WO2020248969A1 - 一种雄性不育保持系植物及其用途 - Google Patents

一种雄性不育保持系植物及其用途 Download PDF

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WO2020248969A1
WO2020248969A1 PCT/CN2020/095082 CN2020095082W WO2020248969A1 WO 2020248969 A1 WO2020248969 A1 WO 2020248969A1 CN 2020095082 W CN2020095082 W CN 2020095082W WO 2020248969 A1 WO2020248969 A1 WO 2020248969A1
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plant
gene
plants
male
seed
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PCT/CN2020/095082
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French (fr)
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赖锦盛
赵海铭
宋伟彬
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中国农业大学
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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
    • A01H1/022—Genic fertility modification, e.g. apomixis
    • A01H1/023—Male sterility
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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
    • 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
    • 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
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
    • A01H6/4684—Zea mays [maize]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C12N15/8289—Male sterility

Definitions

  • This application belongs to the field of plant genetic breeding and seed production. Specifically, this application relates to a male sterile maintainer plant, a method for producing the male sterile maintainer plant, and the use of the plant for the expansion of male sterile plants and male sterile maintainer plants .
  • the present invention also relates to nucleic acid molecules, vectors and host cells (for example, Agrobacterium) used to produce the male sterile maintainer plants.
  • Heterosis makes the hybrids' biomass, resistance to diseases and insects, and tolerance to stress (drought, high temperature, low temperature, saline-alkali, etc.) capabilities have been considerably improved compared to their parents. For example, the yields of hybrid corn and hybrid rice are much higher than their homozygous parents.
  • the methods commonly used to produce hybrids are: planting the female parent and the male parent together; removing the tassels of the female parent and retaining the tassels of the male parent; harvesting seeds from the female parent is a hybrid.
  • Self-pollination refers to the phenomenon that the pollen of a plant pollinates its own pistil.
  • same-flower pollination for example, Phaseolus vulgaris
  • neighbor-flower pollination and same-plant cross-pollination.
  • Same flower pollination is the pollination between the stamens and pistils of the same flower.
  • Adjacent flower pollination is the pollination between different flowers in an inflorescence (individual).
  • Cross-pollination of the same plant is pollination between different flowers of the same plant.
  • Corn is a monoecious plant, and the female and male flowers are located in different parts of the plant. Maize can reproduce by self-pollination or cross-pollination. Under natural conditions, natural pollination is completed when the wind blows the pollen from the male ears to the female ears.
  • a homozygous maize inbred line is usually developed first, and then the two inbred lines are crossed, and the yield and stress resistance of the hybrid offspring are evaluated to determine whether it has commercial potential. Among them, each inbred line may have one or more excellent traits that another inbred line lacks, or supplement one or more undesirable traits of another inbred line.
  • the first generation of seeds crossed between two inbred lines is F1 generation seeds. F1 generation plants are obtained after F1 generation seeds germinate. F1 generation plants are more robust than the two inbred line parents (parents) and possess more organisms. the amount.
  • Hybrids (F1) can be produced by artificially detasseling the female parent, that is, the unpolished female parent (which can be sown in the field with the male parent, such as sowing 5 rows of female parent and one row of male parent) tassels are removed, and the male parent is retained. This tassel. Subsequently, as long as the foreign corn pollen is isolated, the female ears of the female parent can only receive the pollen from the male parent, and the resulting seed is the hybrid (F1), which can be used for agricultural production. However, in the actual process of producing hybrids, changes in the environment may result in tasselization or incomplete emasculation after emasculation.
  • the stable male sterility system provides a simple and efficient means of emasculation. By using a male sterile system, heavy emasculation can be avoided in some cases.
  • the method includes three main materials, namely (1) male sterile lines (also referred to as sterile lines): they are male sterile materials; (2) male sterile maintainers (also referred to as maintainers): they can Provide pollen for the sterile line, so that the offspring of the sterile line are still sterile lines; (3) the male sterile restorer line (also referred to as the restorer line for short): it can restore the fertility of the sterile line.
  • F1 is produced by crossing the sterile line and the restorer line, which is a hybrid for agricultural production.
  • Plant male sterility can be divided into three types: cytoplasmic male sterility, nuclear male sterility, and nuclear-cytoplasmic male sterility.
  • Cytoplasmic male sterile plants are characterized by cytoplasmic inheritance, and single cytoplasmic genes S and N are usually used to represent male sterility and male fertility respectively, which are difficult to be applied in agricultural production.
  • Nuclear male sterile plants show nuclear inheritance; in most cases, male sterility is controlled by a pair of recessive genes (msms), and normal fertility is controlled by relatively dominant genes (MsMs) or (Msms) .
  • Nuclear-cytoplasmic interactive male sterility (CMS) plants show nuclear-cytoplasmic interaction inheritance.
  • the plant will be male sterile. If the cytoplasm contains the fertile gene N, no matter whether the gene in the nucleus is a fertile gene (RfRf) or a sterile gene (rfrf), the plant will be male fertile. Similarly, if there is a fertile gene (RfRf) or (Rfrf) in the nucleus, no matter whether the gene in the cytoplasm is the fertile gene N or the sterile gene S, the plant will be male fertile.
  • CMS nuclear-plasma interactive male sterility
  • the genetic composition of the restorer line is S(RfRf) or N(RfRf), and the F1 produced after crossing with the male sterile line is restored to male fertility, namely: S(rfrf)( ⁇ ) ⁇ S(RfRf) ⁇ S(Rfrf)(F1)(fertile), or S(rfrf)( ⁇ ) ⁇ N(RfRf) ⁇ S(Rfrf)(F1)(fertile).
  • the obtained F1 plants self-bred to produce F2, and F2 can also be widely used in agricultural production.
  • Male sterile lines can avoid manual detasseling, save manpower, reduce seed costs, and ensure seed purity.
  • crops such as rice, corn, sorghum, onion, castor, sugar beet, and rapeseed have used nucleocytoplasmic male sterile lines for hybrid seed production.
  • the nucleo-cytoplasmic male sterile lines of other crops are also under extensive research.
  • the CMS system also has its shortcomings: one is that it is observed that individual CMS materials are susceptible to disease, and the other is that the recovery system is difficult to find. These problems hinder the widespread application of CMS system in seed production.
  • the nuclear gene that controls male sterility is a recessive gene, and only when it is homozygous (msms), the plant will be male sterile.
  • msms the only way to obtain male sterile plants
  • Msms heterozygous plants
  • the method includes: firstly constructing a transgenic vector containing a pollen cell lethal gene and a dominant gene to restore plant fertility; then, transferring the vector into male sterile plants, and the vector is in the transgenic plants Exist in a heterozygous state. Due to the existence of the gene to restore fertility, the transgenic plant is male fertile. Moreover, when it is crossed with a male sterile plant, since the pollen (Msms) containing the fertility restorer gene also contains the lethal gene, the pollen containing the fertility restorer gene will all abort.
  • the transgenic plant can only produce pollen (ms) without the restorer gene, and cross with the female gametes (ms) of the male sterile plant.
  • the offspring produced are all recessive homozygous individuals (msms). That is, when such a plant is crossed with a male sterile plant, its offspring all maintain the homozygous recessive state of the recessive sterile plant.
  • the disadvantage of the above method is that due to the existence of heteroandrogynous fertilization (the embryo and endosperm are fertilized by sperm developed from different male gametophytes, that is, the sperm genotypes that form the embryo and endosperm are different), the endosperm is obtained after screening There is still a certain proportion of fertile seeds in the offspring seeds, and these fertile seeds are difficult to distinguish from sterile seeds and cannot fully meet the needs of actual production.
  • isolated refers to a state obtained by artificial means, which is different from the natural state.
  • a certain substance or component "isolated” from nature it may be that the natural environment in which it is located has changed, or the substance has been isolated from the natural environment, or both.
  • isolation a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolation. of.
  • isolated does not exclude the mixing of artificial or synthetic materials, nor does it exclude the presence of other impure materials that do not affect the activity of the material.
  • the term "extrinsic traits” refers to the heritable and observable phenotypic characteristics of individuals (including seeds and plants), which are usually composed of one or more pairs of alleles control.
  • the "external traits" of seeds and plants mainly include the external morphological characteristics of roots, stems, leaves, flowers, fruits, and seeds, such as seed color, seed size, plant color, plant wilting degree, stem morphology, leaf morphology, etc.
  • the external traits of seeds mainly refer to the external morphological characteristics of seeds, including, for example, seed color or seed size.
  • the external characteristics of plants mainly refer to the external morphological characteristics of roots, stems, leaves, flowers, and fruits, including, for example, the color of the plant or the degree of wilting of the plant.
  • male sterility refers to a phenomenon in which male cells or tissues of plants lose their physiological functions. Generally, in sexually reproducing plants (for example, maize), male sterility is manifested by the abnormal development of male tissues (for example, stamens) and the inability to produce pollen with normal functions, but the development of female tissues (for example, pistils) is normal. Can accept normal pollen and be fertilized.
  • male sterility gene refers to a gene capable of controlling male sterility traits in plants. In the present application, preferably, the male sterility gene is a nuclear male sterility gene.
  • the nuclear gene that controls male sterility is a recessive gene, and the plant will only show male sterility when it is homozygous (msms).
  • msms a maize male sterile line
  • ms45ms45 a homozygous recessive male sterility gene
  • the male sterility gene is a recessive gene, which causes plant male sterility in a homozygous state.
  • the term "restorer gene” refers to a gene capable of restoring the male fertility of plants that have been caused by male sterility genes. When the restorer gene is introduced into a male sterile line plant, the plant will restore male fertility. In this application, when the male sterility gene is a recessive gene, the restorer gene may be a dominant allele of the recessive gene.
  • the term "selected genes that regulate seed color” refers to genes that can affect the color of seeds by affecting the synthesis of pigments, anthocyanins, and the like.
  • genes include, for example, the Lc gene (Ludwig SR et al., Proc Natl Acad Sci, 1989 Sep; 86(18): 7092-6; Wang Juan et al., Genomics and Applied Biology, Issue 02, 2009).
  • Lc gene is a regulatory gene related to anthocyanin synthesis, and its heterologous expression in a variety of plants (for example, corn) can affect anthocyanin synthesis, increase anthocyanin content, and affect seed color. The expression of this gene can make corn seeds appear purple.
  • the Lc gene can affect the color of plants. The expression of this gene can make corn plants appear purple. Therefore, the Lc gene is also a "screening gene that regulates plant color”.
  • the term "selected gene that regulates seed size” refers to a gene whose expression level can affect seed size.
  • genes include, for example, the CWI-2 gene (W.H. Cheng et al., Plant Cell. 1996 Jun; 8(6):971-983).
  • the CWI-2 gene encodes a cell wall invertase specifically expressed in endosperm. After the gene is silenced or mutated, it will affect the normal development of seed endosperm, resulting in smaller seeds, but does not affect the germination rate and plant development.
  • selected genes that regulate plant color refers to genes that can affect plant color by affecting the synthesis of chloroplasts, pigments, anthocyanins, etc.
  • genes include, for example, the Oy1 gene (Ruairidh J.H. Sawers et al., Plant Molecular Biology, volume 60, pages 95-106 (2006)).
  • Oy1 gene is a regulatory gene related to chloroplast synthesis, which can affect the color of plants (for example, corn plants) by affecting chloroplast synthesis. The expression of this gene can make corn plants appear yellow.
  • the term "selected genes that regulate the degree of plant wilting” refers to genes that can affect plant wilting by regulating the stomata opening, water transport, etc. of the plant.
