育成种子或植株的载体和/或试剂，其中包括为利用基因突变或基因工程技术影响参与植物配子或胚发育过程的MTL蛋白而诱导配子发育成种子或植株的载体和/或试剂，MTL蛋白为如SEQ ID NO:29所示的MTL蛋白，与MTL蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与MTL蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白。
根据本发明的又一个方面，提供一种使用上述试剂盒所产生的植物。该植物的生殖细胞的减数分裂转变为类似有丝分裂从而可以产生与杂交种基因型和染色体倍性一致的配子。
进一步地，植物的配子能够被诱导发育成植株或种子。
进一步地，植物为基因突变或基因工程改造植物，植物被利用基因突变或基因工程技术调节参与植物中减数分裂的蛋白实现将生殖细胞的减数分裂转变为类似有丝分裂；被利用基因突变或基因工程技术影响参与植物中配子或胚发育过程的第四蛋白而诱导配子发育成种子或植株；其中蛋白包括第一蛋白、第二蛋白和第三蛋白，其中，
第一蛋白参与DNA双链断裂形成的蛋白，第一蛋白选自以下蛋白：
如SEQ ID NO:13所示的PAIR1蛋白，与PAIR1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与PAIR1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:14所示的PAIR2蛋白，与PAIR2蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与PAIR2蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:15所示的PAIR3蛋白，与PAIR3蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与PAIR3蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:16所示的PRD1蛋白，与PRD1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与PRD1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:17所示的PRD2蛋白，与PRD2蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与PRD2蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:18所示的SPO11-1蛋白，与SPO11-1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与SPO11-1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:19所示的SPO11-2蛋白，与SPO11-2蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与SPO11-2蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:20所示的SDS蛋白，与SDS蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与SDS蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:21所示的CRC1蛋白，与CRC1蛋白具有至少30％、35％、40％、45％、 50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与CRC1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:22所示的P31 comet蛋白，与P31 comet蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与P31comet蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:23所示的MTOPVIB蛋白，与MTOPVIB蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与MTOPVIB蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:24所示的DFO蛋白，与DFO蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与DFO蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
第二蛋白参与控制减数分裂期姐妹染色体间的黏连，第二蛋白选自以下蛋白：
如SEQ ID NO:25所示的REC8蛋白，与REC8蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与REC8蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
第三蛋白参与减数分裂的第二次分裂，第三蛋白选自以下蛋白：
如SEQ ID NO:26所示的OSD1蛋白，与OSD1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与OSD1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:27所示的TAM蛋白，与TAM蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与TAM蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；如SEQ ID NO:28所示的TDM1蛋白，与TDM1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与TDM1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:28所示的TDM1蛋白，与TDM1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白， 或者与TDM1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
第四蛋白选自以下蛋白：
如SEQ ID NO:29所示的MTL蛋白，与MTL蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与MTL蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白。
根据本发明的又一个方面，提供一种保持植物杂种优势的方法。该方法包括以下步骤：S1，利用基因编辑技术将杂交种在F1代时生殖细胞的减数分裂转变为类似有丝分裂从而得到F1代的二倍体雌配子；以及S2，利用基因突变和基因工程技术影响参与植物配子或胚发育过程诱导二倍体雌配子发育成种子，其中涉及的蛋白为MTL蛋白。
进一步地，S1包括取杂交F1代种子，利用基因编辑技术将生殖细胞的减数分裂转变为类似有丝分裂从而得到F1代的二倍体雌配子。
进一步地，S1包括利用基因编辑技术将杂交种的亲本进行编辑，获得经编辑的各基因都为杂合突变的植株，然后通过亲本之间杂交获得杂交种，在杂交种中筛选在两个亲本中经编辑的多个基因都为纯合突变的植株，进而获得生殖细胞的减数分裂转变为类似有丝分裂的F1代的二倍体雌配子。
进一步地，S1包括利用基因编辑技术敲除REC8、OSD1、PAIR1基因实现将生殖细胞的减数分裂转变为类似有丝分裂。
进一步地，S2包括授以二倍体雌配子单倍体诱导系花粉诱导二倍体雌配子发育成种子。
进一步地，S2包括利用基因编辑技术敲除MTL基因而产生单倍体诱导系花粉。
进一步地，S2包括采用其他植株的单倍体诱导系花粉诱导二倍体雌配子发育成种子。
进一步地，杂交种在F1代时同时敲除了REC8、OSD1、PAIR1和MTL基因。
进一步地，植物包括水稻、玉米、高粱、小米、大麦和小麦。
应用本发明的技术方案，杂交种可产生与自身基因型完全一致的克隆种子，使杂交种可以被长期利用，解决之前在利用杂种优势中由于花期不一致等原因导致亲本间杂交困难、制种产量低、杂交种成本高等问题。
附图说明
构成本申请的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1A示出了现有技术中三系杂交育种技术流程示意图；
图1B示出了现有技术中两系杂交育种技术流程示意图；
图2和图3示出了本发明的F1代基因型保持情况示意图；
图4A示出了实施例1中F1代植株春优84的细胞倍性检测结果；以及
图4B示出了实施例1中杂种优势固定植株的细胞倍性检测结果。
图5示出了实施例1中父本C84、母本16A、杂交种春优84(CY84)以及基因型和染色体倍性固定植株的全基因测序结果。
具体实施方式
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将参考附图并结合实施例来详细说明本发明。
本发明中涉及的专业名词解释如下：
杂种优势：heterosis，是指杂种第一代在体型、生长率繁殖力及行为特征方面均比亲本优越的现象。
减数分裂：生殖细胞分裂时，染色体只复制一次，细胞连续分裂两次，这是染色体数目减半的一种特殊分裂方式。
有丝分裂：mitosis，又称做间接分裂，是E.Strasburger(1880)年发现于植物，特点是细胞在分裂的过程中有纺锤体和染色体出现，使已经在S期复制好的子染色体被平均分配到子细胞，这种分裂方式普遍见于高等动植物(动物和高等植物)。
染色体倍性(数)：chromosome ploidy，是指细胞中包含的染色体组数或基因组数，如单倍体染色、多倍体染色。
二倍体雌配子：配子是指生物进行有性生殖时由生殖系统所产生的成熟性细胞，简称生殖细胞，配子分为雄配子(male gamete)和雌配子(female gamete)；通常生殖细胞分裂时，染色体只复制一次，细胞连续分裂两次，染色体数目减半，但是如果雌配子生成时染色体数目并未减半，而是与本物种体细胞染色体组数一致，则称之为二倍体雌配子。
单倍体：体细胞染色体组数等于本物种配子染色体组数的个体或细胞。
孤雌生殖：parthenogenesis，也称单性生殖，即卵不经过受精也能发育成正常的新个体。
在本发明中，杂交种是指基因型杂合的植株或种子，其有性生殖的后代会发生遗传分离。
根据本发明一种典型的实施方式，提供一种利用植物杂种优势的方法。该方法包括以下步骤：S1，利用基因突变或基因工程技术将杂交种的生殖细胞的减数分裂转变为类似有丝分 裂从而得到与杂交种基因型和染色体倍性一致的配子；以及S2，利用基因突变和基因工程技术影响参与植物配子或胚发育过程，其中涉及的蛋白为MTL蛋白。
其中，基因突变包括随机诱变和定向诱变；随机诱变包括化学诱变、物理诱变和生物诱变；定向诱变包括基因编辑技术，优选的，基因编辑技术包括CRISPR/Cas基因编辑技术、CRISPR/Cpf1基因编辑技术、TALEN基因编辑技术、归巢核酸内切酶基因编辑技术和ZFN基因编辑技术；基因工程技术包括转基因技术诱导基因的特异表达、易位表达或基因沉默。
具体的，物理诱变常用方法包括：射线(紫外线、X光线、Y射线，中子线)，激光微束，离子束，微波，超声波，热力等。化学诱变常用方法：浸渍法、涂抹法、滴液法、注射法、施入法和熏蒸法，化学诱变剂包括：烷化剂、碱基类似物，氯化锂、亚硝基化合物、叠氮化物、抗生素、羟胺、吖啶、硫酸二乙酯(DFS)、5-溴尿嘧啶(5-BU)、氮芥(Nm)、N'广甲基N'亚硝基胍(NTG)等。生物诱变方法包括：空间条件处理诱变，病原微生物诱变，组培诱变、转基因诱变。
作为实例应用，可以是TILLING(定向诱导基因组局部突变，Targeting Induced Local Lesions IN Genomes)，由McCallum et al.,Plant Physiology,2000,123,439-442所述)。利用标准技术进行定向诱变，该标准技术是本领域所已知的并且利用同源重组，优选与核酸酶例如TALEN或CRISPR组合。
根据本发明一种典型的实施方式，该方法包括以下步骤：S1，利用基因突变或基因工程技术将杂交种的生殖细胞的减数分裂转变为类似有丝分裂从而得到与杂交种基因型和染色体倍性一致的配子；以及S2，诱导配子发育成种子或植株。
应用本发明的技术方案，杂交种可产生与自身基因型完全一致的克隆种子或植株，使杂交种可以被长期利用，解决之前在利用杂种优势中由于花期不一致等原因导致亲本间杂交困难、制种产量低、杂交种成本高等问题。
根据本发明一种典型的实施方式，S1包括取杂交种，利用基因突变或基因工程技术将其生殖细胞的减数分裂转变为类似有丝分裂从而得到与杂交种基因型和染色体倍性一致的配子，例如，具体操作可以是：S1包括取杂交F1代种子，利用基因工程技术将生殖细胞的减数分裂转变为类似有丝分裂从而得到F1代的二倍体配子。具体的做法可以是：取杂交F1代种子，通过导入基因编辑系统编辑参与减数分裂相关的关键基因获得基因编辑后的F1代植株，该基因编辑后的F1代植株的雌配子即为二倍体配子，优选的，参与减数分裂相关的关键基因是REC8、OSD1、PAIR1三个基因。
根据本发明一种典型的实施方式，S1包括利用基因突变或基因工程技术将杂交种的亲本进行编辑，然后通过亲本之间杂交获得杂交种，进而获得生殖细胞的减数分裂转变为类似有丝分裂的杂交种配子。例如，具体操作可以是：S1包括利用基因工程技术对杂交种的亲本进行编辑，获得参与减数分裂相关的关键基因都为杂合突变状态的杂合突变体，然后通过亲本之间杂交获得杂交种，在杂交种中筛选参与减数分裂相关的关键基因都为纯合突变的植株，进而获得生殖细胞的减数分裂转变为类似有丝分裂的F1代的二倍体雌配子。具体的做法可以 是：分别取杂交种的父本和母本，通过导入基因编辑系统编辑上述三个参与减数分裂相关的关键基因，获得基因编辑后的上述三个基因都为杂合状态的亲本植株，然后将这两个亲本进行杂交，所结的种子会出现不同的基因型，从中挑选上述三个基因都为纯合突变的种子，这样的植株即本发明希望得到F1代种子，该F1代种子的雌配子即为二倍体雌配子。
根据本发明一种典型的实施方式，S1包括利用基因突变或基因工程技术编辑参与植物中减数分裂的蛋白实现将生殖细胞的减数分裂转变为类似有丝分裂；其中蛋白包括第一蛋白、第二蛋白和第三蛋白，其中，
第一蛋白参与DNA双链断裂形成的蛋白，第一蛋白选自以下蛋白：
如SEQ ID NO:13(MKLKMNKACDIASISVLPPRRTGGSSGASASGSVAVAVASQPRSQPLSQSQQSFSQGASASLLHSQSQFSQVSLDDNLLTLLPSPTRDQRFGLHDDSSKRMSSLPASSASCAREESQLQLAKLPSNPVHRWNPSIADTRSGQVTNEDVERKFQHLASSVHKMGMVVDSVQSDVMQLNRAMKEASLDSGSIRQKIAVLESSLQQILKGQDDLKALFGSSTKHNPDQTSVLNSLGSKLNEISSTLATLQTQMQARQLQGDQTTVLNSNASKSNEISSTLATLQTQMQADIRQLRCDVFRVFTKEMEGVVRAIRSVNSRPAAMQMMADQSYQVPVSNGWTQINQTPVAAGRSPMNRAPVAAGRSRMNQLPETKVLSAHLVYPAKVTDLKPKVEQGKVKAAPQKPFASSYYRVAPKQEEVAIRKVNIQVPAKKAPVSIIIESDDDSEGRASCVILKTETGSKEWKVTKQGTEEGLEILRRARKRRRREMQSIVLAS)所示的PAIR1蛋白，与PAIR1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与PAIR1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:14(MVMAQKTKEAEITEQDSLLLTRNLLRIAIYNISYIRGLFPEKYFNDKSVPALEMKIKKLMPMDTESRRLIDWMEKGVYDALQKKYLKTLLFCICEKEEGPMIEEYAFSFSYPNTSGDEVAMNLSRTGSKKNSATFKSNAAEVTPDQMRSSACKMIRTLVSLMRTLDQMPEERTILMKLLYYDDVTPEDYEPPFFKCCADNEAINIWNKNPLKMEVGNVNSKHLVLALKVKSVLDPCDDNNVNSEDDNMSLDNESDQDNDFSDTEVRPSEAERYIVAPNDGTCKGQNGTISEDDTQDPVHEEELTAQVREWICSRDTESLEVSDVLVNFPDISMEMVEDIMERLLKDGLLSRAKKDSYSVNKIADPTTPHIKKEVIMQNVSPTEGTKNSNGDLMYMKALYHALPMDYVSVGKLHGKLDGEASQNMVRKLIEKMVQDGYVKNSANRRLGKAVIHSEVTNRKLLEIKKILEVDIAEQMAIDTNAEPGEPERKDHLSGHEMRDGSTMGCLQSVGSDLTRTRELPEPQQNVSMQSGQEASTVDKDPSRTPTSVREASVCSLESGVLGQKVRKSLAGAGGTQCSQDKRFRKASTVKEPILQYVKRQKSQVQVQVQ)所示的PAIR2蛋白，与PAIR2蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与PAIR2蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:15(MEVELTNIQKATSSDYWSLASNQYPCGKFPKVSVGVTIPRTSSVSRGRDAASTAAFEKNLSQGTDGRSRPPKMDNASLQVSPEAANHGGSAKEVPKPVPAKVSVSQPDDNAIEQTGTFSFGTRREQDSHLDQLDRPPLVSSQGKRQVESADKNKPNSEMLRMKLWEILGGTSQNKEAVASPNPEDIETPCQPKSQIANGPSSGRQKVFTSPVPYNIKTPAQFNSQTANKPSS DPIESDSDSPQVVEVRPITRSLGRKKEPTGSTHQDKSGSAKKPLSTHRSTPKQKILDNVFAFNDKCTPKTVGKSANGESGSLRNLRSLSRRAKVEPKKAHCSDRISHKTTQDDMERKVPSKYIPSEKKGEKTNSFSSLSRTGKTAESCSRSPKRERRVNTMANVGARKMQLSENLLVKTLNDGEHKLSSPQLTSFKSKGKCSSISPQQKENDNTHIPEASDRTAARNSFNSTPSPAANPSPVLRKYSWEHDENPAINGKSGQKDASPLADRFSDMPDDFASPTFAANIKISPHRSKMLDDDLFSSKYPKGVNRSRSTSFTSDPESEPLDKMEKTNELPGSESPNSQEERQNRKQPHLSPLSPIESEGAQISIPSFRKGYKSHKWLSDVDSPDKSSIEHLGRKSHLKEGRKGKRQLTSPTHFATSGTQETMSDKEPEKVPENYLTRAFDQLVVVLGRFQTKIKSETRNKSSKILAATGEIIRQHLEGVEGQMQADVDKLVNAGKSKRKRLESTFEEQQEKLRILHEKFKEEVNQQLLGCKNSVEDFEAYHAELKGVADKQKASHKKLLQNAEKTVGAQLSDAETKIAEVQKRARKRMKGLKFVLKELIAETAE)所示的PAIR3蛋白，与PAIR3蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与PAIR3蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:16(MEMVLIMSFRVLLYHRLTAQTGPFKLHCLGILLNSTKDAATYIGDKQSLYLNLVNNLRLPSDEIRGEILFVLYKLSLLNATPWDDICDNDNVDLSAIGRSLLQFSLEVLLKTQNDDVRLNCIALLLTLAKKGAFDILLLSDPSLINSAEAEDNVPLNDSLVILFAEAVKGSLLSTNIEVQTGTLELIFHFLSSDANIFVLKTLIDQNVADYVFEVLRLSGMRNHLLQSSNASQFLTKLLYVSGNNDPLVISSIKVLSILANSEERFKEKLAIAVSTLLPVLHYVSEIPFHPVQSQVLRLVCISIINCSGILSLSQEEQIACTLSAILRRHGNGELGMSSETFALVCSMLVEILKLPSADDIQKLPSFIVEASKHAISLTFSHEYDCLFLIPHSLLLLKEALIFCLEGNKDQILRKKSLEDSIIETCETYLLPWLESAIVDGNDEETLSGILQIFQIILSRASDNKSFKFAEMLASSSWFSLSFGFMGLFPTDHVKSAVYLVISSIVDKVLGISYGETIRDACIYLPPDPAELLYLLGQCSSEDFNLASCQCAILVILYVCSFYNERLAADNQILASVEQYILLNGAKFPHEIPGSLMLTLLVHLYAFVRGISFRFGIPHSPEAEKTLFHAMTHKEWDLLLIRVHLIALKWLFQNEELMEPLSFHLLNFCKFFCEDRTVMLSSSTQLVDIQLIAELVYSGETCISSLLVSLLSQMIKESAEDEVLSVVNVITEILVSFPCTSDQFVSCGIVDALGSIYLSLCSSRIKSVCSLLIFNILHSASAMTFTCDDDAWLALTMKLLDCFNSSLAYTSSEQEWKILIGILCLILNHSANKVLIEPAKAIILNNCLALLMDGIVQEACAKGPSLFQHNQETTFGELLILMLLLIFFSVRSLQAILEASIDWQEFLQYSDDTESSSVLGIPCHDLCRLMHFGPSPVKLIASQCLLELLNRISDQRSCLNAELRCSAKYLKSMIAVTEGMVFDQDSRVAENCGACLTVILGWERFGSREKAVIRESKWSRLILEEFAVALTAPGLTSKSFSNQQKIAANIALSLLQLSQVPDWLTSLFSDSLISGIVANLSARNVTAEIVTLFSELMAKNYLNQEHIAGLHNLFQVCRRQAYEGGGGSKAQPSEQKAAAARCADDVRALLFGMMLEQRACSRATVEMEQQRLLREIDSFFFQESSLREQNSVK)所示的PRD1蛋白，与PRD1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与PRD1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:17(MAPPASRPPTPTPTPTANAAASSSRIESPSLRAALAMALIHYNRLPSRAAAAAAPSPQALLNWKRKAKDRKREILRLREELKLLQDGARGEEMEPPVASCRCHFFDGCGDLPPPTDGDAGEHWVDDVLRRRFVRLVRWKDKRRRLDRSLPTSSLMEYNTEDEVQQLSLSIDFLVELSDGLFAKREAGSSFTTFSHQAVDFILASLKNILSSEREKEIIEEIINGLVARLMKRMCTTPENAGSVDCSDAQFSLQHLFRKLGNEEFVGQRIILAISQKISNVSEKLLLADPFDDGFPE MHSNMFIMIQLIEFLISDSFNNWLCRDHFDRKLFEEWVRSILKARKDLEVLDGRNGLYVVYIERVIGRLAREVAPAAHQGKLDLEVLSKLLY)所示的PRD2蛋白，与PRD2蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与PRD2蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:18(MAGREKRRRVAALDGEERRRRQEEAATLLHRIRGLVRWVVAEVAAGRSPTVALHRYQNYCSSASAAAASPCACSYDVPVGTDVLSLLHRGSHASRLNVLLRVLLVVQQLLQQNKHCSKRDIYYMYPSIFQEQAVVDRAINDICVLFKCSRHNLNVVPVAKGLVMGWIRFLEGEKEVYCVTNVNAAFSIPVSIEAIKDVVSVADYILIVEKETVFQRLANDKFCERNRCIVITGRGYPDIPTRRFLRYLVEQLHLPVYCLVDADPYGFDILATYKFGSLQLAYDANFLRVPDIRWLGVFTSDFEDYRLPDCCLLHLSSEDRRKAEGILSRCYLHREAPQWRLELEAMLQKGVKFEIEALSACSISFLSEEYIPKKIKQGRHI)所示的SPO11-1蛋白，与SPO11-1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与SPO11-1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:19(MAEAGVAAASLFGADRRLCSADILPPAEVRARIEVAVLNFLAALTDPAAPAISALPLISRGAANRGLRRALLRDDVSSVYLSYASCKRSLTRANDAKAFVRVWKVMEMCYKILGEGKLVTLRELFYTLLSESPTYFTCQRHVNQTVQDVVSLLRCTRQSLGIMASSRGALIGRLVVQGPEEEHVDCSILGPSGHAITGDLNVLSKLIFSSDARYIIVVEKDAIFQRLAEDRIYSHLPCILITAKGYPDLATRFILHRLSQTYPNMPIFALVDWNPAGLAILCTYKYGSISMGLESYRYACNVKWLGLRGDDLQLIPQSAYQELKPRDLQIAKSLLSSKFLQDKHRAELTLMLETGKRAEIEALYSHGFDFLGKYVARKIVQGDYI)所示的SPO11-2蛋白，与SPO11-2蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与SPO11-2蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:20(MPPTMLASVPTRPRSHPFRRRRGAAAAAPPLLPDQIAAAAAAAAKRPAESSTSASSCFHSEVISATSTTCPTSLAAAQRPEKRPRYQDVDEEQPAASECSEIIGGARPRAAEVEVSESSCLASVLESYLACPEQLANDAETTAYSSAREDLTLSETEEEEEEEEVRSGPCICTDCSFSPLHESSSSSDDDNAVPSPTFSLFLALAEQFVPFTHPKTPTATDVALQAGEGKRFEDLDNEVSYERFRRRERRGVVARDYIEVYSSMLGSYGRAVVEQRVVMVNWIMEHSQAMKLQPETVFMGIGLMDRFLTRGYVKGSRNLQLLGIACTTLATRIEENQPYNCILQKAFKVGINTYSRSEVVAMEWLVQEVLDFQCFVTTTHHFLWFYLKAANADDRVEDLAKYLALLSLLDHKHLSFWPSTVAAAVVALACLATNNESSCHLVMETHMRTKNDDLPECLMSLEWLTNYAS)所示的SDS蛋白，与SDS蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与SDS蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:21(MSAPMEVSFSAPPPPDAASAAAAAPSLVPAVSAAAVAATTVSCSPQPPTGSPSADDRILVSVEVLLHATSTARAEDVCAAVERMLEARSLSYVDGPVPIPNDDPFLLANVKRIQICDTDEWTENHKVLLFWQVRPVVHVFQLSEDGPGEEPGEDDTLSSFNEWALPAK EFDGLWESLLYEVGLKQRLLRYAASALLFTEKGVDPCLVSWNRIVLLHGPPGTGKTSLCKALAQKLSIRFKSRYSMCQLIEVNAHSLFSKWFSESGKLVAKLFQKIQEMVEEESNLVFVLIDEVESLAAARQAAISGSEPSDSIRVVNALLTQMDKLKSWPNVIILTTSNITTAIDIAFVDRADIKAYVGPPTLQARYEILRSCLQELLRVGILTHTQGGNSLCLLSYFSLMENQHCPEVADPHGSVHLSGLLHKAAEICEGLSGRTLRKLPFLAHASVANPSCCDASAFLHALIQTAQRELSESRG)所示的CRC1蛋白，与CRC1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与CRC1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:22(MERATTSGGGGGGSQPPRGVGLPLVEVQAAAASLRRSEVFYVVKELLGFVLYMHHQIPAVLQNLENEFASLKEEMTEMALPPGEMKPSDQRKYNTRKREVRRRIKKQEKLMNGLSSVFSALQKALDEVPSIEGVLLILGGSLVRPLFVYDITISHGRFDAGSANERGASKLAQSVSRKAIRALISSGAGSLSYTGPTKLFVLVRCPCTLNLPLDFLPKRDFRYSKKVVPLQMCIKCNIAGIQIDNQQITSIVDASRCTSESTISEVIWFQCKHTIRGLPCKASLEE)所示的P31 