  • genes include, for example, the Wi2 gene (Chris Rock et al., American Journal of Botany, 86(12):1796-800(2000)).
  • the Wi2 gene is a regulatory gene related to the synthesis of plant root cork. It can affect the wilting degree of plants (for example, corn) by affecting plant water transport. The expression of this gene can make corn leaves wilt.
  • tissue-preferred promoter refers to a promoter that can preferentially initiate transcription in certain plant tissues (for example, stamens, pollen sacs, filaments, and pollen).
  • growth phase preferred promoter refers to a promoter that can preferentially initiate transcription in certain growth and development stages (for example, sporogenous tissue, microspores, and microgametophytes).
  • tissue-specific promoter refers to a promoter that specifically initiates transcription only in certain plant tissues.
  • the term “selectable marker gene” refers to a gene whose encoded product is capable of allowing transformed or transfected host cells to grow normally under selective pressure or exhibit other visual characteristics. Such selection pressure includes, but is not limited to, the addition of selective agents (for example, antibiotics or herbicides) or lack of nutrients.
  • the term “antibiotic resistance gene” refers to a gene whose encoded product is capable of allowing transformed or transfected host cells to grow normally under the selective pressure of antibiotics or exhibit other visual characteristics.
  • the term “herbicide resistance gene” refers to a gene whose encoded product enables transformed or transfected host cells to grow normally under the selective pressure of the herbicide or exhibit other visual characteristics.
  • heterospermous fertilization refers to a phenomenon in which the embryo and endosperm of the same seed are fertilized by sperm formed by two different male gametophytes during double fertilization. In this case, the embryo and endosperm of the same seed can have different genotypes.
  • a plant can be all or part of a plant, such as roots, stems, leaves, embryos, root tips, pollen, or anthers.
  • the inventor of the present application has obtained a maintainer plant capable of maintaining the sterility of a male sterile line plant.
  • the maintainer line plant can be simultaneously obtained when crossed with a male sterile line plant.
  • the distinguishable offspring of the sterile line and the maintainer line significantly improve the propagation efficiency and breeding efficiency of the sterile line plants.
  • the present application also provides a method for producing the maintainer plant and a method for multiplying the sterile plant by using the maintainer plant.
  • the maintainer plant contains a double selection gene, and after the maintainer plant is crossed with a male sterile plant, the double selection method can be more accurate Distinguish the offspring of sterile lines and maintainers to further improve breeding efficiency.
  • the inventor of the present application completed the present invention.
  • an isolated nucleic acid molecule which comprises a first polynucleotide and a second polynucleotide, and the first polynucleotide comprises a nucleotide that restores a gene.
  • the restorer gene can restore the male fertility of plants that cause male sterility due to the male sterility gene;
  • the second polynucleotide includes the core of the screening gene capable of regulating the external traits of the seed and/or plant
  • the nucleotide sequence, the external traits are selected from seed color, seed size, plant color, plant wilting degree, stem morphology, leaf morphology, and any combination thereof.
  • the extrinsic traits are selected from seed color, plant color, degree of plant wilting, and any combination thereof.
  • the extrinsic trait is a combination of the extrinsic traits of the seed (e.g., seed color or seed size) and the extrinsic traits of the plant (e.g., plant color or degree of plant wilting). In certain embodiments, the extrinsic trait is a combination of seed color and plant color. In certain embodiments, the extrinsic trait is a combination of seed size and plant color. In certain embodiments, the extrinsic trait is a combination of seed color and plant wilting degree. In certain embodiments, the extrinsic trait is a combination of seed size and plant wilting degree.
  • the simultaneous use of two or more external traits is preferable and advantageous.
  • a screening gene that regulates the external traits of seeds such as seed color or seed size
  • a screening gene that regulates external traits of plants such as plant color or the degree of plant wilting
  • the extrinsic traits of the seed and the extrinsic traits of the plant can be used to screen the offspring of the hybrid to ensure the purity of the offspring.
  • the second polynucleotide includes: (a) the first screening gene capable of regulating the external traits of the seed (for example, seed color or seed size), and (b) capable of regulating The second screening gene for the plant's external traits (such as plant color or plant wilting degree).
  • the first screening gene and the second screening gene are the same.
  • the second polynucleotide contains a screening gene capable of controlling the extrinsic traits of the seed and the extrinsic traits of the plant.
  • the screening gene is the Lc gene, which can control the color of seeds and the color of plants.
  • the Lc gene encodes a protein with an amino acid sequence of SEQ ID NO: 5.
  • the nucleotide sequence of the Lc gene is SEQ ID NO: 4.
  • the first screening gene and the second screening gene are different.
  • the second polynucleotide includes:
  • the second polynucleotide includes: Lc gene and Wi2 gene. In certain embodiments, the second polynucleotide includes: a nucleotide encoding an interfering RNA of the CWI-2 gene and the Lc gene. In certain embodiments, the second polynucleotide includes: a nucleotide encoding an interfering RNA of the CWI-2 gene and an Oy1 gene. In certain embodiments, the second polynucleotide includes: nucleotides encoding interfering RNA of the CWI-2 gene and the Wi2 gene.
  • the nucleotide sequence encoding the interfering RNA of the CWI-2 gene has the nucleotide sequence shown in SEQ ID NO: 18.
  • the Oy1 gene encodes a protein with an amino acid sequence of SEQ ID NO: 7.
  • the nucleotide sequence of the Oy1 gene is SEQ ID NO: 6.
  • the Wi2 gene encodes a protein with an amino acid sequence of SEQ ID NO: 9.
  • the nucleotide sequence of the Wi2 gene is SEQ ID NO: 8.
  • the second polynucleotide further comprises an expression control element, such as a promoter and an enhancer, operably linked to the nucleotide sequence of the screening gene.
  • the expression control element is selected from the group consisting of promoters, enhancers, regulatory sequences, inducible elements, and any combination thereof.
  • the promoter is selected from: a constitutive promoter, an inducible promoter, a tissue-preferred promoter, a tissue-specific promoter, and a growth-phase-preferred promoter.
  • the promoters that can be used in this application are not limited to the promoters listed above. It is easy to understand that in the embodiments of the present application, any promoter known to those skilled in the art can be used according to actual needs.
  • the male sterility gene is a recessive male sterility gene, which causes plant male sterility in a homozygous state.
  • the male sterility gene is selected from ms1, ms2, ms3, ms4, ms5, ms6, ms7, ms8, ms9, ms10, ms11, ms12, ms13, ms14, ms15, ms16, ms17, ms18 , Ms19, ms20, ms21, ms22, ms23, ms24, ms25, ms26, ms27, ms28, ms29, ms30, ms31, ms32, ms33, ms34, ms35, ms36, ms37, ms38, ms43, ms45, ms47, ms48, ms49 , Ms50, ms52, and any combination thereof.
  • the male sterility gene is ms45.
  • the restorer gene is selected from Ms1, Ms2, Ms3, Ms4, Ms5, Ms6, Ms7, Ms8, Ms9, Ms10, Ms11, Ms12, Ms13, Ms14, Ms15, Ms16, Ms17, Ms18, Ms19 , Ms20, Ms21, Ms22, Ms23, Ms24, Ms25, Ms26, Ms27, Ms28, Ms29, Ms30, Ms31, Ms32, Ms33, Ms34, Ms35, Ms36, Ms37, Ms38, Ms43, Ms45, Ms47, Ms48, Ms50 , Ms52, and any combination thereof.
  • the restorer gene is Ms45.
  • the restorer gene and the male sterility gene should correspond, so that it can save the sterility traits caused by the male sterility gene.
  • the male sterility gene is a recessive gene
  • the restorer gene may be a dominant allele of the recessive gene, which can restore the male fertility of the plant.
  • the male sterility gene is ms45
  • the restorer gene is Ms45
  • the restorer gene encodes a protein with an amino acid sequence of SEQ ID NO: 2.
  • the nucleotide sequence of the restorer gene is SEQ ID NO:1.
  • the first polynucleotide further comprises an expression control element operably linked to the nucleotide sequence of the restorer gene, such as a promoter and an enhancer.
  • the expression control element is selected from the group consisting of promoters, enhancers, regulatory sequences, inducible elements, and any combination thereof.
  • the promoter is selected from: a constitutive promoter, an inducible promoter, a tissue-preferred promoter, a tissue-specific promoter, and a growth-phase-preferred promoter.
  • the promoters that can be used in this application are not limited to the promoters listed above. It is easy to understand that in the embodiments of the present application, any promoter known to those skilled in the art can be used according to actual needs.
  • the nucleotide sequence of the promoter is SEQ ID NO: 15.
  • the first polynucleotide sequence comprises or consists of SEQ ID NO: 3.
  • the first polynucleotide and the second polynucleotide are covalently linked with or without linking nucleotides.
  • the length of the linking nucleotide is no more than 10kb, no more than 5kb, no more than 1kb, no more than 500bp, no more than 100bp, no more than 50bp, no more than 10bp, no more than 5bp, or shorter .
  • the first nucleotide sequence and the second nucleotide sequence are genetically linked.
  • the isolated nucleic acid molecule further comprises a third polynucleotide comprising the nucleotide sequence of a selectable marker gene.
  • the selectable marker gene is an antibiotic resistance gene or a herbicide resistance gene, such as a bialaphos resistance gene (bar gene).
  • Selectable marker genes that can be used in the embodiments of this application include, but are not limited to, neomycin resistance genes (such as genes encoding neomycin phosphotransferase), hygromycin resistance genes (such as hygromycin phosphotransferase encoding genes) Gene), chloramphenicol resistance gene, streptomycin resistance gene, spectinomycin resistance gene, bleomycin resistance gene, sulfonamide resistance gene, bromoxynil resistance gene, glyphosate resistance Genes, bialaphos resistance gene and glufosinate resistance gene.
  • the selectable marker gene is a herbicide resistance gene.
  • the herbicide resistance gene is the bar gene.
  • the nucleotide sequence of the selectable marker gene is SEQ ID NO: 10.
  • a vector which contains the isolated nucleic acid molecule as described above.
  • the vector can be a cloning vector, transfer vector, or expression vector.
  • the vector is a plasmid (e.g., pCAMBAI3301), cosmid, phage, and the like.
  • the vector is capable of expressing the isolated nucleic acid molecule as described above in plant cells (for example, corn).
  • a host cell which comprises the isolated nucleic acid molecule as described above or the vector as described above.
  • the host cell is an Agrobacterium cell (e.g., Agrobacterium EHA105) or a plant cell (e.g., corn).
  • the plant cell is a cell of a monocot plant or a dicot plant.
  • the plant cell is a cell selected from the group consisting of corn (Zea mays), rape (Brassica napus), rice (Oryza sativa), Arabidopsis thaliana, and barley (Hordeum vulgare) , Wheat (Triticumaestivum), sorghum (Sorghum bicolor), soybean (Glycine max), alfalfa (Medicago sativa), tobacco (Nicotiana tabacum), cotton (Gossypium hirsutum), sunflower (Helianthus annuus) or sugar cane (Saccharum officinarum).
  • the application also provides a tissue culture of the host cell, and protoplasts produced from the tissue culture.
  • a plant or plant seed is provided, wherein the plant or plant seed contains the nucleic acid molecule as described above in the genome.
  • the plant or plant seed further includes the male sterility gene (for example, ms45) in the genome.
  • the male sterility gene is a homozygous recessive male sterility gene (e.g., ms45ms45).