comet蛋白，与P31 comet蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与P31 comet蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:23(MASSPPPSTASPTSSSPYRKLLHSLIYWAVQRCRMSESPCRLTVSVKRSPEPAGSSPLRISVSDTGVGSKLEEFLELDALARETPVEKWDGTLLITTTGIDDKAIYRYQFNLQEDTSSSTRFTKLATMYKSRAIFSGTEVCLCLPTEADVDDLILWLVGFVRKIFVLRASNLACELFVAQTDSAGSGDVCLSQDSDDVHISITTSSIDRLVSGLKDYALSHANTSDRCEACYMNRDRLKIGTGTAKYVDKRKAKGQLVEVVIMIAPTSSDLSCWMTNCSSTQVLHFVEFIPCPISQSSLSALMSIDWQSYGFKFKGGFIDDDGNAELQWDNMAFSHVDIAIHTYHEGAVDEWKSSQPERHLLRKALKSALFGLKADHAEDFLSCHGQKVREYVPDLAESIAGLILSSNDQEFQDECIALLGLGSDQDLTEGAVRSCIGEKMNRIIEMNDTKENVEHNAPYLFECERFDEDYSLLDEDDPDEDMIFDF)所示的MTOPVIB蛋白，与MTOPVIB蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与MTOPVIB蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:24(MRHNIKFKSKGTLKIRNTAQISLWKKCSDSMIADQTYLFINRVQDRRFDEESLRILELSLVAMNVKSFLEVRSRLRDFMRSESVVIFGELTGESMVAKLSVLEFFARAFALLGDMESCLAMRYEALNLRQLKSPSCLWLGVSHSEWTKFAVQSMENGFPSIAGKASENALLSLKKDSLIEPKSEDNSDILDAAEKVRRLRDSAASLTSSHSGIFIYIVSSLKFAVCNRLLTTF)所示的DFO蛋白，与DFO蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与DFO蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
第二蛋白参与控制减数分裂期姐妹染色体间的黏连，第二蛋白选自以下蛋白：
如SEQ ID NO:25(MFYSHQLLARKAPLGQIWMAATLHSKINRKRLDKLDIIKICEEILNPSVPMALRLSGILMGGVAIVYERKVKALYDDVSRFLIEINEAWRVKPVADPTVLPKGKTQAKYEAVTLPENIMDMDVEQPMLFSEADTTRFRGMRLEDLDDQYINVNLDDDDFSRAENHHQADAENITLADNFGSGLGETDVFNRFERFDITDDDATFNVTPDGHPQVPSNLVPSPPRQEDSPQQQENHHAASSPLHEEAQQGGASVKNEQEQQKMKGQQPAKSSKRKKRRKDDEVMMDNDQIMIPGNVYQTWLKDPSSLITKRHRINSKVNLIRSIKIRDLMDLPLVSLISSLEKSPLEFYYPKELMQLWKECTEVKSPKAPSSGGQQSSSPEQQQRNLPPQAFPTQPQVDNDREMGFHPVDFADDIEKLRGNTSGEYGRDYDAFHSDHSVTPGSPGLSRRSASSSGGSGRGFTQLDPEVQLPSGRSKRQHSSGKSFGNLDPVEEEFPFEQELRDFKMRRLSDVGPTPDLLEEIEPTQTPYEKKSNPIDQVTQSIHSYLKLHFDTPGASQSESLSQLAHGMTTAKAARLFYQACVLATHDFIKVNQLEPYGDILISRGPKM)所示的REC8蛋白，与REC8蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与REC8蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
第三蛋白参与减数分裂的第二次分裂，第三蛋白选自以下蛋白：
如SEQ ID NO:26(MPEVRNSGGRAALADPSGGGFFIRRTTSPPGAVAVKPLARRALPPTSNKENVPPSWAVTVRATPKRRSPLPEWYPRSPLRDITSVVKAVERKSRLGNAAVRQQIQLSEDSSRSVDPATPVQKEEGVPQSTPTPPTQKALDAAAPCPGSTQAVASTSTAYLAEGKPKASSSSPSDCSFQTPSRPNDPALADLMEKELSSSIEQIEKMVRKNLKRAPKAAQPSKVTIQKRTLLSMR)所示的OSD1蛋白，与OSD1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与OSD1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:27(MSSSSRNLSQENPIPRPNLAKTRTSLRDVGNRRAPLGDITNQKNGSRNPSPSSTLVNCSNKIGQSKKAPKPALSRNWNLGILDSGLPPKPNAKSNIIVPYEDTELLQSDDSLLCSSPALSLDASPTQSDPSISTHDSLTNHVVDYMVESTTDDGNDDDDDEIVNIDSDLMDPQLCASFACDIYEHLRVSEVNKRPALDYMERTQSSINASMRSILIDWLVEVAEEYRLSPETLYLAVNYVDRYLTGNAINKQNLQLLGVTCMMIAAKYEEVCVPQVEDFCYITDNTYLRNELLEMESSVLNYLKFELTTPTAKCFLRRFLRAAQGRKEVPSLLSECLACYLTELSLLDYAMLRYAPSLVAASAVFLAQYTLHPSRKPWNATLEHYTSYRAKHMEACVKNLLQLCNEKLSSDVVAIRKKYSQHKYKFAAKKLCPTSLPQELFL)所示的TAM蛋白，与TAM蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与TAM蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:28(MCPCVERRAPPGVYYTPPPARTSDHVAAMPMTERRRPPYSCSSSSERRDPFHIVHKVPSGDSPYVRAKHAQLIDKDPNRAISLFWTAINAGDRVDSALKDMAVVMKQLGRSDEGIEAIKSFRYLCSFESQDSIDNLLLELYKKSGRIEEEAVLLEHKLQTLEQGMGFGGRVSRAKRVQGKHVIMTIEQEKARILGNLGWVHLQLHNYGIAEQHYRFGFVTKIPNIDYCLVMRALGLERDKNKLCNLAICLMRMSRIPEAKSLLDDVRDSPAESECGDEPFAKSYDRAVEMLA EIESKKPEADLSEKFYAGCSFVNRMKENIAPGTANKNYSDVSSSPASVRPNSAGLYTQPRRCRLFEEETRGAARKLLFGKPQPFGSEQMKILERGEEEPMKRKKLDQNMIQYLHEFVKDTADGPKSESKKSWADIAEEEEAEEEEEERLQGELKTAEM)所示的TDM1蛋白，与TDM1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与TDM1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白。
其中，PAIR1蛋白参与起始减数分裂重组的发生，催化DNA双链缺口的形成，PAIR1基因的缺失会导致重组过程缺失；REC8蛋白负责将新复制的姐妹染色体紧密联系在一起，是保证姐妹(或同源)染色体正确分离并分配到子细胞中的关键调控因子，它的功能缺失，会导致姐妹染色单体在减数第一次分裂末期就分开，移向细胞两极；OSD1基因功能的缺失，则会导致配子的形成直接跳过减数第二次分裂过程。
将上述基因敲除是实现将生殖细胞的减数分裂转变为类似有丝分裂的简便有效的方法。
本发明中所述的抑制蛋白是指通过诱变编码蛋白的基因或其启动子，并且选择部分或全部丢失蛋白的活性，其中包括，通过在植物中表达沉默RNA，来获得对相关蛋白的抑制。
根据本发明一种典型的实施方式，S2包括利用基因突变和基因工程技术影响参与植物配子或胚发育过程，诱导配子发育成种子或植株。另外，S2可以包括授以其它植株的诱导花粉，诱导配子发育成种子或植株，例如S2包括授以二倍体雌配子单倍体诱导系花粉诱导二倍体雌配子发育成种子；又如，S2包括通过物理刺激、生物胁迫或化学药剂处理，诱导配子发育成种子或植株；又如，S2包括通过花药培养或花粉培养诱导配子发育成种子或植株。
优选的，MTL蛋白为如SEQ ID NO:29(MAASYSCRRTCEACSTRAMAGCVVGEPASAPGQRVTLLAIDGGGIRGLIPGTILAFLEARLQELDGPDARLADYFDCIAGTSTGGLITAMLAAPGDHGRPLFAASDINRFYLDNGPLIFPQKRCGMAAAMAALTRPRYNGKYLQGKIRKMLGETRVRDTLTNVVIPTFDVRLLQPTIFSTYDAKSMPLKNALLSDICISTSAAPTYLPAHCFQTTDDATGKVREFDLIDGGVAANNPTMVAMTQITKKIMVKDKEELYPVKPSDCGKFLVLSVGTGSTSDQGMYTARQCSRWGIVRWLRNKGMAPIIDIFMAASSDLVDIHAAVMFQSLHSDGDYLRIQDNTLHGDAATVDAATRDNMRALVGIGERMLAQRVSRVNVETGRYVEVPGAGSNADALRGFARQLSEERRARLGRRNACGGGGEGEPSGVACKR)所示的MTL蛋白，与所述MTL蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述MTL蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白。其中该诱导系花粉可以是来自产生基因型和倍性与杂交种一致配子的植株，也可以是来自于其他植株，优选的，诱导系花粉是来自产生基因型和倍性与杂交种一致雌配子的植株，通过在杂交种同时敲除了REC8、OSD1、PAIR1和MTL基因实现。
根据本发明一种典型的实施方式，植物包括单子叶植物和双子叶植物；优选的，植物包括水稻、玉米、高粱、谷子、大麦、小麦、黑麦、燕麦、荞麦、薏仁、甘蔗、芦笋、竹笋、韭菜、山药、大豆、土豆、豌豆、绿豆、小豆、蚕豆、豇豆、菜豆、小扁豆、蔓豆、鹰嘴豆、 木薯、甘薯、油菜、棉花、甜菜、茄子、花生、茶叶、薄荷、咖啡、芝麻、向日葵、蓖麻、苏子、红花、番茄、辣椒、黄瓜、青菜、生菜、菠菜、大蒜、甘蓝、芥菜、茭白、大葱、冬瓜、西葫芦、丝瓜、白菜、萝卜、洋葱、西瓜、葡萄、胡萝卜、花菜、南瓜、烟草、牧草、象草、狼尾草、苏丹草、兰花、百合、郁金香和苜蓿。
本申请的实现原理如下：
本申请实现无融合生殖的原理是通过绕过减数分裂和受精过程直接形成胚并产生种子，主要分为两大步骤：
第一步：减数分裂是动植物在生殖时期发生的一次特殊的细胞分裂过程。在减数分裂时，来自父母本的遗传信息将发生重组，产生染色体数目减半的配子。
将参与植物减数分裂时期的三个不同重要阶段的基因同时突变后(此三突材料命名为MiMe，Mitosis instead of Meiosis)，植株的减数分裂将转变成类似有丝分裂的过程。
MiMe植株产生的雌、雄配子细胞中染色体数目及基因型与体细胞完全一致，其自交后代都为基因型杂合的四倍体，证明通过同时突变三个基因就可以使杂交植物绕过减数分裂过程，产生与体细胞基因型一致的克隆配子。
第二步：花粉特异性磷脂酶基因(MATRILINEAL,MTL)主要作用于植物雄配子，是控制单倍体诱导的基因，首先在玉米中克隆。通过敲除MTL基因就可获得单倍体诱导材料mtl。双授精过程中，合子中mtl雄配子的基因组被降解，即父本精核并未与受体卵核形成合子，诱导产生卵核单倍体结实。
因而，在植物中同时改造MiMe及MTL这四个内源基因，就获得了可以发生无融合生殖的Fix(Fixation of hybrids)材料，即通过绕过减数分裂和受精过程保留母本基因组的方式获得了细胞倍性为二倍体且基因型与亲本完全一致的植株。由此证明，通过同时改造四个内源基因，就可将无融合生殖特性引入到杂交植物当中，从而实现杂合基因型的固定。
根据本发明一种典型的实施方式，包括以下步骤：1)将配子形成过程中的减数分裂转变为成类似有丝分裂。研究发现，将参与减数分裂时期的REC8、OSD1、PAIR1三个基因同时敲除后(此材料命名为MiMe，Mitosis instead of Meiosis)，染色体复制一次，生殖细胞由原来的分裂两次变为分裂一次，由此形成的配子中，染色体数目并没有减半，与体细胞一致。即，使减数分裂向类似有丝分裂转变，达到染色体加倍的目的；2)产生的雌配子受可以诱导雌配子发育的花粉刺激，即雌配子不与精细胞染色体融合即可发育形成胚，形成与体细胞基因型完全一致的种子。敲除MTL基因就可获得诱导单倍体产生的花粉。以杂交种子为转基因背景，利用基因突变或基因工程技术同时敲除REC8、OSD1、PAIR1及MTL四个基因，该植株产生的雌配子是与体细胞一样的染色体倍性，又由于MTL基因的破坏，产生的花粉可以诱导雌配子发育成种子或植株，这样得到的种子或植株并未发生基因分离(性状或育性的分离)，基因型与母细胞(用以转基因的背景材料杂交种)是一模一样的，最终达到固定杂种优势的目的。
图2和图3清楚的示出了本发明的F1子代基因型及染色体倍性与杂种母细胞保持一致。
根据一种典型的实施方式，提供一种保持有杂种优势的植物或种子。该植物或种子通过上述任一种方法制备得到，该种子能够很好的固定杂交优势。
根据一种典型的实施方式，提供一种用于使植物保持杂种优势的试剂盒。该试剂盒包括能够使植物生殖细胞的减数分裂转变为类似有丝分裂的载体和/或试剂，和使配子发育成种子或植株的载体和/或试剂。优选的，能够使植物生殖细胞的减数分裂转变为类似有丝分裂的载体和/或试剂以及诱导植物配子发生孤雌生殖的载体和/或试剂为随机诱变或定向诱变的载体和/或试剂。其中，随机诱变包括化学诱变、物理诱变和生物诱变；定向诱变包括CRISPR/Cas基因编辑技术、CRISPR/Cpf1基因编辑技术、TALEN基因编辑技术、归巢核酸内切酶基因编辑技术、ZFN基因编辑技术；基因工程技术包括转基因技术诱导基因的特异表达、易位表达或基因沉默。