  • the isolated nucleic acid molecule is integrated in the genome of the plant or plant seed. In certain embodiments, the isolated nucleic acid molecule is integrated in the genome of the plant or plant seed and is located on a chromosome different from the male sterility gene. In certain embodiments, the nucleic acid molecule is present in the genome of the plant or plant seed in a heterozygous form.
  • the term "existing in a hybrid form" has the meaning commonly understood by those skilled in the art. For example, it may mean that the same allele (ie, heterozygous) does not exist at the gene locus corresponding to the position where the nucleic acid molecule is integrated into the plant genome. For example, the nucleic acid molecule only exists in one chromatids, and its sister chromatids do not contain the nucleic acid molecules.
  • Any method known to those skilled in the art can be used to integrate the nucleic acid molecule into the genome of the plant or plant seed. Such methods include, but are not limited to, stable transformation methods, transient transformation methods, virus-mediated methods, and Agrobacterium-mediated methods.
  • the plant or plant seed is male fertile. In certain embodiments, the plant or plant seed can be used as a maintainer for a male sterile plant containing the male sterility gene.
  • the plant or plant seed is a plant or plant seed of a monocotyledonous or dicotyledonous plant.
  • the plant or plant seed is a plant or plant seed of corn, rape, rice, Arabidopsis, barley, wheat, sorghum, soybean, alfalfa, tobacco, cotton, sunflower, or sugar cane.
  • a method for obtaining plants comprising: (1) introducing the nucleic acid molecule as described above or the vector as described above into plant cells, and (2) introducing the Plant cells are grown into plants.
  • any method known to those skilled in the art can be used to introduce the nucleic acid molecule or vector into the plant cell. Such methods include, but are not limited to, stable transformation methods, transient transformation methods, virus-mediated methods, and Agrobacterium-mediated methods. In addition, any method known to those skilled in the art can be used to cultivate the plant cells into plants. For example, see the method described in McCormicketal. (1986) Plant Cell Reports 5: 81-84.
  • step (1) an Agrobacterium-mediated method is used. In some embodiments, in step (1), Agrobacterium is used to introduce the nucleic acid molecule or vector into the plant cell.
  • the plant cell contains the male sterility gene in its genome, and preferably, the plant cell is male sterile before introducing the nucleic acid molecule or vector. In certain embodiments, the plant cell contains a homozygous recessive male sterility gene in its genome.
  • the nucleic acid molecule in step (1), is integrated into the genome of the plant cell. In certain embodiments, the nucleic acid molecule is located on a chromosome different from the male sterility gene after being integrated into the genome of the plant cell.
  • the plant cell is a cell of a monocot plant or a dicot plant. In certain embodiments, the plant cell is a cell of a plant selected from the group consisting of corn, rape, rice, Arabidopsis, barley, wheat, sorghum, soybean, alfalfa, tobacco, cotton, sunflower, or sugar cane.
  • the plant comprises a homozygous recessive male sterility gene, and the nucleic acid molecule or vector, and it is male fertile.
  • the nucleic acid molecule or vector is present in the genome in a heterozygous form in the plant.
  • the nucleic acid molecule is integrated in the genome of the plant and is located on a chromosome different from the male sterility gene.
  • the plant can be used as a maintainer plant of a male sterile plant comprising the male sterility gene.
  • the method further includes:
  • step (3) Pollinate the male sterile plant containing the male sterility gene with the plant of step (2) to produce offspring seeds or plants;
  • a method of obtaining seeds or plants of the progeny of male sterile plants and maintainer plants comprising: combining the above-mentioned plants or the above-mentioned method The plant is used as the male parent and is crossed with the male sterile plant containing the male sterility gene as the female parent, and produces offspring seeds or plants.
  • the method includes:
  • male sterile plant containing the male sterility gene as a female parent; preferably, the male sterility gene is a homozygous recessive male sterility gene (for example, ms45ms45);
  • step (3) Pollinate the plants of step (1) with the plants of step (2) to produce offspring seeds;
  • the offspring seeds or plants that show the external traits regulated by the screening gene are male fertile and can be used as maintainers; and, the offspring seeds or plants that do not show the external traits regulated by the screening gene are male non Fertile, can be used as a male sterile line.
  • the plant used as the male parent in step (2) may contain: (a) a first screening gene capable of regulating the external traits of the seed (such as seed color or seed size), and (b) ) A second screening gene capable of regulating the extrinsic traits of the plant (such as plant color or plant wilting degree). Therefore, after crossing the plant of step (2) with the plant of step (1), the external traits of the seed (such as seed color or seed size) and the external traits of the plant (such as plant color or plant wilting degree) can be used. ) To screen the hybrid offspring to ensure the purity of the hybrid offspring.
  • the method includes:
  • male sterile plant containing the male sterility gene as a female parent; preferably, the male sterility gene is a homozygous recessive male sterility gene;
  • the second polynucleotide includes: (a) capable of regulating the external traits of seeds (such as seed color) (Or seed size) of the first screening gene, and (b) the second screening gene that can regulate the external traits of the plant (such as plant color or plant wilting degree);
  • step (3) Pollinate the plant of step (1) with the plant of step (2) to produce two progeny seeds; wherein, the first progeny seed shows the external traits of the seed regulated by the first screening gene; and, The two progeny seeds do not show the external traits of the seeds regulated by the first screening gene;
  • the method further includes the following steps:
  • Such plants are removed from the second progeny plants, which show the extrinsic traits of the plants regulated by the second screening gene, so that the remaining second progeny plants are male sterile and can be used as male sterility Line plant
  • the present application provides a method for preparing hybrid seeds, the method comprising:
  • Seeds are harvested from male sterile plants, which are hybrid seeds.
  • the method of the present application can use two or more external traits to screen and distinguish the progeny of male sterile plants and maintainer plants, which avoids the possibility of heteromale fertilization, genetic mutation, and genetic mutation during the breeding process. Loss, chromosome recombination, chromosome translocation, etc. lead to "false positive” or “false negative” traits, thereby further improving the purity of the offspring of the male sterile line and the maintainer plant, increasing the breeding efficiency and the production The quality and purity of hybrid seeds.
  • the first screening gene and the second screening gene are the same.
  • the second polynucleotide contains a screening gene capable of controlling the extrinsic traits of the seed and the extrinsic traits of the plant.
  • the screening gene is the Lc gene, which can control the color of seeds and the color of plants.
  • the Lc gene encodes a protein with an amino acid sequence of SEQ ID NO: 5.
  • the nucleotide sequence of the Lc gene is SEQ ID NO: 4.
  • the first screening gene and the second screening gene are different.
  • the second polynucleotide includes:
  • the second polynucleotide includes: Lc gene and Wi2 gene. In certain embodiments, the second polynucleotide includes: a nucleotide encoding an interfering RNA of the CWI-2 gene and the Lc gene. In certain embodiments, the second polynucleotide includes: a nucleotide encoding an interfering RNA of the CWI-2 gene and an Oy1 gene. In certain embodiments, the second polynucleotide includes: nucleotides encoding interfering RNA of the CWI-2 gene and the Wi2 gene.
  • the nucleotide sequence encoding the interfering RNA of the CWI-2 gene has the nucleotide sequence shown in SEQ ID NO: 18.
  • the Oy1 gene encodes a protein with an amino acid sequence of SEQ ID NO: 7.
  • the nucleotide sequence of the Oy1 gene is SEQ ID NO: 6.
  • the Wi2 gene encodes a protein with an amino acid sequence of SEQ ID NO: 9.
  • the nucleotide sequence of the Wi2 gene is SEQ ID NO: 8.
  • a product is provided, which is made from the plant or part thereof as described above.
  • the product is a food product, and it is made from the edible part (eg, seed) of the plant as described above.
  • a tissue culture or a protoplast produced therefrom wherein the tissue culture comprises the host cell (such as plant cell) as described above or is obtained by the method as described above The cells of the plant or the progeny seeds or cells of the plant obtained by the method described above.
  • the plant is a plant of a monocotyledonous plant or a dicotyledonous plant.
  • the plant is a plant selected from the group consisting of corn, rape, rice, Arabidopsis, barley, wheat, sorghum, soybean, alfalfa, tobacco, cotton, sunflower, or sugar cane.
  • An object of the present invention is to provide a method.
  • the method provided by the present invention is used to maintain the homozygous recessive state of male sterile plants, and the method includes:
  • (b) Provide a second plant, which contains the same homozygous recessive allele as the first plant to make the plant male sterile, and contains the following construct (for example, the transgenic element Ms45- Lc or Ms45-Oy1 or Ms45-Wi2) exists in a heterozygous state in the second plant (heterozygous Ms45-Lc/- or Ms45-Oy1/- or Ms45-Wi2/- inserts are only present in one staining list In the body, its sister chromatid does not contain transgenic elements), and the construct contains:
  • the first nucleotide sequence which when expressed in the first plant will restore the male fertility of the first plant
  • the second nucleotide sequence when it exists in a heterozygous state, can affect the color of the plant (for example, the Lc gene makes the plant and/or the grain purple, or the Oy1 gene makes the plant yellow) or affects the morphology of the plant (For example, the Wi2 gene makes the top leaves of the plant wilt), the plant or grain containing the construct can be distinguished from the plant or grain not containing the construct by the naked eye or instrument; the first nucleotide sequence (for example, SEQ ID NO The Ms45 gene expression element shown in: 3) and the second nucleotide sequence (for example, the Lc gene shown in SEQ ID NO: 4 or the Oy1 gene shown in SEQ ID NO: 6 or SEQ ID NO: 8 The Wi2 gene shown) is closely connected, and these two nucleotide sequences exist at the same time in the plant (for example, the transgenic elements Ms45-Lc, Ms45-Oy1 or Ms45-Wi2 in the examples);
  • the above method is a method of propagating male sterile lines of plants
  • the plant, the first plant and the second plant are all dicotyledonous plants or monocotyledonous plants;
  • the plant, the first plant and the second plant can be not only corn (Zea mays), but also rice (Oryza sativa), sorghum (Sorghum bicolor), wheat (Triticumaestivum), soybean (Glycine max), cotton (Gossypiumhirsutum), sunflower (Helianthus annuus) and other crops.
  • the first nucleotide sequence includes a restorer gene for controlling male fertility, such as the wild-type allele Ms45 of ms45 in Table 1.
  • This restorer gene for controlling male fertility is not limited to the genes listed in Table 1.
  • the restorer gene for controlling male fertility in corn or other species can also achieve the purpose of the present invention, and therefore is also within the protection scope of the present invention.
  • the first plant is the maize male sterile mutant ms45, specifically the ms45 homozygous recessive inbred line Zheng 58 ( ⁇ 58(ms45ms45)), which is derived from ms45 Backcross offspring of homozygous recessive mutants (Maize Genetics Cooperation Stock Center, 905I) and Zheng 58 (zheng58).
  • the first nucleotide sequence is an Ms45 gene expression element in the embodiment of the present invention, and the Ms45 gene expression element expresses the protein Ms45 in the first plant.
  • the protein Ms45 is as follows a) or b):
  • the Ms45 gene expression element includes Ms45 gene promoter, Ms45 gene 5'UTR, Ms45 gene exon, Ms45 gene intron, Ms45 gene 3'UTR and Ms45 gene terminator;
  • the Ms45 gene expression element is a DNA molecule shown in 1) or 2) or 3) as follows:
  • the coding region includes the DNA molecule of SEQ ID NO:1;
  • the second nucleotide sequence includes a gene for controlling the color of a plant or controlling the color of a plant kernel or controlling the degree of plant wilting;
  • the second nucleotide sequence when the second nucleotide sequence exists in a heterozygous state in the second plant, it affects the color of the second plant or the color of the grain or the degree of plant wilting:
  • the above-mentioned gene controlling plant color or controlling the color of plant grain is the key gene Lc for anthocyanin synthesis; the above-mentioned key gene Lc for anthocyanin synthesis expresses the protein LC in the second plant.