根据一种典型的实施方式，能够使植物生殖细胞的减数分裂转变为类似有丝分裂的载体和/或试剂为利用基因工程技术抑制参与植物中减数分裂重组的蛋白实现将生殖细胞的减数分裂转变为类似有丝分裂的载体和/或试剂，其中蛋白包括第一蛋白、第二蛋白和第三蛋白，其中，
第一蛋白参与DNA双链断裂形成的蛋白，第一蛋白选自以下蛋白：
如SEQ ID NO:13所示的PAIR1蛋白，与PAIR1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与PAIR1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:14所示的PAIR2蛋白，与PAIR2蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与PAIR2蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:15所示的PAIR3蛋白，与PAIR3蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与PAIR3蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:16所示的PRD1蛋白，与PRD1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与PRD1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:17所示的PRD2蛋白，与PRD2蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与PRD2蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、 90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:18所示的SPO11-1蛋白，与SPO11-1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与SPO11-1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:19所示的SPO11-2蛋白，与SPO11-2蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与SPO11-2蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:20所示的SDS蛋白，与SDS蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与SDS蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:21所示的CRC1蛋白，与CRC1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与CRC1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:22所示的P31 comet蛋白，与P31 comet蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与P31 comet蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:23所示的MTOPVIB蛋白，与MTOPVIB蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与MTOPVIB蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:24所示的DFO蛋白，与DFO蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与DFO蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
第二蛋白参与控制减数分裂期姐妹染色体间的黏连，第二蛋白选自以下蛋白：
如SEQ ID NO:25所示的REC8蛋白，与REC8蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与REC8蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
第三蛋白参与减数分裂的第二次分裂，第三蛋白选自以下蛋白：
如SEQ ID NO:26所示的OSD1蛋白，与OSD1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与OSD1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:27所示的TAM蛋白，与TAM蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与TAM蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:28所示的TDM1蛋白，与TDM1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与TDM1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白。
进一步地，使配子发育成种子或植株的载体和/或试剂，其中包括为利用基因突变或基因工程技术影响参与植物配子或胚发育过程的MTL蛋白而诱导配子发育成种子或植株的载体和/或试剂，MTL蛋白为如SEQ ID NO:29所示的MTL蛋白，与MTL蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与MTL蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白。
为了方便出售和使用，优选的，试剂盒包含了用于在杂交种同时敲除REC8、OSD1、PAIR1和MTL基因的载体和/或试剂。
根据一种典型的实施方式，提供一种植物。该植物的生殖细胞的减数分裂转变为类似有丝分裂从而可以产生与杂交种基因型和染色体倍性一致的配子；例如，该植物的生殖细胞的减数分裂转变为类似有丝分裂从而可以产生与杂交种染色体倍性及基因型一致的配子。优选的，植物能够诱导配子发育成植株或种子。
根据一种典型的实施方式，植物为基因突变或基因工程改造植物，植物利用基因突变或基因工程技术调节参与植物中减数分裂的蛋白实现将生殖细胞的减数分裂转变为类似有丝分裂；利用基因突变或基因工程技术影响参与植物中配子发育过程的MTL蛋白而诱导配子发育成种子或植株；其中蛋白包括第一蛋白、第二蛋白和第三蛋白，其中，
第一蛋白参与DNA双链断裂形成的蛋白，第一蛋白选自以下蛋白：
如SEQ ID NO:13所示的PAIR1蛋白，与PAIR1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与PAIR1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:14所示的PAIR2蛋白，与PAIR2蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与PAIR2蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:15所示的PAIR3蛋白，与PAIR3蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与PAIR3蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:16所示的PRD1蛋白，与PRD1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与PRD1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:17所示的PRD2蛋白，与PRD2蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与PRD2蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:18所示的SPO11-1蛋白，与SPO11-1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与SPO11-1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:19所示的SPO11-2蛋白，与SPO11-2蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与SPO11-2蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:20所示的SDS蛋白，与SDS蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与SDS蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:21所示的CRC1蛋白，与CRC1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与CRC1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:22所示的P31 comet蛋白，与P31 comet蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白， 或者与P31 comet蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:23所示的MTOPVIB蛋白，与MTOPVIB蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与MTOPVIB蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:24所示的DFO蛋白，与DFO蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与DFO蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
第二蛋白参与控制减数分裂期姐妹染色体间的黏连，第二蛋白选自以下蛋白：
如SEQ ID NO:25所示的REC8蛋白，与REC8蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与REC8蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
第三蛋白参与减数分裂的第二次分裂，第三蛋白选自以下蛋白：
如SEQ ID NO:26所示的OSD1蛋白，与OSD1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与OSD1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:27所示的TAM蛋白，与TAM蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与TAM蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；如SEQ ID NO:28所示的TDM1蛋白，与TDM1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与TDM1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
如SEQ ID NO:28所示的TDM1蛋白，与TDM1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与TDM1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
MTL蛋白为如SEQ ID NO:29所示的MTL蛋白，与MTL蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与MTL蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、 85％、90％、95％或98％的序列相似性的蛋白。
下面将结合实施例进一步说明本发明的有益效果，以下实施例中没有详细描述的步骤或试剂均可采用本领域的常规技术手段或常规试剂实现。
实施例1
1.在本实施例中，使用的F 1杂交种是已审定的、商品化杂交稻品种春优84。春优84是利用早花时晚粳不育系春江16A与籼粳中间型广亲和恢复系C84配组育成的粳不籼恢亚种间杂交稻新组合。该杂交稻具有产量潜力大、制种产量高、综合农艺性状优良、抗瘟性较好、适应性广等优点。本实施例中所用的遗传转化背景材料是用杂交稻F 1种子诱导获得的愈伤，未经过有性繁殖阶段，因此，经转基因后获得的转基因T 0代材料在遗传背景上是与杂交稻F 1植株一致的。
2.多基因敲除载体的构建。
主要步骤如下(具体细节也可参见CN201510485573.2)：
1)单个目的SK-gRNA的构建：
选择以下四个位点作为CRISPR-Cas9基因编辑系统敲除REC8、OSD1、PAIR1及MTL的位点(下划线表示的PAM序列):
OSD1基因敲除位点(SEQ ID NO：1)：CTGCCGCCGACGAGCAACA AGG
PAIR1基因敲除位点(SEQ ID NO：2)：AAGCAACCCAGTGCACCGC TGG
REC8基因敲除位点(SEQ ID NO：3)：CCCATGGCACTAAGGCTCT CCG
MTL基因敲除位点(SEQ ID NO：4)：GGTCAACGTCGAGACCGGC AGG
设计两个互补DNA序列分别为：在正向序列前加GGCA，在反向互补序列前加AAAC；
SK-gRNA上有两个AarI酶切位点，用AarI酶切后，形成带有粘性末端的载体；将设计的靶序列正向及反向引物变性退火后，T4连接酶连入之前构建的中间载体SK-gRNA，形成单个目的gRNA；
2)多个gRNA的串联及最终双元表达载体的构建：
利用BglII和BamHI，NheI和XbaI，SalI和XhoI是同尾酶的特性，进行gRNA的聚合：SK-gRNA OSD1用KpnI和XhoI进行酶切，作为载体；SK-gRNA PAIR1用SalI和XbaI酶切提供PAIR1sgRNA片段，SK-gRNA REC8用NheI和BamHI酶切提供REC8sgRNA片段，SK-gRNA MTL用BglII和KpnI进行酶切提供MTL sgRNA片段，进行4个之内gRNA的一步法快速聚合；最终将聚合好的gRNA OSD1-gRNA REC8-gRNA PAIR1-gRNA MTL片段用KpnI和BglII进行酶切并回收片段，连接到表达Cas9蛋白的双元载体pC1300-Cas9中(KpnI和BamHI位点间)，最终获得可以同时敲除REC8、OSD1、PAIR1及MTL四个基因的多基因敲除载体 pC1300-Cas9-gRNA OSD1-gRNA REC8-gRNA PAIR1-gRNA MTL，用于转基因制备水稻多突变体。
3.转基因植株的获得。
将多基因敲除的双元表达载体pC1300-Cas9-gRNA OSD1-gRNA REC8-gRNA PAIR1-gRNA MTL通过电击的方法转入农杆菌(AgroBacteriumtumefaciens)株系EHA105中，利用农杆菌介导法将此双元表达载体转入水稻春优84的愈伤中。转化的具体方法是将杂交稻春优84种子的胚灭菌后，接种到诱导愈伤组织的培养基中。培养1周后，挑选生长旺盛，颜色浅黄，比较松散的胚性愈伤组织，用作转化的受体。用含有pC1300-Cas9-gRNA OSD1-gRNA REC8-gRNA PAIR1-gRNA MTL质粒的EHA105菌株侵染水稻愈伤组织，在黑暗处25℃培养3天后，在含有50mg/l潮霉素的选择培养基上筛选抗性愈伤组织和转基因植株。挑选在潮霉素选择培养基上正常生长的转基因植株。
4.测序鉴定四突变体
利用分子生物学手段鉴定目的基因突变情况。CTAB法单株提取转基因植物基因组DNA，PCR扩增目的条带。所用引物对：
OSD1-F(SEQ ID NO：5):atctccaggatgcctgaagtgag
OSD1-R(SEQ ID NO：6):cctagactgctactcttgctagtgat
PAIR1-F(SEQ ID NO：7):ctgtacctgtgcatctaattacag
PAIR1-R(SEQ ID NO：8):ccccatcttatgtactgagcttgccag
REC8-F(SEQ ID NO：9):gcgacgcttcactcgaagatca
REC8-R(SEQ ID NO：10):cgccatgcctcgttgatctcaa
MTL-F(SEQ ID NO：11):acagtgactagtgacaaacgatcg
MTL-R(SEQ ID NO：12):gatcgcgtcagcatgatgcgtgtac
得到的PCR产物送测序公司，分别用OSD1-F,PAIR1-F,REC8-F,MTL-F作为测序引物测序。所得结果与野生型序列进行比对。测序结果为双峰的，用简并密码子策略分析(http://dsdecode.scgene.com/进行峰图分析)，直接获得突变信息。从中筛选出四个基因都为双等位突变的四突变体。
5.在子一代中鉴定倍性和基因型固定的植株。
1)在鉴定得到的四突变体植物的子一代植株中，利用流式细胞术进行细胞倍性的筛选，筛选得到的细胞倍性与母体植株一致的植株。
具体方法如下：
取一定量的植物组织放入玻璃培养皿中，加入1～2ml植物裂解缓冲液LB01，用刀片切碎(此操作始终在冰上进行)；吸取培养皿内的解离液，50μm尼龙网过滤至离心管中；1200rpm，4℃，离心5min；弃上清，加入450μl的LB01、25uL的预冷PI(1mg/ml)和RNase A(1mg/ml)避光染色10min，上机检测，筛选出二倍体植株。
图4A示出了F1代植株春优84的细胞倍性检测结果；以及图4B示出了杂种优势固定植株的细胞倍性检测结果。
2)全基因组测序。
选取两个亲本春江16A和C84，春优84及倍性固定的子一代(随机挑选了4株)植株的叶子，提取DNA进行全基因组测序。根据全基因组测序结果显示(图5)：春江16A和C84之间存在许多不同的纯合基因型，杂交种春优84在这些位点的基因型都为既有春江16A又有C84基因型的杂合状态，检测的4株植株的基因型与春优84一致，都为杂合状态，从分子生物学角度证明基因型与杂种母细胞完全一致。
实施例2
1.在本实施例中，使用保持系春江16B与籼粳中间型广亲和恢复系C84。本实施例中所用的遗传转化背景材料是用亲本种子诱导获得的愈伤。
2.多基因敲除载体的构建。
主要步骤如下
1)单个目的SK-gRNA的构建：
选择以下四个位点作为CRISPR-Cas9基因编辑系统敲除REC8、OSD1、PAIR1及MTL的位点(下划线表示的PAM序列):
OSD1基因敲除位点(SEQ ID NO：1)：CTGCCGCCGACGAGCAACA AGG
PAIR1基因敲除位点(SEQ ID NO：2)：AAGCAACCCAGTGCACCGC TGG
REC8基因敲除位点(SEQ ID NO：3)：CCCATGGCACTAAGGCTCT CCG
MTL基因敲除位点(SEQ ID NO：4)：GGTCAACGTCGAGACCGGC AGG
设计两个互补DNA序列分别为：在正向序列前加GGCA，在反向互补序列前加AAAC；
SK-gRNA上有两个AarI酶切位点，用AarI酶切后，形成带有粘性末端的载体；将设计的靶序列正向及反向引物变性退火后，T4连接酶连入之前构建的中间载体SK-gRNA，形成单个目的gRNA；
4)多个gRNA的串联及最终双元表达载体的构建：
利用BglII和BamHI，NheI和XbaI，SalI和XhoI是同尾酶的特性，进行gRNA的聚合： SK-gRNA OSD1用KpnI和XhoI进行酶切，作为载体；SK-gRNA PAIR1用SalI和XbaI酶切提供PAIR1sgRNA片段，SK-gRNA REC8用NheI和BamHI酶切提供REC8sgRNA片段，SK-gRNA MTL用BglII和KpnI进行酶切提供MTL sgRNA片段，进行4个之内gRNA的一步法快速聚合；最终将聚合好的gRNA OSD1-gRNA REC8-gRNA PAIR1-gRNA MTL片段用KpnI和BglII进行酶切并回收片段，连接到表达Cas9蛋白的双元载体pC1300-Cas9中(KpnI和BamHI位点间)，最终获得可以同时敲除REC8、OSD1、PAIR1及MTL四个基因的多基因敲除载体pC1300-Cas9-gRNA OSD1-gRNA REC8-gRNA PAIR1-gRNA MTL，用于转基因制备水稻多突变体。
3.转基因植株的获得。
将多基因敲除的双元表达载体pC1300-Cas9-gRNA OSD1-gRNA REC8-gRNA PAIR1-gRNA MTL通过电击的方法转入农杆菌(AgroBacteriumtumefaciens)株系EHA105中，利用农杆菌介导法将此双元表达载体转入春江16B和C84的愈伤中。转化的具体方法是将种子的胚灭菌后，接种到诱导愈伤组织的培养基中。培养1周后，挑选生长旺盛，颜色浅黄，比较松散的胚性愈伤组织，用作转化的受体。用含有pC1300-Cas9-gRNA OSD1-gRNA REC8-gRNA PAIR1-gRNA MTL质粒的EHA105菌株侵染水稻愈伤组织，在黑暗处25℃培养3天后，在含有50mg/l潮霉素的选择培养基上筛选抗性愈伤组织和转基因植株。挑选在潮霉素选择培养基上正常生长的转基因植株。
4.测序鉴定四个基因都为杂合突变的春江16B和C84材料，再进行杂交，在后代筛选出四个基因都突变的杂交植株
利用分子生物学手段鉴定目的基因突变情况。CTAB法单株提取转基因植物基因组DNA，PCR扩增目的条带。所用引物对：
OSD1-F(SEQ ID NO：5):atctccaggatgcctgaagtgag
OSD1-R(SEQ ID NO：6):cctagactgctactcttgctagtgat
PAIR1-F(SEQ ID NO：7):ctgtacctgtgcatctaattacag
PAIR1-R(SEQ ID NO：8):ccccatcttatgtactgagcttgccag
REC8-F(SEQ ID NO：9):gcgacgcttcactcgaagatca
REC8-R(SEQ ID NO：10):cgccatgcctcgttgatctcaa
MTL-F(SEQ ID NO：11):acagtgactagtgacaaacgatcg
MTL-R(SEQ ID NO：12):gatcgcgtcagcatgatgcgtgtac
得到的PCR产物送测序公司，分别用OSD1-F,PAIR1-F,REC8-F,MTL-F作为测序引物测序。所得结果与野生型序列进行比对，可直接获得突变信息。
筛选到杂合突变的春江16B和C84材料后，相互杂交并在F1代中筛选出双等位突变的杂 交植株。
5.收取杂交植株的种子，在子一代中鉴定倍性和基因型固定的植株。
1)在鉴定得到的3突变体植物的子一代植株中，利用流式细胞术进行细胞倍性的筛选，筛选得到的细胞倍性与母体植株一致的植株。
具体方法如下：
取一定量的植物组织放入玻璃培养皿中，加入1～2ml植物裂解缓冲液LB01，用刀片切碎(此操作始终在冰上进行)；吸取培养皿内的解离液，50μm尼龙网过滤至离心管中；1200rpm，4℃，离心5min；弃上清，加入450μl的LB01、25uL的预冷PI(1mg/ml)和RNase A(1mg/ml)避光染色10min，上机检测，筛选出二倍体植株。
2)全基因组测序。
选取两个亲本春江16B和C84，春优84及倍性固定的子一代(随机挑选了2株)植株的叶子，提取DNA进行全基因组测序。根据全基因组测序结果显示：春江16B和C84之间存在许多不同的纯合基因型，杂交种春优84在这些位点的基因型都为既有春江16B又有C84基因型的杂合状态，检测的2株植株的基因型与春优84一致，都为杂合状态，从分子生物学角度证明基因型与杂种母细胞完全一致。
实施例3
1.在本实施例中，使用的F 1杂交种是已审定的、商品化杂交稻品种春优84。春优84是利用不育系春江16A与籼粳中间型广亲和恢复系C84配组育成的粳不籼恢亚种间杂交稻新组合。该杂交稻具有产量潜力大、制种产量高、综合农艺性状优良、抗瘟性较好、适应性广等优点。本实施例中所用的遗传转化背景材料是用杂交稻F 1种子诱导获得的愈伤，未经过有性繁殖阶段，因此，经转基因后获得的转基因T 0代材料在遗传背景上是与杂交稻F 1植株一致的。
2.多基因敲除载体的构建。
主要步骤如下
1)单个目的SK-gRNA的构建：
选择以下3个位点作为CRISPR-Cas9基因编辑系统敲除REC8、OSD1及PAIR1的位点(下划线表示的PAM序列):
OSD1基因敲除位点(SEQ ID NO：1)：CTGCCGCCGACGAGCAACA AGG
PAIR1基因敲除位点(SEQ ID NO：2)：AAGCAACCCAGTGCACCGC TGG
REC8基因敲除位点(SEQ ID NO：3)：CCCATGGCACTAAGGCTCT CCG
设计两个互补DNA序列分别为：在正向序列前加GGCA，在反向互补序列前加AAAC；
SK-gRNA上有两个AarI酶切位点，用AarI酶切后，形成带有粘性末端的载体；将设计的靶序列正向及反向引物变性退火后，T4连接酶连入之前构建的中间载体SK-gRNA，形成单个目的gRNA；
2)三个gRNA的串联及最终双元表达载体的构建：
利用BglII和BamHI，NheI和XbaI，SalI和XhoI是同尾酶的特性，进行gRNA的聚合；最终将聚合好的gRNA OSD1-gRNA REC8-gRNA PAIR1片段用KpnI和BglII进行酶切并回收片段，连接到表达Cas9蛋白的双元载体pC1300-Cas9中(KpnI和BamHI位点间)，最终获得可以同时敲除REC8、OSD1及PAIR1三个基因的多基因敲除载体pC1300-Cas9-gRNA OSD1-gRNA REC8-gRNA PAIR1，用于转基因制备水稻多突变体。
3.转基因植株的获得。
将多基因敲除的双元表达载体pC1300-Cas9-gRNA OSD1-gRNA REC8-gRNA PAIR1-gRNA通过电击的方法转入农杆菌(AgroBacteriumtumefaciens)株系EHA105中，利用农杆菌介导法将此双元表达载体转入水稻春优84的愈伤中。转化的具体方法是将杂交稻春优84种子的胚灭菌后，接种到诱导愈伤组织的培养基中。培养1周后，挑选生长旺盛，颜色浅黄，比较松散的胚性愈伤组织，用作转化的受体。用含有pC1300-Cas9-gRNA OSD1-gRNA REC8-gRNA PAIR1质粒的EHA105菌株侵染水稻愈伤组织，在黑暗处25℃培养3天后，在含有50mg/l潮霉素的选择培养基上筛选抗性愈伤组织和转基因植株。挑选在潮霉素选择培养基上正常生长的转基因植株。
4.测序鉴定三突变体
利用分子生物学手段鉴定目的基因突变情况。CTAB法单株提取转基因植物基因组DNA，PCR扩增目的条带。所用引物对：
OSD1-F(SEQ ID NO：5):atctccaggatgcctgaagtgag
OSD1-R(SEQ ID NO：6):cctagactgctactcttgctagtgat
PAIR1-F(SEQ ID NO：7):ctgtacctgtgcatctaattacag
PAIR1-R(SEQ ID NO：8):ccccatcttatgtactgagcttgccag
REC8-F(SEQ ID NO：9):gcgacgcttcactcgaagatca
REC8-R(SEQ ID NO：10):cgccatgcctcgttgatctcaa
得到的PCR产物送测序公司，分别用OSD1-F,PAIR1-F,REC8-F作为测序引物测序。所得结果与野生型序列进行比对。测序结果为双峰的，用简并密码子策略分析(http://dsdecode.scgene.com/进行峰图分析)，可直接获得突变信息。
其中，这3个位点都为双等位突变的突变体，就是可以产生与体细胞基因型和染色体倍性一致的配子的植株。
5.以三突变体为母本，授以其他单倍体诱导系植株花粉，诱导雌配子发育成种子，大量获得保持杂种优势的杂交种。
6.在子一代中鉴定倍性和基因型固定的植株。
1)在鉴定得到的三突变体植物的子一代植株中，利用流式细胞术进行细胞倍性的筛选，筛选得到的细胞倍性与母体植株一致的植株。
具体方法如下：
取一定量的植物组织放入玻璃培养皿中，加入1～2ml植物裂解缓冲液LB01，用刀片切碎(此操作始终在冰上进行)；吸取培养皿内的解离液，50μm尼龙网过滤至离心管中；1200rpm，4℃，离心5min；弃上清，加入450μl的LB01、25uL的预冷PI(1mg/ml)和RNase A(1mg/ml)避光染色10min，上机检测，筛选出二倍体植株。
2)全基因组测序。
选取两个亲本春江16A和C84，春优84及倍性固定的子一代(随机挑选了4株)植株的叶子，提取DNA进行全基因组测序。根据全基因组测序结果显示：春江16A和C84之间存在许多不同的纯合基因型，杂交种春优84在这些位点的基因型都为既有春江16A又有C84基因型的杂合状态，检测的4株植株的基因型与春优84一致，都为杂合状态，从分子生物学角度证明基因型与杂种母细胞完全一致。
实施例4