  • the second nucleotide sequence exists in a heterozygous state in the second plant, it affects whether the seed or plant color of the second plant is purple; if the second nucleotide sequence is contained, the color of the seed or plant is purple , Does not contain, the color of the seed or plant is not purple;
  • the above-mentioned gene for controlling plant color is the gene Oy1 related to chlorophyll synthesis; the above-mentioned Oy1 gene expresses the protein Oy1 in the second plant.
  • the second nucleotide sequence exists in a heterozygous state in the second plant, it affects whether the plant color of the second plant is yellow; if the second nucleotide sequence is contained, the plant color is yellow, and does not contain, The plant color is not yellow;
  • the above-mentioned gene for controlling the wilting degree of a plant is the Wi2 gene in the embodiment of the present invention; the above-mentioned Wi2 gene expresses the protein Wi2 in the second plant.
  • the second nucleotide sequence When the second nucleotide sequence is present in the second plant in a heterozygous state, it affects whether the plant of the second plant is wilting; if the second nucleotide sequence is contained, the plant is wilting, and if it does not, the plant is Not wilting.
  • the above-mentioned protein LC is the following a) or b):
  • the protein Oy1 is as follows a) or b):
  • the protein Wi2 is the following a) or b):
  • the gene Lc is a DNA molecule shown in 1) or 2) or 3) as follows:
  • the coding region includes the DNA molecule of SEQ ID NO: 4;
  • the gene Oy1 is a DNA molecule shown in 1) or 2) or 3) as follows:
  • the coding region includes the DNA molecule of SEQ ID NO: 6;
  • the gene Wi2 is a DNA molecule shown in 1) or 2) or 3) as follows:
  • the coding region includes the DNA molecule of SEQ ID NO: 8;
  • the present invention relates to nucleotide sequences for controlling male fertility, controlling plant or seed color, and controlling plant wilting, and a method for expanding male sterility of plants developed by using these nucleotide sequences and transgenic technology.
  • One purpose is to provide an efficient seed marking method, which can be used to multiply male sterile seeds of plants, save manpower for hybrid seed production, reduce costs, and ensure seed purity.
  • Another object is to provide a method for efficiently distinguishing fertile grains from sterile grains by the color of plants or seeds or the degree of plant wilting.
  • Another object is to provide a transgenic plant that can maintain the sterility of male sterile plants.
  • Another object of the present invention is to provide a DNA construct.
  • the construct provided by the present invention is the construct described in the above method.
  • the above-mentioned DNA construct can restore the fertility of the male sterile mutant, and at the same time change the color of the plant or the color of the seed of the plant or change the degree of plant wilting.
  • the present invention constructs a plant expression vector containing the Ms45 gene expression element shown in SEQ ID NO: 3, and transfers the vector into the male sterility mutant ms45 to restore the fertility of the mutant.
  • the present invention constructs a vector for controlling corn anthocyanin synthesis gene Lc (Ludwig et al. 1993).
  • the vector can affect the synthesis of anthocyanins, and overexpression of this gene makes plants or grains appear purple.
  • the invention constructs a vector for regulating the Oy1 gene of chloroplast synthesis. This vector affects chloroplast synthesis, and overexpression of this gene makes the plant yellow.
  • the invention constructs a vector for regulating and controlling the gene Wi2 related to the synthesis of root cork. This vector affects water transport, and overexpression of this gene causes plants to wilt (top leaves wilt).
  • the third object of the present invention is to provide any of the following substances.
  • the above-mentioned second plant can maintain the sterility of the male sterile plant.
  • the above-mentioned plant can be all or part of the plant, such as root, stem, leaf, embryo, root tip, pollen or anther.
  • a key of the present invention is to construct the male restorer gene Ms45, control plant color or seed color or control plant wilting, etc., into a vector, which can restore the fertility of the male sterile mutant ms45. At the same time, make the color of the plant containing the transgenic sequence the desired color or the desired state of plant wilting, that is, to mark the plant or grain containing the restorer gene to distinguish between fertile individuals (maintainer lines) and sterile individuals (sterile lines) .
  • the inventors used the control of plant male fertility, overexpression of the nucleotide sequence of the anthocyanin synthesis-related gene Lc, and transgenic technology to invent a highly efficient propagation of plant males.
  • the genes that control the male fertility of plants include the male sterility genes listed in Table 1 and other male sterility genes as well as the male sterility genes of other species.
  • the inventors first constructed a plant transformation vector that contained expression elements for restoring male fertility genes and expression elements for overexpressing anthocyanin synthesis-related genes Lc, and transferred the vector into HiIIA ⁇ HiIIB corn hybrids. Under normal conditions of hybrids, the plants are green.
  • the male sterile plants are then used to backcross the obtained transgenic plants, so that the nucleotide sequence of the Ms45 restorer gene for controlling the male fertility of the plant and the control plant or seed color (Lc gene) is introduced into the male sterile plant ms45. Due to the presence of the restorer gene Ms45, the transgenic plants appear to be fertile.
  • transgenic heterozygous plants When transgenic heterozygous plants (Msmsms) are crossed with male sterile plants (msms), the following two progeny will be produced, one is a male sterile individual with a green plant and a yellow seed (the sterile line, the genotype is msms), Male sterile normal grains that do not contain transgenic sequences (sterile line ms45ms45), the sterile line can be restored to fertility by any wild-type plant, and can be used as a sterile line during seed production; the other is a purple plant Or the fertile grain of the seed (maintainer line, genotype Msmsms), the maintainer line is recessively homozygous at the site of male fertility control. Because it contains complementary transgenic sequences, the plant appears to be fertile, and because it contains A nucleotide sequence that controls the color of a plant or seed (anthocyanin synthesis gene), which is different from the wild type.
  • the inventors used the nucleotide sequence of controlling plant male fertility, overexpression of the anthocyanin synthesis-related gene Oy1, and transgenic technology to invent a highly efficient multiplication plant male sterile A new method of breeding.
  • the genes that control the male fertility of plants include the male sterility genes listed in Table 1 and other male sterility genes as well as the male sterility genes of other species.
  • the inventors first constructed a plant transformation vector, which contains the expression element for restoring male fertility genes and the expression element for overexpressing the chlorophyll synthesis-related gene Oy1, and transferred the vector into the HiIIA ⁇ HiIIB corn hybrid. Under normal conditions, the plant is green.
  • the male sterile plants are used to backcross the obtained transgenic plants, so that the nucleotide sequence of the Ms45 restorer gene for controlling the male fertility of the plant and the plant color (Oy1 gene) is introduced into the male sterile plant ms45. Due to the presence of the restorer gene Ms45, the transgenic plants appear to be fertile. When the transgenic heterozygous plant (Msmsms) is crossed with the male sterile plant (msms), the following two progeny will be produced.
  • Msmsms transgenic heterozygous plant
  • msms male sterile plant
  • sterile line the genotype is msms
  • ms45ms45 the sterile line
  • the sterile line can be restored to fertility by any wild-type plant, and can be used as a sterile line during seed production;
  • the other is the fertile yellow plant Grains (maintainer line, genotype Msmsms), the maintainer line is recessively homozygous at the site of male fertility control, because it contains complementary transgenic sequences, the plant appears to be fertile, because it also contains control plants or seed color
  • the nucleotide sequence of the gene related to chloroplast synthesis, the color of the plant is different from the wild type.
  • the inventors used the nucleotide sequence of the gene Wi2 related to the control of plant male fertility, overexpression and regulation of root cork synthesis and transgenic technology to invent a highly efficient multiplication plant A new method of male sterility.
  • the genes that control the male fertility of plants include the male sterility genes listed in Table 1 and other male sterility genes as well as the male sterility genes of other species.
  • the inventors first constructed a plant transformation vector, which contains the expression element of the gene to restore male fertility and the expression element of the gene Wi2 related to overexpression and regulation of root cork synthesis, and transferred the vector into HiIIA ⁇ HiIIB corn hybrids , Under normal conditions of this hybrid, the plants are in a non-wilting state. Then, the male sterile plants are used to backcross the obtained transgenic plants, so that the nucleotide sequence that controls the plant male fertility Ms45 restorer gene and regulates root cork synthesis (Wi2 gene) is introduced into the male sterile plant ms45. Due to the presence of the restorer gene Ms45, the transgenic plants appear to be fertile.
  • the transgenic heterozygous plant (Msmsms) is crossed with the male sterile plant (msms)
  • the following two progeny will be produced, one is a non-wilting state (or the degree of wilting is lower than the maintainer line) of male sterile individuals (sterile lines)
  • the genotype is msms
  • male sterile normal grains without transgenic sequences (sterile line ms45ms45)
  • the sterile line can be restored to fertility by any wild-type plant, and can be used as a sterile line during seed production ;
  • the other is a fertile grain with wilted leaves of the plant (maintainer line, genotype Msmsms), which is recessively homozygous at the site of male fertility control, and because it contains complementary transgenic sequences, the plant behaves as It is fertile, because it also contains the nucleotide sequence that controls the color of the plant or seed (anthocyanin synthesis gene),
  • the experiment of the present invention proves that the method provided by the present invention can multiply plant nuclear male sterile seeds and bring convenience to hybrid seed production.
  • the system mainly uses a nucleotide sequence that can distinguish the color of plants or seeds or regulate plant wilting and a wild-type allele of a nuclear male sterility gene and transgenic technology.
  • Transgenic seeds can be distinguished by color
  • transgenic plants can be distinguished by color
  • plants can be distinguished by the degree of wilting.
  • the application provides a method for multiplying male sterile plants and maintainer plants for multiplying male sterile plants. After the maintainer plants provided in the present application are crossed with the male sterile plants, they can be harvested at the same time and effectively distinguish the progeny of the male sterile plants and the maintainer plants. Thus, the method and maintainer plants provided in the present application can realize the efficient expansion of male sterile plants and improve the breeding efficiency. In addition, through the combined use of the extrinsic traits of seeds and the extrinsic traits of plants, the method provided in this application can screen the hybrid progeny two or more times, which further improves the progeny and progeny of male sterile plants. Maintaining the respective purity of the progeny of the line plant improves the breeding efficiency and the quality and purity of the hybrid seeds produced.
  • the method of the present application combines the marking and screening of seeds and plants, and can determine sterile plants and fertile plants after sowing and before dispersing powder (such as seedling stage), remove mixed fertile plants in time, and significantly improve breeding efficiency And the quality and purity of the hybrid seeds produced.
  • Figure 1 shows the male flower phenotypes of male sterile maize plants ms45 (A) and wild-type maize plants Ms45 (B).
  • Figure 2 shows the grains of the hybrid progeny that do not contain the transgenic element Ms45-Lc (A, the color of the grain is yellow) and the hybrid progeny grains that contain the transgenic element Ms45-Lc (B, the color of the seed is purple).
  • Figure 3 shows the hybrid seedlings (which are not purple) that do not contain the transgenic element Ms45-Lc and the hybrid seedlings (which are purple) that contain the transgenic element Ms45-Lc.
  • Figure 4 shows a schematic diagram of the pMs45-Lc vector structure.
  • Figure 5 shows the hybrid progeny plants containing the transgenic element Ms45-Lc (A, the plants are purple and are male fertile) and the hybrid progeny plants that do not contain the transgenic element Ms45-Lc (B, the plants are non-purple, and Are male sterile).