1.在本实施例中，使用的F 1杂交种是已审定的、商品化杂交稻品种春优84。春优84是利用不育系春江16A与籼粳中间型广亲和恢复系C84配组育成的粳不籼恢亚种间杂交稻新组合。该杂交稻具有产量潜力大、制种产量高、综合农艺性状优良、抗瘟性较好、适应性广等优点。本实施例中所用的遗传转化背景材料是用杂交稻F 1种子诱导获得的愈伤，未经过有性繁殖阶段，因此，经转基因后获得的转基因T 0代材料在遗传背景上是与杂交稻F 1植株一致的。
2.多基因敲除载体的构建。
主要步骤如下
1)单个目的SK-gRNA的构建：
选择以下3个位点作为CRISPR-Cas9基因编辑系统敲除REC8、OSD1及PAIR1的位点(下划线表示的PAM序列):
OSD1基因敲除位点(SEQ ID NO：1)：CTGCCGCCGACGAGCAACA AGG
PAIR1基因敲除位点(SEQ ID NO：2)：AAGCAACCCAGTGCACCGC TGG
REC8基因敲除位点(SEQ ID NO：3)：CCCATGGCACTAAGGCTCT CCG
设计两个互补DNA序列分别为：在正向序列前加GGCA，在反向互补序列前加AAAC；
SK-gRNA上有两个AarI酶切位点，用AarI酶切后，形成带有粘性末端的载体；将设计的靶序列正向及反向引物变性退火后，T4连接酶连入之前构建的中间载体SK-gRNA，形成单个目的gRNA；
2)三个gRNA的串联及最终双元表达载体的构建：
利用BglII和BamHI，NheI和XbaI，SalI和XhoI是同尾酶的特性，进行gRNA的聚合；最终将聚合好的gRNA OSD1-gRNA REC8-gRNA PAIR1片段用KpnI和BglII进行酶切并回收片段，连接到表达Cas9蛋白的双元载体pC1300-Cas9中(KpnI和BamHI位点间)，最终获得可以同时敲除REC8、OSD1及PAIR1三个基因的多基因敲除载体pC1300-Cas9-gRNA OSD1-gRNA REC8-gRNA PAIR1，用于转基因制备水稻多突变体。
3.转基因植株的获得。
将多基因敲除的双元表达载体pC1300-Cas9-gRNA OSD1-gRNA REC8-gRNA PAIR1-gRNA MTL通过电击的方法转入农杆菌(AgroBacteriumtumefaciens)株系EHA105中，利用农杆菌介导法将此双元表达载体转入水稻春优84的愈伤中。转化的具体方法是将杂交稻春优84种子的胚灭菌后，接种到诱导愈伤组织的培养基中。培养1周后，挑选生长旺盛，颜色浅黄，比较松散的胚性愈伤组织，用作转化的受体。用含有pC1300-Cas9-gRNA OSD1-gRNA REC8-gRNA PAIR1质粒的EHA105菌株侵染水稻愈伤组织，在黑暗处25℃培养3天后，在含有50mg/l潮霉素的选择培养基上筛选抗性愈伤组织和转基因植株。挑选在潮霉素选择培养基上正常生长的转基因植株。
4.测序鉴定三突变体
利用分子生物学手段鉴定目的基因突变情况。CTAB法单株提取转基因植物基因组DNA，PCR扩增目的条带。所用引物对：
OSD1-F(SEQ ID NO：5):atctccaggatgcctgaagtgag
OSD1-R(SEQ ID NO：6):cctagactgctactcttgctagtgat
PAIR1-F(SEQ ID NO：7):ctgtacctgtgcatctaattacag
PAIR1-R(SEQ ID NO：8):ccccatcttatgtactgagcttgccag
REC8-F(SEQ ID NO：9):gcgacgcttcactcgaagatca
REC8-R(SEQ ID NO：10):cgccatgcctcgttgatctcaa
得到的PCR产物送测序公司，分别用OSD1-F,PAIR1-F,REC8-F作为测序引物测序。所得结果与野生型序列进行比对。测序结果为双峰的，用简并密码子策略分析(http://dsdecode.scgene.com/进行峰图分析)，可直接获得突变信息。
其中，这3个位点都为双等位突变的突变体，就是可以产生与体细胞基因型和染色体倍性一致的配子的植株。
5.在三突变体发育到一定阶段后，分别通过无菌操作取花药或花粉接种到人工配置的花药培养基上，诱导其形成愈伤组织，进而通过组织培养获得了植株。
6.在组织培养的植株中鉴定倍性和基因型固定的植株。
1)在鉴定得到的3突变体植物的子一代植株中，利用流式细胞术进行细胞倍性的筛选，筛选得到的细胞倍性与母体植株一致的植株。
具体方法如下：
取一定量的植物组织放入玻璃培养皿中，加入1～2ml植物裂解缓冲液LB01，用刀片切碎(此操作始终在冰上进行)；吸取培养皿内的解离液，50μm尼龙网过滤至离心管中；1200rpm，4℃，离心5min；弃上清，加入450μl的LB01、25uL的预冷PI(1mg/ml)和RNase A(1mg/ml)避光染色10min，上机检测，筛选出二倍体植株。
2)全基因组测序。
选取两个亲本春江16A和C84，春优84及倍性固定的子一代(随机挑选了4株)植株的叶子，提取DNA进行全基因组测序。根据全基因组测序结果显示：春江16A和C84之间存在许多不同的纯合基因型，杂交种春优84在这些位点的基因型都为既有春江16A又有C84基因型的杂合状态，检测的4株植株的基因型与春优84一致，都为杂合状态，从分子生物学角度证明基因型与杂种母细胞完全一致。
实施例5
1.在本实施例中，使用的F 1杂交种是已审定的、商品化杂交稻品种春优84。春优84是利用不育系春江16A与籼粳中间型广亲和恢复系C84配组育成的粳不籼恢亚种间杂交稻新组合。该杂交稻具有产量潜力大、制种产量高、综合农艺性状优良、抗瘟性较好、适应性广等优点。本实施例中所用的遗传转化背景材料是用杂交稻F 1种子诱导获得的愈伤，未经过有性繁殖阶段，因此，经转基因后获得的转基因T 0代材料在遗传背景上是与杂交稻F 1植株一致的。
2.多基因敲除载体的构建。
主要步骤如下
1)单个目的SK-gRNA的构建：
选择以下3个位点作为CRISPR-Cas9基因编辑系统敲除REC8、OSD1及PAIR1的位点(下划线表示的PAM序列):
OSD1基因敲除位点(SEQ ID NO：1)：CTGCCGCCGACGAGCAACA AGG
PAIR1基因敲除位点(SEQ ID NO：2)：AAGCAACCCAGTGCACCGC TGG
REC8基因敲除位点(SEQ ID NO：3)：CCCATGGCACTAAGGCTCT CCG
设计两个互补DNA序列分别为：在正向序列前加GGCA，在反向互补序列前加AAAC；
SK-gRNA上有两个AarI酶切位点，用AarI酶切后，形成带有粘性末端的载体；将设计的靶序列正向及反向引物变性退火后，T4连接酶连入之前构建的中间载体SK-gRNA，形成单个目的gRNA；
2)三个gRNA的串联及最终双元表达载体的构建：
利用BglII和BamHI，NheI和XbaI，SalI和XhoI是同尾酶的特性，进行gRNA的聚合；最终将聚合好的gRNA OSD1-gRNA REC8-gRNA PAIR1片段用KpnI和BglII进行酶切并回收片段，连接到表达Cas9蛋白的双元载体pC1300-Cas9中(KpnI和BamHI位点间)，最终获得可以同时敲除REC8、OSD1及PAIR1三个基因的多基因敲除载体pC1300-Cas9-gRNA OSD1-gRNA REC8-gRNA PAIR1，用于转基因制备水稻多突变体。
3.转基因植株的获得。
将多基因敲除的双元表达载体pC1300-Cas9-gRNA OSD1-gRNA REC8-gRNA PAIR1-gRNA MTL通过电击的方法转入农杆菌(AgroBacteriumtumefaciens)株系EHA105中，利用农杆菌介导法将此双元表达载体转入水稻春优84的愈伤中。转化的具体方法是将杂交稻春优84种子的胚灭菌后，接种到诱导愈伤组织的培养基中。培养1周后，挑选生长旺盛，颜色浅黄，比较松散的胚性愈伤组织，用作转化的受体。用含有pC1300-Cas9-gRNA OSD1-gRNA REC8-gRNA PAIR1质粒的EHA105菌株侵染水稻愈伤组织，在黑暗处25℃培养3天后，在含有50mg/l潮霉素的选择培养基上筛选抗性愈伤组织和转基因植株。挑选在潮霉素选择培养基上正常生长的转基因植株。
4.测序鉴定三突变体
利用分子生物学手段鉴定目的基因突变情况。CTAB法单株提取转基因植物基因组DNA，PCR扩增目的条带。所用引物对：
OSD1-F(SEQ ID NO：5):atctccaggatgcctgaagtgag
OSD1-R(SEQ ID NO：6):cctagactgctactcttgctagtgat
PAIR1-F(SEQ ID NO：7):ctgtacctgtgcatctaattacag
PAIR1-R(SEQ ID NO：8):ccccatcttatgtactgagcttgccag
REC8-F(SEQ ID NO：9):gcgacgcttcactcgaagatca
REC8-R(SEQ ID NO：10):cgccatgcctcgttgatctcaa
得到的PCR产物送测序公司，分别用OSD1-F,PAIR1-F,REC8-F作为测序引物测序。所得结果与野生型序列进行比对。测序结果为双峰的，用简并密码子策略分析(http://dsdecode.scgene.com/进行峰图分析)，可直接获得突变信息。
其中，这3个位点都为双等位突变的突变体，就是可以产生与体细胞基因型和染色体倍性一致的配子的植株。
5.化学诱导孤雌生殖
取同时敲除REC8、OSD1、PAIR1三个基因的水稻材料，水稻开花之前，按一般杂交技术剪颖杀雄，然后将稻穗浸没于处理液5～50mg/L马来酰肼或2～20mg/L 6-苄氨基腺嘌呤中2-3min，严密套袋避免花粉进入。处理后20天，取幼胚或谷粒培养，获得孤雌生殖的植株。
6.在子一代中鉴定倍性和基因型固定的植株。
1)在鉴定得到的三突变体植物的子一代植株中，利用流式细胞术进行细胞倍性的筛选，筛选得到的细胞倍性与母体植株一致的植株。
具体方法如下：
取一定量的植物组织放入玻璃培养皿中，加入1～2ml植物裂解缓冲液LB01，用刀片切碎(此操作始终在冰上进行)；吸取培养皿内的解离液，50μm尼龙网过滤至离心管中；1200rpm，4℃，离心5min；弃上清，加入450μl的LB01、25uL的预冷PI(1mg/ml)和RNase A(1mg/ml)避光染色10min，上机检测，筛选出二倍体植株。
2)全基因组测序。
选取两个亲本春江16A和C84，春优84及倍性固定的子一代(随机挑选了4株)植株的叶子，提取DNA进行全基因组测序。根据全基因组测序结果显示：春江16A和C84之间存在许多不同的纯合基因型，杂交种春优84在这些位点的基因型都为既有春江16A又有C84基因型的杂合状态，检测的4株植株的基因型与春优84一致，都为杂合状态，从分子生物学角度证明基因型与杂种母细胞完全一致。
实施例6
1.突变体诱变与筛选
在大豆品种中黄39中，通过EMS诱变，在后代通过高通量测序技术筛选得到REC8和OSD1分别为杂合突变的植株，通过杂合植株间的杂交与后代筛选，得到REC8和OSD1都为杂合突变的植株；在大豆品种齐黄34中，通过EMS诱变，在后代通过高通量测序技术筛选得到SPO11-1和CENH3基因分别为杂合突变的植株，通过杂合植株间的杂交与后代筛选，得到SPO11-1和CENH3都为杂合突变的植株；将REC8和OSD1杂合突变的植株和SPO11-1和CENH3都为杂合突变的植株进行杂交，在后代中筛选得到四个基因都为杂合突变的植株。
2.转基因载体构建
构建了卵细胞特异表达的EC1.2基因启动子驱动野生型CENH3表达的双元载体，将该载体转化到4个基因都为杂合突变的植株中；在植株的自交后代中鉴定并筛选得到REC8、OSD1、SPO11-1和CENH3基因都为纯合突变、且有EC1.2::CENH3转基因成分的单株，收获该植株上的自交种子。
3.在子一代中鉴定倍性和基因型固定的植株。
1)在子一代植株中，利用流式细胞术进行细胞倍性的筛选，筛选得到的细胞倍性与母体植株一致的植株。
具体方法如下：
取一定量的植物组织放入玻璃培养皿中，加入1～2ml植物裂解缓冲液LB01，用刀片切碎(此操作始终在冰上进行)；吸取培养皿内的解离液，50μm尼龙网过滤至离心管中；1200rpm，4℃，离心5min；弃上清，加入450μl的LB01、25uL的预冷PI(1mg/ml)和RNase A(1mg/ml)避光染色10min，上机检测，筛选出二倍体植株。
2)基因型检测。
选取倍性固定的子一代(随机挑选了4株)以及其上一代植株的叶子，提取DNA；在上一代杂交材料中随机筛选出16处杂合状态的位点，并设计出检测引物。对子一代植株进行基因型检测，检测发现该4株植株在16个位点处的基因型与上一代完全一致，即都为杂合状态，从分子生物学角度证明杂合基因型没有发生重组与分离。
实施例7
1.在本实施例中，使用的F 1杂交种是玉米杂交种佳禾158，是由LD140×LD975配组而成的。
2.多基因敲除载体的构建。
主要步骤如下：
1)单个目的SK-gRNA的构建：
选择以下四个位点作为CRISPR-Cas9基因编辑系统敲除玉米REC8、OSD1、PAIR1及MTL的位点(下划线表示的PAM序列):
ZmOSD1基因敲除位点(SEQ ID NO：30)：TCTGCCTGTACTGGAGTTAT TGG
ZmPAIR1基因敲除位点(SEQ ID NO：31)：GGATTGCTGCGACAGCGGCT GGG
ZmREC8基因敲除位点(SEQ ID NO：32)：GGAAGTCCCACGAGTAATTA TGG
ZmMTL基因敲除位点(SEQ ID NO：33)：GGAAGGCGAGGATGGTTCCC GGG
2)多个gRNA的串联及最终双元表达载体的构建：
利用BglII和BamHI，NheI和XbaI，SalI和XhoI是同尾酶的特性，进行gRNA的聚合：SK-gRNA ZmOSD1用KpnI和XhoI进行酶切，作为载体；SK-gRNA ZmPAIR1用SalI和XbaI酶切提供ZmPAIR1sgRNA片段，SK-gRNA ZmREC8用NheI和BamHI酶切提供ZmREC8sgRNA片段，SK-gRNA ZmMTL用BglII和KpnI进行酶切提供ZmMTL sgRNA片段，进行4个之内gRNA的一步法快速聚合；最终将聚合好的gRNA ZmOSD1-gRNA ZmREC8-gRNA ZmPAIR1-gRNA ZmMTL片段用KpnI和BglII进行酶切并回收片段，连接到表达Cas9蛋白的 双元载体pC1300-Cas9中(KpnI和BamHI位点间)，最终获得可以同时敲除玉米REC8、OSD1、PAIR1及MTL四个基因的多基因敲除载体pC1300-Cas9-gRNA ZmOSD1-gRNA ZmREC8-gRNA ZmPAIR1-gRNA ZmMTL，用于转基因制备玉米多突变体。
3.转基因植株的获得。
将上一步骤获得的玉米多基因敲除载体通过电击的方法转入根癌农杆菌菌株LBA4404中，利用农杆菌介导法将此双元表达载体转入玉米杂交种佳禾158愈伤中。玉米授粉后人工套袋9-12天，取雌穗剥去苞叶，每剥去一层苞叶喷洒75％的酒精，进行表面消毒，用刀片在超净工作台下挑取1.0-1.2mm大小的幼胚置于高渗透液中备用，置于渗透液时间不超过1小时。将农杆菌培养至OD600值至0.8时，离心收集菌体，用1mol/L悬液，重悬后加入乙酰丁香酮至终浓度为200μmol/L，用该菌液侵染幼胚5min，然后转移到共培养基中，25℃暗培养7天，将幼胚转移至含有15mg/l潮霉素的选择培养基及后期的再生培养基上筛选抗性愈伤组织和转基因植株。
4.测序鉴定四突变体
CTAB法单株提取转基因玉米基因组DNA，利用Hi-Tom鉴定目的基因突变情况(具体细节可参见CN201710504178.3)。
5.在子一代中鉴定倍性和基因型固定的玉米植株。
1)在鉴定得到的四突变体玉米的子一代植株中，利用流式细胞术进行细胞倍性的筛选，筛选得到的细胞倍性与母体植株一致的植株。
具体方法如下：
取一定量的植物组织放入玻璃培养皿中，加入1～2ml植物裂解缓冲液LB01，用刀片切碎(此操作始终在冰上进行)；吸取培养皿内的解离液，50μm尼龙网过滤至离心管中；1200rpm，4℃，离心5min；弃上清，加入450μl的LB01、25uL的预冷PI(1mg/ml)和RNase A(1mg/ml)避光染色10min，上机检测，筛选出二倍体植株。
2)全基因组测序。
选取两个亲本LD140和LD975，佳禾158及倍性固定的子一代玉米植株的叶子，提取DNA进行全基因组测序。检测的子一代玉米植株的基因型与佳禾158一致，都为杂合状态，从分子生物学角度证明基因型与杂种母细胞完全一致。
实施例8
1.在本实施例中，使用的F 1杂交种是番茄杂交种艾丽莎，其母本是耐低温的自交系“S以2-4”，父本是优质抗病自交系“S28”。
2.多基因敲除载体的构建。
主要步骤如下：
1)单个目的SK-gRNA的构建：
选择以下四个位点作为CRISPR-Cas9基因编辑系统敲除番茄REC8、OSD1、SPO11及MTL的位点(下划线表示的PAM序列):
SlOSD1基因敲除位点(SEQ ID NO：34)：CAGAAGCAGGGAGAATGGC AGG
SlSPO11基因敲除位点(SEQ ID NO：35)：TGAGGATCTCGCTCGAGGT AGG
SlREC8基因敲除位点(SEQ ID NO：36)：GCACAGGAGGAACCTGCTA AGG
SlMTL基因敲除位点(SEQ ID NO：37)：TGATTGCCGGAACGAGCAC CGG
2)多个gRNA的串联及最终双元表达载体的构建：
利用BglII和BamHI，NheI和XbaI，SalI和XhoI是同尾酶的特性，进行gRNA的聚合：SK-gRNA SlOSD1用KpnI和XhoI进行酶切，作为载体；SK-gRNA SlSPO11用SalI和XbaI酶切提供SlSPO11sgRNA片段，SK-gRNA SlREC8用NheI和BamHI酶切提供SlREC8sgRNA片段，SK-gRNA SlMTL用BglII和KpnI进行酶切提供SlMTL sgRNA片段，进行4个之内gRNA的一步法快速聚合；最终将聚合好的gRNA SlOSD1-gRNA SlREC8-gRNA SlPAIR1-gRNA SlMTL片段用KpnI和BglII进行酶切并回收片段，连接到表达Cas9蛋白的双元载体pC1300-Cas9中(KpnI和BamHI位点间)，最终获得可以同时敲除番茄REC8、OSD1、SPO11及MTL四个基因的多基因敲除载体pC1300-Cas9-gRNA SlOSD1-gRNA SlREC8-gRNA SlSPO11-gRNA SlMTL，用于转基因制备番茄多突变体。
3.转基因植株的获得。
通过叶盘法将上一步骤获得的番茄多基因敲除载体通过电击的方法转入农杆菌菌株EHA105中，利用农杆菌介导法将此双元表达载体转入番茄杂交种艾丽莎愈伤中。
番茄种子经过无菌处理播于1/2MS培养基上，暗培养2-3天待发芽后，光照培养，10-12天后，当幼苗子叶充分展开，但还未有真叶生成时，选用子叶为外植体，子叶切除两端，中间部分横向一分为二，切成的小块即为叶盘。叶盘接种于预培养培养基中，叶片正面朝上，预培养2天。用制备好的农杆菌菌液浸泡预培养的子叶叶盘，充分浸润5分钟，无菌滤纸将叶盘适当吸干，叶片背面朝上，暗培养48-72小时，培养温度为28℃。将与农杆菌共培养后的叶盘转接到脱菌培养基中，光照培养。5天后将叶盘转接于筛选培养基中，每14天转接一次。当抗性芽长至2cm左右时，从外植体上切下，转入生根培养基中，待根系发达后，移栽到土壤中。
4.测序鉴定四突变体
CTAB法单株提取转基因番茄基因组DNA，利用Hi-Tom鉴定目的基因突变情况(具体细节可参见CN201710504178.3)。
5.在子一代中鉴定倍性和基因型固定的番茄植株。
1)在鉴定得到的四突变体番茄的子一代植株中，利用流式细胞术进行细胞倍性的筛选，筛选得到的细胞倍性与母体植株一致的植株。
具体方法如下：
取一定量的植物组织放入玻璃培养皿中，加入1～2ml植物裂解缓冲液LB01，用刀片切碎(此操作始终在冰上进行)；吸取培养皿内的解离液，50μm尼龙网过滤至离心管中；1200rpm，4℃，离心5min；弃上清，加入450μl的LB01、25uL的预冷PI(1mg/ml)和RNase A(1mg/ml)避光染色10min，上机检测，筛选出二倍体植株。
2)全基因组测序。
选取两个亲本“S以2-4”和“S28”，番茄杂交种艾丽莎及倍性固定的子一代番茄植株的叶子，提取DNA进行全基因组测序。检测的子一代番茄植株的基因型与艾丽莎一致，都为杂合状态，从分子生物学角度证明基因型与杂种母细胞完全一致。
另外，本实施例中所用的所有载体和试剂包含在本实施例的试剂盒之中。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (30)

  1. 一种保持植物杂种优势的方法，其特征在于，包括以下步骤：
    S1，利用基因突变或基因工程技术将杂交种的生殖细胞的减数分裂转变为类似有丝分裂从而得到与杂交种基因型和染色体倍性一致的配子；以及
    S2，利用基因突变和基因工程技术影响参与植物配子或胚发育过程，其中涉及的蛋白为MTL蛋白。
  2. 根据权利要求1所述的方法，其特征在于，所述基因突变包括随机诱变和定向诱变；其中，所述随机诱变包括化学诱变、物理诱变和生物诱变；所述定向诱变包括基因编辑技术，所述基因编辑技术包括CRISPR/Cas基因编辑技术、CRISPR/Cpf1基因编辑技术、TALEN基因编辑技术、归巢核酸内切酶基因编辑技术和ZFN基因编辑技术；所述基因工程技术包括转基因技术诱导基因的特异表达、易位表达或基因沉默。
  3. 根据权利要求1所述的方法，其特征在于，所述S1包括取杂交种，利用基因突变或基因工程技术将其生殖细胞的减数分裂转变为类似有丝分裂从而得到与杂交种基因型和染色体倍性一致的配子。
  4. 根据权利要求1所述的方法，其特征在于，所述S1包括利用基因突变或基因工程技术将杂交种的亲本进行编辑，然后通过亲本之间杂交获得杂交种，进而获得生殖细胞的减数分裂转变为类似有丝分裂的杂交种配子。
  5. 根据权利要求1所述的方法，其特征在于，所述S1包括利用基因突变或基因工程技术编辑参与植物中减数分裂的蛋白实现将生殖细胞的减数分裂转变为类似有丝分裂；其中所述蛋白包括第一蛋白、第二蛋白和第三蛋白，其中，
    所述第一蛋白为参与DNA双链断裂形成的蛋白，所述第一蛋白选自以下蛋白：
    如SEQ ID NO:13所示的PAIR1蛋白，与所述PAIR1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述PAIR1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:14所示的PAIR2蛋白，与所述PAIR2蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述PAIR2蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:15所示的PAIR3蛋白，与所述PAIR3蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述PAIR3蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:16所示的PRD1蛋白，与所述PRD1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述PRD1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:17所示的PRD2蛋白，与所述PRD2蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述PRD2蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:18所示的SPO11-1蛋白，与所述SPO11-1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述SPO11-1蛋白具、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:19所示的SPO11-2蛋白，与所述SPO11-2蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述SPO11-2蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:20所示的SDS蛋白，与所述SDS蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述SDS蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:21所示的CRC1蛋白，与所述CRC1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述CRC1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:22所示的P31 comet蛋白，与所述P31 comet蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述P31 comet蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:23所示的MTOPVIB蛋白，与所述MTOPVIB蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述MTOPVIB蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:24所示的DFO蛋白，与所述DFO蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致 性的蛋白，或者与所述DFO蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    所述第二蛋白参与控制减数分裂期姐妹染色体间的黏连，所述第二蛋白选自以下蛋白：
    如SEQ ID NO:25所示的REC8蛋白，与所述REC8蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述REC8蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    所述第三蛋白参与减数分裂的第二次分裂，所述第三蛋白选自以下蛋白：
    如SEQ ID NO:26所示的OSD1蛋白，与所述OSD1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述OSD1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:27所示的TAM蛋白，与所述TAM蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述TAM蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:28所示的TDM1蛋白，与所述TDM1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述TDM1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白。
  6. 根据权利要求1所述的方法，其特征在于，所述S2包括利用基因突变和基因工程技术影响参与植物配子或胚发育过程，诱导所述配子发育成种子或植株。
  7. 根据权利要求6所述的方法，其特征在于，所述S2包括授以其它植株的诱导花粉，诱导所述配子发育成种子或植株。
  8. 根据权利要求1所述的方法，其特征在于，所述S2包括通过物理刺激、生物胁迫或化学药剂处理，诱导所述配子发育成种子或植株。
  9. 根据权利要求1所述的方法，其特征在于，所述S2包括通过花药培养或花粉培养诱导所述配子发育成种子或植株。
  10. 根据权利要求6所述的方法，其特征在于，所述MTL蛋白为如SEQ ID NO:29所示的MTL蛋白，与所述MTL蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述MTL蛋白具有至 少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白。
  11. 根据权利要求1所述的方法，其特征在于，所述植物包括单子叶植物和双子叶植物。
  12. 根据权利要求1所述的方法，其特征在于，所述植物包括水稻、玉米、高粱、谷子、大麦、小麦、黑麦、燕麦、荞麦、薏仁、甘蔗、芦笋、竹笋、韭菜、山药、大豆、土豆、豌豆、绿豆、小豆、蚕豆、豇豆、菜豆、小扁豆、蔓豆、鹰嘴豆、木薯、甘薯、油菜、棉花、甜菜、茄子、花生、茶叶、薄荷、咖啡、芝麻、向日葵、蓖麻、苏子、红花、番茄、辣椒、黄瓜、青菜、生菜、菠菜、大蒜、甘蓝、芥菜、茭白、大葱、冬瓜、西葫芦、丝瓜、白菜、萝卜、洋葱、西瓜、葡萄、胡萝卜、花菜、南瓜、烟草、牧草、象草、狼尾草、苏丹草、兰花、百合、郁金香和苜蓿。
  13. 一种保持有杂种优势的植物或种子，其特征在于，通过如权利要求1至11中任一项所述的方法制备得到。
  14. 一种用于权利要求1的方法的使植物保持杂种优势的试剂盒，其特征在于，所述试剂盒包括能够使植物生殖细胞的减数分裂转变为类似有丝分裂的载体和/或试剂，和使配子发育成种子或植株的载体和/或试剂。
  15. 根据权利要求14所述的试剂盒，其特征在于，所述能够使植物生殖细胞的减数分裂转变为类似有丝分裂的载体和/或试剂为通过基因突变或基因工程技术将杂交种的生殖细胞的减数分裂转变为类似有丝分裂的载体和/或试剂，优选为随机诱变或定向诱变的载体和/或试剂。
  16. 根据权利要求15所述的试剂盒，其特征在于，所述随机诱变包括化学诱变、物理诱变和生物诱变；所述定向诱变包括CRISPR/Cas基因编辑技术、CRISPR/Cpf1基因编辑技术、TALEN基因编辑技术、归巢核酸内切酶基因编辑技术、ZFN基因编辑技术；所述基因工程技术包括转基因技术诱导基因的特异表达、易位表达或基因沉默。