  • Figure 6 schematically shows the selection and breeding process of maintainer plants, sterile plants and their hybrid offspring.
  • Figure 7 shows the hybrid progeny plants containing the transgenic element Ms45-Oy1 (A, the plant is shown in yellow) and the hybrid progeny plants not containing the transgenic element Ms45-Oy1 (B, the plant is shown in green).
  • Figure 8 shows the hybrid progeny plants containing the transgenic element Ms45-Wi2 (A, the plant appears to be wilting) and the plants that do not contain the transgenic element Ms45-Wi2 hybrid (B, the plant appears to be normal).
  • the male sterility gene ms45 and its restorer gene Ms45 in Table 1 were used for experiments.
  • the male sterility restorer gene Ms45 is derived from the corn variety B73, and its nucleotide sequence is shown in SEQ ID NO: 1, and the amino acid sequence of the encoded Ms45 protein is shown in SEQ ID NO: 2.
  • B73 genomic DNA refers to the B73 genomic sequence (www.maizesequence.org)
  • design amplification primers to amplify the restorer gene Ms45 are as follows:
  • Ms45F 5'tgaattcTGCTGAGTTCTCCTTGGGTTATCC 3'(SEQ ID NO: 11),
  • Ms45R 5'tcccgggGGTTGCGCATGAAATAGGGGT 3'(SEQ ID NO: 12).
  • the designed upstream primer Ms45F contains an EcoRI restriction site at the 5'end
  • the downstream primer Ms45R contains a SmaI restriction site at the 5'end for amplification.
  • the amplification reaction system is: template DNA 2 ⁇ L, primer Ms45F 0.5 ⁇ L, primer Ms45R 0.5 ⁇ L, dNTP 1.6 ⁇ L, 10 ⁇ Buffer 2 ⁇ L, high-fidelity taq enzyme 0.3 ⁇ L, ddH 2 O 13.1 ⁇ L.
  • the reaction conditions were: denaturation at 95°C for 5 minutes; 32 cycles (denaturation at 95°C for 45s, annealing at 59°C for 45s, and extension at 72°C for 3 minutes); and extension at 72°C for 10 minutes.
  • the full length of the amplified target product is about 3500 bp. It is recovered and connected to the T-easy sequencing vector, and then used for transformation and sequencing. The sequencing results confirmed that the amplified product was a 3518 bp DNA fragment composed of EcoRI restriction site, SEQ ID NO: 3 and SmaI restriction site, which was used as the Ms45 gene construct.
  • the sequence shown in SEQ ID NO: 3 contains the promoter (SEQ ID NO: 15) of the Ms45 gene and the nucleotide sequence of the coding region.
  • the nucleotide sequence of the Lc gene is shown in SEQ ID NO: 4, and the amino acid sequence of the encoded Lc protein is shown in SEQ ID NO: 5.
  • Add NcoI restriction site and protective base to the 5'end of SEQ ID NO: 4 add BstEII restriction site and protective base to the 3'end, and artificially synthesize this sequence to use as a Lc gene construct.
  • the nucleotide sequence of the Oy1 gene is shown in SEQ ID NO: 6, and the amino acid sequence of the encoded Oy1 protein is shown in SEQ ID NO: 7. Add NcoI restriction site and protective base to the 5'end of SEQ ID NO: 6, add BstEII restriction site and protective base to the 3'end, and artificially synthesize this sequence to use as an Oy1 gene construct.
  • the Wi2 gene can regulate root cork, and its expression can affect the water transport of the plant and make it easier for the plant to wilt.
  • the nucleotide sequence of the Wi2 gene is shown in SEQ ID NO: 8, and the amino acid sequence of the coded Wi2 protein is shown in SEQ ID NO: 9.
  • the plasmid pCAMBAI3301 (International Agricultural Molecular Biology Application Center CAMBIA, Australia) was used to construct the following recombinant expression vector, which contains the selectable marker gene bar (the nucleotide sequence of which is shown in SEQ ID NO: 10).
  • the Lc gene construct was cloned between the BstEII and NcoI restriction sites of the pCAMBAI3301 plasmid, and the Ms45 gene construct was cloned into the EcoRI and SmaI restriction sites of the pCAMBAI3301 plasmid. between.
  • the constructed vector was named pMs45-Lc.
  • the schematic diagram of the structure of the vector is shown in Figure 4.
  • the vector contains the Ms45 gene, the Lc gene and the selectable marker gene bar of the vector itself.
  • the Oy1 gene construct was cloned between the BstEII and NcoI restriction sites of the pCAMBAI3301 plasmid, and the Ms45 gene construct was cloned into one of the EcoRI and SmaI restriction sites of the pCAMBAI3301 plasmid between.
  • the constructed vector was named pMs45-Oy1.
  • the vector includes Ms45 gene, Oy1 gene and the selection marker gene bar of the vector itself.
  • the Wi2 gene construct was cloned between the BstEII and NcoI restriction sites of the pCAMBAI3301 plasmid, and the Ms45 gene construct was cloned into one of the EcoRI and SmaI restriction sites of the pCAMBAI3301 plasmid between.
  • the constructed vector was named pMs45-Wi2.
  • the vector includes Ms45 gene, Wi2 gene and the selection marker gene bar of the vector itself.
  • the vectors pMs45-Lc, pMs45-Oy1 and pMs45-Wi2 obtained above were respectively transformed into Agrobacterium EHA105 to obtain recombinant Agrobacterium EHA105/pMs45-Lc, EHA105/pMs45-Oy1 and EHA105/pMs45-Wi2.
  • the vector pMs45-Oy1-CWI-2 (which carries the Ms45 gene, the Oy1 gene, the nucleotide (SEQ ID NO: 18) of the interfering RNA encoding the CWI-2 gene, and the vector itself was prepared by a method similar to the above
  • the selectable marker gene bar of the vector and pMs45-Lc-CWI-2 (it carries the Ms45 gene, the Lc gene, the nucleotide encoding the interfering RNA of the CWI-2 gene (SEQ ID NO: 18) and the selectable marker gene bar of the vector itself ), and prepared recombinant Agrobacterium EHA105/pMs45-Oy1-CWI-2 and EHA105/pMs45-Lc-CWI-2.
  • Example 2 Obtaining transgenic corn
  • HiIIA and HiIIB Planting corn varieties HiIIA and HiIIB (Armstrong C L, Green C E and Phillips R L. Development and availability of germplasm with high Type II culture formation response. Maize Genetics Cooperation News Letter, 1991, 65: 92-93) in the field, to When dispersing pollen, they are separately bagged, pollinated, and hybridized. Two hybridization methods are used: HiIIA as the female parent and HiIIB as the male parent; or, HiIIA as the male parent and HiIIB as the female parent.
  • the pollen of the T0 transgenic plants was used to pollinate the female parent HiIIA and HiIIB and ms45 male sterile materials (obtained from Maize Genetics Cooperation Stock Center, 905I), and observe the phenotype.
  • EHA105/pMs45-Lc, EHA105/pMs45-Oy1 and EHA105/pMs45-Wi2 were cultured on YEP (containing Kana33mg/L and Str100mg/L antibiotics) medium one week in advance.
  • the selection medium contains 1.5 mg/L of bialaphos . After subculturing two weeks later, the concentration of bialaphos can rise to 3mg/L.
  • the T0 generation transgenic pMs45-Lc corn, pMs45-Oy1 corn and pMs45-Wi2 corn and their progeny were evaluated for the overall plant morphology (for example, pollen, plant and kernel phenotype).
  • the above-mentioned transgenic corn was crossed with ms45 homozygous recessive male sterile material (obtained from Maize Genetics Cooperation Stock Center, 905I) to obtain hybrid offspring.
  • the bar gene is detected to determine whether the hybrid offspring contains the transgenic elements Ms45-Lc, Ms45-Oy1 or Ms45-Wi2.
  • the bar gene detection method is: PCR amplify the genome of the hybrid offspring with the following primers Bar669F (SEQ ID NO: 13) and Bar669R (SEQ ID NO: 14). If the amplified product contains a target fragment with a size of about 669 bp, then The hybrid offspring is the offspring that contains the transgenic element; if the amplified product does not contain the 669bp target fragment, the hybrid offspring is the hybrid offspring that does not contain the transgenic element.
  • Bar669F 5'TCTCGGTGACGGGCAGGAC 3'(SEQ ID NO: 13);
  • Bar669R 5'TGACGCACAATCCCACTATCCTT 3'(SEQ ID NO: 14).
  • the hybrid offspring containing the transgenic element Ms45-Lc, the hybrid offspring containing the transgenic element Ms45-Oy1 and the hybrid offspring containing the transgenic element Ms45-Wi2 were obtained respectively.
  • the plant color of the hybrid offspring is shown in Figure 5, among which the hybrid offspring containing the transgenic element Ms45-Lc is shown in Figure 5A, and the plant appears purple (test fertility); and The hybrid progeny of Ms45-Lc without transgenic elements is shown in Figure 5B, and the plants appear to be non-purple (test for sterility).
  • the results of the seed color of the hybrid offspring are shown in Figure 2.
  • Figure 2B is the hybrid offspring grain containing the transgenic element Ms45-Lc, the color of the grain is purple (test fertility)
  • Figure 2A is the hybrid offspring grain without the transgenic element Ms45-Lc , The color of the grain is yellow (not purple, sterility detection).
  • the results of the seedling color of the hybrid offspring are shown in Figure 3. Among them, the hybrid offspring seedlings containing the transgenic element Ms45-Lc are purple (test fertility), and the hybrid offspring seedlings that do not contain the transgenic element Ms45-Lc are non-purple (test sterile ).
  • the color of the hybrid offspring plants is shown in Figure 7.
  • the hybrid offspring containing the transgenic element Ms45-Oy1 is shown in Figure 7A, and the plants appear yellow (test fertility);
  • the hybrid progeny containing the transgenic element Ms45-Oy1 is shown in Figure 7B, and the plants appear green (test for sterility).
  • the degree of wilting of the hybrid offspring plants is shown in Figure 8.
  • the hybrid offspring containing the transgenic element Ms45-Wi2 is shown in Figure 8A, and the plants appear to be wilting (test fertility); and The plants that do not contain the transgenic element Ms45-Wi2 hybrid progeny are shown in Figure 8B, and the plants appear to be normal (test for sterility).
  • MS salt was purchased from Phyto Technology Laboratories, and the product number is M524.
  • transgenic pMs45-Oy1-CWI-2 corn (which contains transgenic elements).
  • Ms45-Oy1-CWI-2) and transgenic pMs45-Lc-CWI-2 corn which contains the transgenic element Ms45-Lc-CWI-2).
  • the homozygous recessive mutant of ms45 (Maize Genetics Cooperation Stock Center, 905I) was used as the female parent and crossed with Zheng 58 (Grain Planting Institute, Henan Academy of Agricultural Sciences), and the obtained F1 continued to backcross with the maize inbred line Zheng 58. Genotype analysis was performed on the obtained BC1 (backcross generation) population, and the plants that were heterozygous at the Ms45 locus were screened to continue backcrossing with Zheng 58.
  • Ms45ms45 ms45 homozygous recessive inbred line Zheng 58
  • the above method for screening the genotype of the Ms45 locus is as follows: PCR amplification of the plant genome is performed with the following primers Ms45F1 (SEQ ID NO: 16) and Ms45R1 (SEQ ID NO: 17), and the amplification results are sequenced.
  • the target fragment size of Ms45 is 859bp
  • the target fragment size of ms45 is 811bp.
  • the genotype of the locus is heterozygous Ms45/ms45; if the amplified product does not contain the 811bp fragment, the genotype of the locus is Ms45/Ms45 dominantly pure If the amplified product does not contain the 859bp target fragment, the genotype of the locus is ms45/ms45 recessive homozygous.
  • Ms45F1 5'CTTGAGCGACAGCGGGAACT 3'(SEQ ID NO: 16);
  • Ms45R1 5'TGTTGTTTCTTGGCAAAGGTCAG 3'(SEQ ID NO: 17).
  • Example 2 The T0 generation in Example 2 was transformed into pMs45-Lc maize as the male parent, and crossed with Zheng 58 (ms45ms45) as the female parent obtained above, and the purple seeds were selected from the hybrid progeny to be sown in the field and then sprayed with 200 mM diprofen .
  • Zheng 58 Zheng 58
  • the purple seeds and plants are always selected to cross with the female parent.
  • the transgenic sites (Ms45-Lc) of the purple seeds and plants in the offspring are all heterozygous. Use purple grains and pollen from plants to pollinate the female parent.
  • the transgenic site Ms45-lc of the pollen-providing plant is heterozygous and ms45 The points are invisible homozygous, that is, maintainer plants.
  • Example 2 The T0 generation in Example 2 was transformed into pMs45-Oy1 maize as the male parent, and crossed with Zheng 58 (ms45ms45) as the female parent obtained above. Yellow plants were selected from the hybrid progeny to be sown in the field and sprayed with 200 mM biprofen . Continue backcrossing the surviving plants with the female parent Zheng 58. In the process of backcrossing, the yellow plants are always selected to cross with the female parent. After 5-6 generations of backcrossing, the transgenic sites (Ms45-Oy1) of the yellow plants in the offspring are all heterozygous. Use the pollen of yellow plants to pollinate the female parent. If the normal plants (green plants) obtained are all sterile, the transgenic site Ms45-Oy1 of the pollen-providing plant is heterozygous and the ms45 site is invisible homozygous, that is, maintaining Department of plants.
  • Example 2 The T0 generation in Example 2 was transformed into pMs45-Wi2 maize as the male parent and crossed with Zheng 58 (ms45ms45) as the female parent obtained above, and wilted plants were selected from the hybrid offspring (the degree of wilting is shown in Figure 8A, the main performance is (Curl the heart leaf) after sowing to the field, spray 200mM bialaphos. Continue backcrossing the surviving plants with the female parent. In the process of backcrossing, wilting plants are always selected to cross with the female parent. After 5-6 generations of backcrossing, the transgenic sites (Ms45-Wi2) of wilting plants in the generations are all heterozygous.
  • the obtained maintainer plants (Ms45-Lc heterozygous and ms45ms45) were used as the male parent and crossed with Zheng 58 as the female parent.
  • the progeny produced not only had male sterile lines (ms45ms45), but also maintained lines (Ms45- Lc heterozygous and ms45ms45).
  • the grains of the male sterile line (ms45ms45) appeared normal, while the grains of the maintainer line (Ms45-Lc heterozygous and ms45ms45) were purple (the grains shown in B in Figure 2). See Figure 6 for the specific breeding process.
  • the obtained maintainer plants (Ms45-Oy1 heterozygous and ms45ms45) were used as the male parent and crossed with Zheng 58 as the female parent.
  • the progeny produced not only had male sterile lines (ms45ms45), but also maintained lines (Ms45- Oy1 heterozygous and ms45ms45).
  • the male sterile line plants (ms45ms45) were normal and showed green (shown in Figure 7B), while the maintainer lines (Ms45-Oy1 heterozygous and ms45ms45) plants were yellow.
  • the obtained maintainer plants (Ms45-Wi2 heterozygous and ms45 homozygous) were used as the male parent and crossed with Zheng 58 as the female parent.
  • the offspring produced not only had male sterile lines (ms45ms45), but also maintained lines ( Ms45-Wi2 heterozygous and ms45ms45).
  • the male sterile plants (ms45ms45) were normal (shown in Fig. 8B), while the maintainers (Ms45-Wi2 heterozygous and ms45ms45) plants showed a wilting state.
  • the obtained male sterile line Zheng 58 (ms45ms45) and the maintainer line obtained above (Ms45-Lc heterozygous and ms45ms45, Ms45-Oy1 heterozygous and ms45ms45 and Ms45-Wi2 heterozygous And ms45ms45) for seeding.
  • the two materials are sown separately, and each row is planted with 1 row of maintainer lines and 5 rows of sterile lines are planted. Ensure that no other corn is sown within 300 meters of the breeding, so that the sterile lines and maintainer lines are naturally pollinated in the field.
  • Maintainers can only accept their own pollen and will produce two kinds of offspring.
  • One is the offspring that exhibit the external traits of the transgenic element (for example, purple grains and purple plants, yellow plants, wilting plants).
  • the transgenic elements of these offspring may be homozygous or heterozygous, which is difficult to distinguish. Therefore, these grains or plants are discarded.
  • the second offspring with normal external traits does not contain transgenic elements and can be used as the offspring of the sterile line and retained.
  • the sterile line material receives the pollen from the maintainer line and produces two kinds of offspring.
  • One is the offspring that exhibit the external traits of the transgenic elements (for example, purple grains and purple plants, yellow plants, wilting plants).
  • the transgenic elements of these offspring are heterozygous and can be used as maintainer offspring and retained.
  • the other is the offspring with normal external traits (for example, yellow kernels, green plants, non-wilting plants), which does not contain transgenic elements, and can be used as the offspring of sterile lines and retained.
  • Maintainer lines can be used for the next year to continue to propagate sterile lines and maintainer lines, most of the sterile lines are used for the production of commercial seeds, and the remaining small part is used for the next year to continue to propagate sterile lines and maintainer lines.
  • the production process is shown in Figure 6.
  • maintainer plants heterozygous for Ms45-Oy1-CWI-2 and homozygous for ms45 were obtained. Both can be used for large-scale expansion of male sterile lines (ms45ms45).
  • the sterile line produced in Example 3 is a recessive homozygous sterile line controlled by the nucleus, and the sterile line can be restored to fertility by any wild-type plant (Ms45Ms45). Therefore, as long as selecting an inbred line (for example, Chang 7-2) with high combining ability with a male sterile line (for example, Zheng 58) for hybridization, a hybrid with excellent agronomic traits can be produced.
  • an inbred line for example, Chang 7-2
  • a male sterile line for example, Zheng 58
  • Zheng 58 and Chang 7-2 were planted alternately in the field, and no other corn was planted within 300 meters of the breeding ground.
  • the ears of the sterile line can only accept the pollen of the wild-type inbred line, and the wild-type inbred line can only be selfed. In this way, the seeds produced on the ears of the sterile line are both dominant hybrids.
  • Example 5 Quality assessment of breeding hybrids of sterile lines
  • the Lc gene was used to double-mark the plant (seed color and plant color), which solved the problem caused by heteromale fertilization and significantly improved the seed purity of the hybrids produced (to 100%) .
  • the maintainer plants (transgenic pMs45-Lc/ms45ms45 corn) and the sterile line plants (ms45ms45 corn) obtained in the above examples were sown separately, and each row of the maintainer line was sown with 5 rows of sterile lines to ensure No other corn is sown within 300 meters around the breeding, allowing the sterile line plants and maintainer lines to be pollinated naturally in the field.
  • the progeny seeds of the sterile line plants are collected, and the first screening is performed according to the color of the seeds, that is, the progeny of the sterile line (the seeds are yellow) and the progeny of the maintainer line (the seeds are purple) are distinguished.
  • the obtained sterile line seeds (as the female parent) and the target corn line seeds (as the male parent, Chang 7-2 inbred line) are sown separately, and each line of the target corn line seeds is sown, and the corresponding sowing is 5 lines. Breed seeds and ensure that no other corn is sown within 300 meters of the breeding.
  • the external characteristics of the sterile line plants were observed, and the plants showing the purple plant color were removed from the sterile line plants. According to statistics, 290 purple seedlings out of 100,000 sterile line plants were removed, that is, the purity of the expanded sterile line was 99.71%.
  • the sterile line plants and the target corn line plants are naturally pollinated in the field. Collect the hybrid seeds produced on the sterile plants. Then, the produced hybrid seeds were sown in the field, and 10,000 plants were randomly selected for genetic testing to determine the proportion of hybrid seeds containing genetically modified components (pMs45-Lc) derived from the maintainer line, and to evaluate the quality of hybrid seeds (purity) ). Experimental results showed that after double screening using seed color and plant color, the purity of the hybrid seeds produced reached 100%, that is, all hybrid seeds did not contain genetically modified components (pMs45-Lc) derived from the maintainer line.
  • the male sterile maintainer line of the present invention with the aid of double screening (seed screening and seedling stage screening), 100% of the progeny sterile line plants and the progeny maintainer plants can be achieved at the seedling stage.
  • the male sterile maintainer line and seed breeding method of the present invention can be used to produce high-purity sterile line offspring seeds and high-purity hybrid seeds.
  • the inventors used the two traits of grain size (interfering RNA of the CWI-2 gene) and plant color (Oy1 gene) to mark the plants, thus, during the seed production process, it is sufficient at the seedling stage.
  • Remove the fertile maintainer lines ie, yellow seedlings; about 3 ⁇
  • heteromale sterile line in the plot of female parent sterile lines ie, yellow seedlings; about 3 ⁇
  • the Ms45 gene (SEQ ID NO: 1), Oy1 gene (SEQ ID NO: 6), and the nucleotides encoding the interfering RNA of CWI-2 gene (SEQ ID NO: 18) was constructed into the vector and prepared to obtain maintainer plants, namely transgenic corn plants pMs45-Oy1-CWI-2/ms45ms45, which can express Ms45 protein, Oy1 protein, and interfering RNA that inhibits CWI-2 gene.
  • the progeny seeds of the sterile line plants are collected, and the first screening is performed according to the size of the seeds, that is, the progeny of the sterile line (seed size is normal) and the progeny of the maintainer line (seed size is small) are distinguished.
  • the obtained sterile line seeds (as the female parent, the seed size is normal) and the target corn line seeds (as the male parent, Chang 7-2 inbred line, the seed size is normal) are sown separately, and each seeding line is one line of target For corn line seeds, 5 rows of sterile line seeds should be sown accordingly, and no other corn seeds should be sown within 300 meters of the breeding area.
  • the external characteristics of the sterile line plants were observed, and the plants showing the yellow plant color were removed from the sterile line plants. According to statistics, 310 yellow seedlings out of 100,000 sterile line plants were removed, that is, the purity of the expanded sterile line was 99.69%.
  • the sterile line plants and the target corn line plants are naturally pollinated in the field. Collect the hybrid seeds produced on the sterile plants. Then, the produced hybrid seeds were sown in the field, and 10,000 plants were randomly selected for genetic testing to determine the proportion of hybrid seeds containing genetically modified components derived from the maintainer line (pMs45-Oy1-CWI-2), and evaluate the hybrid seeds The quality (purity).
  • the experimental results showed that after double screening using seed color and plant color, the purity of hybrid seeds produced reached 100%, that is, all hybrid seeds did not contain genetically modified components derived from the maintainer (pMs45-Oy1-CWI-2 ).
  • the male sterile maintainer line of the present invention with the aid of double screening (seed screening and seedling stage screening), 100% of the progeny sterile line plants and the progeny maintainer plants can be achieved at the seedling stage.
  • the male sterile maintainer line and seed breeding method of the present invention can be used to produce high-purity sterile line offspring seeds and high-purity hybrid seeds.