  17. 根据权利要求14所述的试剂盒，其特征在于，所述能够使植物生殖细胞的减数分裂转变为类似有丝分裂的载体和/或试剂为利用基因突变或基因工程技术编辑参与植物中减数分裂蛋白实现将生殖细胞的减数分裂转变为类似有丝分裂的载体和/或试剂，其中所述蛋白包括第一蛋白、第二蛋白和第三蛋白，其中，
    所述第一蛋白参与DNA双链断裂形成的蛋白，所述第一蛋白选自以下蛋白：
    如SEQ ID NO:13所示的PAIR1蛋白，与所述PAIR1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述PAIR1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:14所示的PAIR2蛋白，与所述PAIR2蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述PAIR2蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:15所示的PAIR3蛋白，与所述PAIR3蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述PAIR3蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:16所示的PRD1蛋白，与所述PRD1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述PRD1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:17所示的PRD2蛋白，与所述PRD2蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述PRD2蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:18所示的SPO11-1蛋白，与所述SPO11-1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述SPO11-1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:19所示的SPO11-2蛋白，与所述SPO11-2蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述SPO11-2蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:20所示的SDS蛋白，与所述SDS蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述SDS蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:21所示的CRC1蛋白，与所述CRC1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述CRC1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:22所示的P31 comet蛋白，与所述P31 comet蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致 性的蛋白，或者与所述P31 comet蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:23所示的MTOPVIB蛋白，与所述MTOPVIB蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述MTOPVIB蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:24所示的DFO蛋白，与所述DFO蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述DFO蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    所述第二蛋白参与控制减数分裂期姐妹染色体间的黏连，所述第二蛋白选自以下蛋白：
    如SEQ ID NO:25所示的REC8蛋白，与所述REC8蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述REC8蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    所述第三蛋白参与减数分裂的第二次分裂，所述第三蛋白选自以下蛋白：
    如SEQ ID NO:26所示的OSD1蛋白，与所述OSD1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述OSD1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:27所示的TAM蛋白，与所述TAM蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述TAM蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:28所示的TDM1蛋白，与所述TDM1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述TDM1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白。
  18. 根据权利要求14所述的试剂盒，其特征在于，所述使配子发育成种子或植株的载体和/或试剂，其中包括为利用基因突变或基因工程技术影响参与植物配子或胚发育过程的MTL蛋白而诱导配子发育成种子或植株的载体和/或试剂，所述MTL蛋白为如SEQ ID NO:29所示的MTL蛋白，与所述MTL蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所 述MTL蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白。
  19. 一种使用权利要求14至18中任意一项的试剂盒所产生的植物，其特征在于，所述植物的生殖细胞的减数分裂转变为类似有丝分裂从而可以产生与杂交种基因型和染色体倍性一致的配子。
  20. 根据权利要求19所述的植物，其特征在于，所述植物的配子能够被诱导发育成植株或种子。
  21. 根据权利要求20所述的植物，其特征在于，所述植物为基因突变或基因工程改造植物，所述植物被利用基因突变或基因工程技术调节参与植物中减数分裂的蛋白实现将生殖细胞的减数分裂转变为类似有丝分裂；被利用基因突变或基因工程技术影响参与植物中配子或胚发育过程的第四蛋白而诱导配子发育成种子或植株；其中所述蛋白包括第一蛋白、第二蛋白和第三蛋白，其中，
    所述第一蛋白参与DNA双链断裂形成的蛋白，所述第一蛋白选自以下蛋白：
    如SEQ ID NO:13所示的PAIR1蛋白，与所述PAIR1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述PAIR1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:14所示的PAIR2蛋白，与所述PAIR2蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述PAIR2蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:15所示的PAIR3蛋白，与所述PAIR3蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述PAIR3蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:16所示的PRD1蛋白，与所述PRD1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述PRD1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:17所示的PRD2蛋白，与所述PRD2蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述PRD2蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:18所示的SPO11-1蛋白，与所述SPO11-1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述SPO11-1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:19所示的SPO11-2蛋白，与所述SPO11-2蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述SPO11-2蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:20所示的SDS蛋白，与所述SDS蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述SDS蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:21所示的CRC1蛋白，与所述CRC1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述CRC1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:22所示的P31comet蛋白，与所述P31comet蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述P31comet蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:23所示的MTOPVIB蛋白，与所述MTOPVIB蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述MTOPVIB蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:24所示的DFO蛋白，与所述DFO蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述DFO蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    所述第二蛋白参与控制减数分裂期姐妹染色体间的黏连，所述第二蛋白选自以下蛋白：
    如SEQ ID NO:25所示的REC8蛋白，与所述REC8蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述REC8蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    所述第三蛋白参与减数分裂的第二次分裂，所述第三蛋白选自以下蛋白：
    如SEQ ID NO:26所示的OSD1蛋白，与所述OSD1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述OSD1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:27所示的TAM蛋白，与所述TAM蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述TAM蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；如SEQ ID NO:28所示的TDM1蛋白，与所述TDM1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述TDM1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    如SEQ ID NO:28所示的TDM1蛋白，与所述TDM1蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述TDM1蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白；
    所述第四蛋白选自以下蛋白：
    如SEQ ID NO:29所示的MTL蛋白，与所述MTL蛋白具有至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列一致性的蛋白，或者与所述MTL蛋白具有至少40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％的序列相似性的蛋白。
  22. 一种保持植物杂种优势的方法，其特征在于，包括以下步骤：
    S1，利用基因编辑技术将杂交种在F1代时生殖细胞的减数分裂转变为类似有丝分裂从而得到F1代的二倍体雌配子；以及
    S2，利用基因突变和基因工程技术影响参与植物配子或胚发育过程诱导所述二倍体雌配子发育成种子，其中涉及的蛋白为MTL蛋白。
  23. 根据权利要求22所述的方法，其特征在于，所述S1包括取杂交F1代种子，利用基因编辑技术将生殖细胞的减数分裂转变为类似有丝分裂从而得到F1代的二倍体雌配子。
  24. 根据权利要求22所述的方法，其特征在于，所述S1包括利用基因编辑技术将杂交种的亲本进行编辑，获得经编辑的各基因都为杂合突变的植株，然后通过亲本之间杂交获得杂交种，在杂交种中筛选在两个亲本中经编辑的多个基因都为纯合突变的植株，进而获得生殖细胞的减数分裂转变为类似有丝分裂的F1代的二倍体雌配子。
  25. 根据权利要求23或24所述的方法，其特征在于，所述S1包括利用基因编辑技术敲除REC8、OSD1、PAIR1基因实现将生殖细胞的减数分裂转变为类似有丝分裂。
  26. 根据权利要求22所述的方法，其特征在于，所述S2包括授以所述二倍体雌配子单倍体诱导系花粉诱导所述二倍体雌配子发育成种子。
  27. 根据权利要求26所述的方法，其特征在于，所述S2包括利用基因编辑技术敲除MTL基因而产生单倍体诱导系花粉。
  28. 根据权利要求26所述的方法，其特征在于，所述S2包括采用其他植株的单倍体诱导系花粉诱导所述二倍体雌配子发育成种子。
  29. 根据权利要求25所述的方法，其特征在于，所述杂交种在F1代时同时敲除了REC8、OSD1、PAIR1和MTL基因。
  30. 根据权利要求22所述的方法，其特征在于，所述植物包括水稻、玉米、高粱、小米、大麦和小麦。
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