  • Example 7 Quality evaluation of breeding hybrids of sterile lines
  • the inventors used the two traits of grain size (interfering RNA of CWI-2 gene) and plant color (Lc gene) to mark the plants, thus, during the seed production process, the seedling stage Remove the fertile maintainer lines (i.e., purple seedlings; about 3 ⁇ ) produced by heteromale fertilization in the female parent sterile line plot, so as to ensure 100% male sterility of all female parents during the pollen period, and then Improve and ensure the purity of the produced hybrids, which satisfies the production requirements well.
  • the seedling stage Remove the fertile maintainer lines (i.e., purple seedlings; about 3 ⁇ ) produced by heteromale fertilization in the female parent sterile line plot, so as to ensure 100% male sterility of all female parents during the pollen period, and then Improve and ensure the purity of the produced hybrids, which satisfies the production requirements well.
  • the Ms45 gene (SEQ ID NO: 1), Lc gene (SEQ ID NO: 4), and the nucleotides encoding the interfering RNA of CWI-2 gene (SEQ ID NO: 18) was constructed into the vector, and the maintainer plant was prepared, namely the transgenic corn plant pMs45-Lc-CWI-2/ms45ms45 corn, which can express Ms45 protein, Lc protein, and interfering RNA that inhibits CWI-2 gene .
  • the obtained maintainer plants (pMs45-Lc-CWI-2/ms45ms45 corn) and the sterile line plants (ms45ms45 corn) are sown separately, and each row of the maintainer line corresponds to 5 rows of sterile lines to ensure that the breeding area is 300 No other corn is sown in the rice, and the sterile line plants and maintainer line plants are naturally pollinated in the field.
  • the progeny seeds of the sterile line plants are collected, and the first screening is performed according to the size of the seeds, that is, the progeny of the sterile line (seed size is normal) and the progeny of the maintainer line (seed size is small) are distinguished.
  • the obtained sterile line seeds (as the female parent, the seed size is normal) and the target corn line seeds (as the male parent, Chang 7-2 inbred line, the seed size is normal) are sown separately, and each seeding line is one line of target For corn line seeds, 5 rows of sterile line seeds should be sown accordingly, and no other corn seeds should be sown within 300 meters of the breeding area.
  • the external characteristics of the sterile line plants were observed, and the plants showing the purple plant color were removed from the sterile line plants. According to statistics, 305 purple seedlings out of 100,000 sterile line plants were removed, that is, the purity of the expanded sterile line was 99.695%.
  • the sterile line plants and the target corn line plants are naturally pollinated in the field. Collect the hybrid seeds produced on the sterile plants. Then, the produced hybrid seeds were sown in the field, and 10,000 plants were randomly selected for genetic testing to determine the proportion of hybrid seeds containing genetically modified components derived from the maintainer line (pMs45-Lc-CWI-2), and to evaluate the hybrid seeds The quality (purity).
  • the experimental results showed that after double screening using seed color and plant color, the purity of the hybrid seeds produced reached 100%, that is, all hybrid seeds did not contain genetically modified components derived from the maintainer (pMs45-Lc-CWI-2 ).
  • the male sterile maintainer line of the present invention with the aid of double screening (seed screening and seedling stage screening), 100% of the progeny sterile line plants and the progeny maintainer plants can be achieved at the seedling stage.
  • the male sterile maintainer line and seed breeding method of the present invention can be used to produce high-purity sterile line offspring seeds and high-purity hybrid seeds.

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Abstract

提供了一种雄性不育保持系植物，产生所述雄性不育保持系植物的方法，以及所述植物用于扩繁雄性不育系植物和雄性不育保持系植物的用途。此外，还提供了用于产生所述雄性不育保持系植物的核酸分子、载体和宿主细胞。

Description

一种雄性不育保持系植物及其用途 技术领域
本申请属于植物遗传育种及种子生产领域。具体而言，本申请涉及一种雄性不育保持系植物，产生所述雄性不育保持系植物的方法，以及所述植物用于扩繁雄性不育系植物和雄性不育保持系植物的用途。此外，本发明还涉及用于产生所述雄性不育保持系植物的核酸分子、载体和宿主细胞(例如，农杆菌)。
背景技术
杂种优势使得杂交种的生物量、抗病虫能力、耐胁迫(干旱、高温、低温、盐碱等)能力较其双亲有相当的提升。例如，杂交玉米、杂交水稻的产量远远高于其纯合的双亲。生产杂交种通常采用的方法为：将雌性亲本和雄性亲本种在一起；并将雌性亲本的雄穗去除，保留雄性亲本的雄穗；从雌性亲本收获种子，即为杂交种。
自然界中的植物存在自花授粉、异花授粉及常异花授粉三种类型。自花授粉是指一株植物的花粉，对自身的雌蕊进行授粉的现象。在两性花的植物中，又可分为同花授粉(例如，菜豆属)、邻花授粉和同株异花授粉。同花授粉为同一花的雄蕊与雌蕊之间进行授粉。邻花授粉为在一个花序(个体)中不同花之间进行授粉。同株异花授粉为同株不同花之间进行授粉。有些植物雄蕊和雌蕊不长在同一朵花里，有些甚至不长在同一棵植株上，无法自花授粉，它们的雌蕊只能得到其他花的花粉，这叫做异花授粉。将天然杂交率高于50％且自交衰退的一类作物归为常异花授粉作物。
玉米为雌雄同株，并且雌花和雄花位于植株的不同部位。玉米既可通过自花授粉也可通过异花授粉繁衍后代。在自然条件下，当风将花粉从雄穗吹到雌穗的花丝上时即完成了天然授粉。在玉米育种中，通常先开发出纯合的玉米自交系，然后将两个自交系进行杂交，对杂交的后代的产量、抗逆性等进行评估，以确定其是否具有商业化潜力。其中，每个自交系都可能具有另一个自交系所缺乏的一种或多种优良性状，或补充另一个自交系的一种或多种不良性状。两个自交系杂交的第一代种子为F1代种子，F1代的种子发芽后获得F1代植株，F1代植株较两个自交系亲本(父母本)更健壮，同时拥有更多的生物量。
可以通过对母本人工去雄来生产杂交种(F1)，即将未散粉的母本(其可与父本在田间间隔播种，如播种5行母本，一行父本)雄穗去除，保留父本雄穗。随后，只要对外来玉米花粉进行隔离，母本雌穗就只能接受父本的花粉，得到的种子即为杂交种(F1)，该杂交 种即可用于农业生产。然而，在生产杂交种的实际过程中由于环境的变化可能导致去雄完成后植株又雄穗化，或者去雄不完全。以上两种情况均能导致母本自交授粉，致使生产的杂交种中混杂了母本自交系的种子。而母本自交系的产量远低于杂交种的产量，这样的种子为不合格产品，既会影响农民收入又会影响制种公司信誉，甚至导致制种公司承担相应的赔偿责任。也可以采用机器对母本进行去雄。机器去雄和手工去雄的可靠性基本相同，但速度更快并且成本更低。然而，较之手工去雄，大多数去雄机器会对植株造成更大的破坏。因此，目前没有令人满意的去雄手段。人们仍在寻找成本更低、去雄更彻底的替换方法。
稳定的雄性不育系统提供了简单、高效的去雄手段。通过使用雄性不育系统，在一些情况下可以避免繁重的去雄工作。该手段包括三个主要材料，即(1)雄性不育系(也简称为不育系)：其为雄性不育材料；(2)雄性不育保持系(也简称为保持系)：其可以为不育系提供花粉，使不育系的后代仍为不育系；(3)雄性不育恢复系(也简称为恢复系)：其可以恢复不育系的育性。不育系与恢复系杂交产生F1，即为用于农业生产的杂交种。
植物的雄性不育可分为三种类型：细胞质雄性不育，细胞核雄性不育，以及核-质互作雄性不育。细胞质雄性不育植株表现为细胞质遗传，通常以单一的细胞质基因S和N分别代表雄性不育和雄性可育，其难以在农业生产上应用。细胞核雄性不育植株表现为细胞核遗传；在多数情况下，雄性不育性为一对隐性基因(msms)所控制，正常可育性为相对的显性基因(MsMs)或(Msms)所控制。核-质互作雄性不育(CMS)植株表现为核-质互作遗传。简言之，仅当细胞质具有不育基因S，且细胞核里有纯合的不育基因(rfrf)时，植株才表现为雄性不育。如果胞质含有可育基因N，则不论细胞核里的基因是可育基因(RfRf)还是不育基因(rfrf)，植株都表现为雄性可育。同样，如果核里具有可育基因(RfRf)或(Rfrf)，则不论胞质中的基因是可育基因N还是不育基因S，植株都表现为雄性可育。
已报道使用核-质互作雄性不育(CMS)材料来进行育种。在此类方法中，雄性不育系的遗传组成为S(rfrf)，其不能产生正常的花粉，但可作为杂交母本。保持系的遗传组成为N(rfrf)，其与所述雄性不育系杂交后所产生的F1仍能保持雄性不育，即：S(rfrf)(♀)×N(rfrf)→S(rfrf)(雄性不育)。恢复系的遗传组成为S(RfRf)或N(RfRf)，其与所述雄性不育系杂交后所产生的F1恢复为雄性可育，即：S(rfrf)(♀)×S(RfRf)→S(Rfrf)(F1)(可育)，或S(rfrf)(♀)×N(RfRf)→S(Rfrf)(F1)(可育)。得到的F1植株自交产生F2，F2在农业生产上也可以广泛应用。雄性不育系可以免除人工去雄，节约人力，降低种子成本，还可保证种子的纯度。目前水稻、玉米、高粱、洋葱、蓖麻、甜菜和油菜 等作物已经利用核质互作雄性不育系进行杂交种子的生产。其他作物的核-质互作雄性不育系也正在进行广泛的研究。然而，CMS系统也有它的缺陷：一是观察到个别CMS材料容易感病，二是恢复系比较难寻找。这些问题阻碍了CMS系统在制种中的广泛应用。
对于细胞核雄性不育系统而言，在多数情况下，控制雄性不育的核基因为隐性基因，其仅在纯合(msms)时，植株才会表现为雄性不育。但由于雄性不育植株无法自交，因此，只能通过用杂合植株(Msms)与其杂交，才能得到雄性不育后代植株(msms)。然而，杂合植株(Msms)与雄性不育植株(msms)的后代果穗上，雄性不育种子(msms)与可育的杂合种子(Msms)同时存在，并且，无法分辨哪些是不育的，哪些是可育的；只能在播种后，植株散粉时才可分辨。这限制了细胞核雄性不育系统在制种中的广泛应用。
近年来，已报道利用转基因的手段来保持雄性不育植株不育性的方法(US6743968)。该方法包括：首先构建一个转基因载体，该载体含有一个花粉细胞致死基因，以及一个恢复植株育性的显性基因；然后，将该载体转入雄性不育植株中，并且该载体在转基因植株中以杂合状态存在。由于恢复育性基因的存在，该转基因植株是雄性可育的。并且，当其与雄性不育植株杂交时，由于含有育性恢复基因的花粉(Msms)同时含有致死基因，因此，含有育性恢复基因的花粉都会败育。因此，该转基因植株只能产生不含恢复基因的花粉(ms)，与雄性不育植株雌配子(ms)进行杂交。所产生的后代均为隐性纯合个体(msms)。也即，当这样的植株与雄性不育植株杂交时，其后代都保持了隐性不育植株的纯合隐性状态。然而，上述方法的缺陷是，因异雄核受精现象(胚和胚乳由不同的雄配子体发育形成的精子受精形成，即形成胚和胚乳的精子基因型不同)的存在，对胚乳进行筛选后得到的后代种子中仍然存在着一定比例的可育种子，并且这些可育种子难以与不育种子区分开，不能完全满足实际生产的需要。
此外，还已报道，利用同时含有Ms45和mn1RNAi的载体来构建雄性不育保持系和保持雄性不育植株不育性的方法(中国专利ZL201210406155.6)。然而，由于异雄核受精等原因，通过对胚乳表型进行筛选获得的种子中，胚乳中不含转基因成分，胚中却含有转基因成分，使得这些植株可育，并且这些可育种子难以与不育种子区分开，只能等到植株散粉时才能确定，但散粉时再去除可育的植株将会对繁殖的杂交种纯度产生影响，不能完全满足实际生产的需要。因此，有必要建立一种播种后、散粉前确定不育株和可育株的方法，以便及时去除混杂的可育单株。
因此，如何改良细胞核雄性不育系统以方便地保持雄性不育系植株的不育性，以及如何获得高纯度的雄性不育后代是育种上急需解决的问题。本领域仍然需要开发更高效的繁育雄性不育系植株的方法。
发明内容
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、植物学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“分离的”是指经人工手段获得的状态，其不同于天然状态。例如，对于某一种从自然界“分离”的物质或成分，可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
如本文中所使用的，术语“外在性状”是指，个体(包括种子和植株)所表现出来的可遗传的、能观察到的表型特征，其通常由一对或多对等位基因控制。种子和植株的“外在性状”主要包括根、茎、叶、花、果实、种子的外部形态特征，例如种子颜色，种子尺寸，植株颜色，植株萎蔫程度，茎形态，叶形态等。通常，种子的外在性状主要是指种子的外部形态特征，包括例如，种子颜色或种子尺寸。植株的外在性状主要是指根、茎、叶、花、果实的外部形态特征，包括例如植株颜色或植株萎蔫程度。
如本文中所使用的，术语“雄性不育”是指植物的雄性细胞或组织丧失生理机能的现象。通常，在有性繁殖的植物中(例如，玉米)，雄性不育表现为雄性组织(例如，雄蕊)发育不正常，不能产生有正常功能的花粉，但雌性组织(例如，雌蕊)发育正常，能接受正常花粉而受精结实。如本文中所使用的，术语“雄性不育基因”是指能够控制植物雄性不育性状的基因。在本申请中，优选地，雄性不育基因是细胞核雄性不育基因。在多数情况下，控制雄性不育的核基因为隐性基因，其仅在纯合(msms)时，植株才会表现为雄性不育。例如，玉米雄性不育系郑58，其具有纯合的隐性雄性不育基因(ms45ms45)。在本申请中，特别优选地，雄性不育基因是隐性基因，其在纯合状态下导致植物雄性不育。
如本文中所使用的，术语“恢复基因”是指，能够恢复因雄性不育基因导致雄性不育的植物的雄性能育性的基因。当将该恢复基因导入雄性不育系植株时，所述植株将恢复雄性能育性。在本申请中，当雄性不育基因是隐性基因时，所述恢复基因可以是所述隐性基因的显性等位基因。
已报道了多种能够导致植物雄性不育的隐性基因以及其对应的能够恢复植物雄性能育性的显性等位基因。例如，已经在玉米中鉴定了多种隐性雄性不育基因以及其对应的显性等位恢复基因，包括但不限于，表1中所示的那些(Skibbe et al.2005)。
表1.玉米中的隐性雄性不育基因以及其对应的显性等位恢复基因
Figure PCTCN2020095082-appb-000001
Figure PCTCN2020095082-appb-000002
如本文中所使用的，术语“调控种子颜色的筛选基因”是指，能够通过影响色素、花青素等的合成而影响种子颜色的基因。此类基因包括例如，Lc基因(Ludwig SR等人，Proc Natl Acad Sci,1989Sep；86(18):7092-6；王娟等人，基因组学与应用生物学，2009年第02期)。Lc基因是一种与花青素合成相关的调节基因，它在多种植物(例如，玉米)中的异源表达可以影响花青素合成，增加花青素的含量，并影响种子的颜色。该基因的表达能够使玉米种子表现为紫色。另外，还已发现，Lc基因能够影响植株的颜色。该基因的表达能够使玉米植株表现为紫色。因此，Lc基因也是“调控植株颜色的筛选基因”。
如本文中所使用的，术语“调控种子尺寸的筛选基因”是指，其表达水平能够影响种子大小的基因。此类基因包括例如，CWI-2基因(W.H.Cheng等人，Plant Cell.1996 Jun；8(6):971–983)。CWI-2基因编码胚乳特异表达的细胞壁转化酶。该基因被沉默或突变后，将影响种子胚乳的正常发育，导致种子变小，但并不影响发芽率和植株发育。
如本文中所使用的，术语“调控植株颜色的筛选基因”是指，能够通过影响叶绿体、色素、花青素等的合成，而影响植株颜色的基因。此类基因包括例如，Oy1基因(Ruairidh J.H.Sawers等人，Plant Molecular Biology,volume 60,pages95-106(2006))。Oy1基因是一种 与叶绿体合成相关的调控基因，它能够通过影响叶绿体合成而影响植株(例如玉米植株)的颜色。该基因的表达能够使玉米植株表现为黄色。
如本文中所使用的，术语“调控植株萎蔫程度的筛选基因”是指，能够通过调控植物气孔开度、水分运输等，而影响植株萎蔫的基因。此类基因包括例如，Wi2基因(Chris Rock等人，American Journal of Botany,86(12):1796-800(2000))。Wi2基因是一种与植物根木栓质合成相关的调节基因，它能够通过影响植物水分运输而影响植物(例如玉米)的萎蔫程度。该基因的表达能够使玉米叶片萎蔫。
如本文中所使用的，术语“组织优选的启动子”是指，可优先地在某些植物组织(例如，雄蕊、花粉囊、花丝和花粉)中起始转录的启动子。如本文中所使用的，术语“生长期优选的启动子”是指，可优先地在某些生长发育阶段(例如，造孢组织、小孢子和小配子体)中起始转录的启动子。如本文中所使用的，术语“组织特异性启动子”是指，仅在在某些植物组织中特异性起始转录的启动子。
如本文中所使用的，术语“选择标记基因”是指，其编码产物能够使转化或转染的宿主细胞在选择压力下正常生长或表现出其他可视特征的基因。此类选择压力包括但不限于，选择剂(例如，抗生素或除草剂)的添加或营养的缺乏。如本文中所使用的，术语“抗生素抗性基因”是指，其编码产物能够使转化或转染的宿主细胞在抗生素的选择压力下正常生长或表现出其他可视特征的基因。如本文中所使用的，术语“除草剂抗性基因”是指，其编码产物能够使转化或转染的宿主细胞在除草剂的选择压力下正常生长或表现出其他可视特征的基因。
如本文中所使用的，术语“异雄核受精”是指，在双受精过程中，同一个种子的胚和胚乳由2个不同的雄配子体形成的精子受精而形成的现象。在这种情况下，同一个种子的胚和胚乳可具有不同的基因型。
在本申请中，植株可为植株的全部或部分，如根、茎、叶、胚、根尖、花粉或花药等。
本申请的发明人通过精心设计和大量实验，获得了一种能够保持雄性不育系植株的不育性的保持系植株，所述保持系植株在与雄性不育系植株杂交时，能够同时获得可区分的不育系和保持系后代，显著提升了不育系植株的扩繁效率和育种效率。基于此，本申请还提供了产生所述保持系植株的方法，以及利用所述保持系植株扩繁不育系植株的方法。此外，在本申请的某些优选实施方案中，所述保持系植株含有双重的筛选基因，并且，在将 所述保持系植株在与雄性不育系植株杂交后，通过双重筛选法可以更准确地区分不育系和保持系后代，进一步提高育种效率。由此，本申请的发明人完成了本发明。
因此，在本申请的第一方面，提供了一种分离的核酸分子，其包含第一多核苷酸和第二多核苷酸，所述第一多核苷酸包含恢复基因的核苷酸序列，所述恢复基因能够恢复因雄性不育基因导致雄性不育的植物的雄性能育性；所述第二多核苷酸包含能够调控种子和/或植株的外在性状的筛选基因的核苷酸序列，所述外在性状选自种子颜色，种子尺寸，植株颜色，植株萎蔫程度，茎形态，叶形态，以及其任何组合。在某些实施方案中，所述外在性状选自种子颜色，植株颜色，植株萎蔫程度，以及其任何组合。
在某些实施方案中，所述外在性状为种子的外在性状(例如种子颜色或种子尺寸)与植株的外在性状(例如植株颜色或植株萎蔫程度)的组合。在某些实施方案中，所述外在性状为种子颜色和植株颜色的组合。在某些实施方案中，所述外在性状为种子尺寸和植株颜色的组合。在某些实施方案中，所述外在性状为种子颜色和植株萎蔫程度的组合。在某些实施方案中，所述外在性状为种子尺寸和植株萎蔫程度的组合。
在本申请中，在某些情况下，两个或更多个外在性状的同时使用是优选和有利的。例如，在某些实施方案中，可以将调控种子的外在性状(例如种子颜色或种子尺寸)的筛选基因和调控植株的外在性状(例如植株颜色或植株萎蔫程度)的筛选基因联合使用，由此，可以利用种子的外在性状与植株的外在性状来对杂交后代进行筛选，从而确保杂交后代的纯度。
因此，在某些实施方案中，所述第二多核苷酸包括：(a)能够调控种子的外在性状(例如种子颜色或种子尺寸)的第一筛选基因，以及，(b)能够调控植株的外在性状(例如植株颜色或植株萎蔫程度)的第二筛选基因。
在某些实施方案中，所述第一筛选基因和第二筛选基因是相同的。在此类实施方案中，所述第二多核苷酸含有能够控制种子的外在性状与植株的外在性状的一种筛选基因。例如，在某些实施方案中，所述筛选基因为Lc基因，其能够控制种子的颜色和植株的颜色。在某些实施方案中，所述Lc基因编码氨基酸序列为SEQ ID NO:5的蛋白。在某些实施方案中，所述Lc基因的核苷酸序列为SEQ ID NO:4。
在某些实施方案中，所述第一筛选基因和第二筛选基因是不同的。例如，在某些实施方案中，所述第二多核苷酸包括：
(1)能够调控种子颜色的第一筛选基因和能够调控植株颜色的第二筛选基因，
(2)能够调控种子颜色的第一筛选基因和能够调控植株萎蔫程度的第二筛选基因，
(3)能够调控种子尺寸的第一筛选基因和能够调控植株颜色的第二筛选基因，或，
(4)能够调控种子尺寸的第一筛选基因和能够调控植株萎蔫程度的第二筛选基因。
在某些实施方案中，所述第二多核苷酸包括：Lc基因和Wi2基因。在某些实施方案中，所述第二多核苷酸包括：编码CWI-2基因的干扰RNA的核苷酸和Lc基因。在某些实施方案中，所述第二多核苷酸包括：编码CWI-2基因的干扰RNA的核苷酸和Oy1基因。在某些实施方案中，所述第二多核苷酸包括：编码CWI-2基因的干扰RNA的核苷酸和Wi2基因。
在某些实施方案中，所述编码CWI-2基因的干扰RNA的核苷酸具有如SEQ ID NO:18所示的核苷酸序列。
在某些实施方案中，所述Oy1基因编码氨基酸序列为SEQ ID NO:7的蛋白。在某些实施方案中，所述Oy1基因的核苷酸序列为SEQ ID NO:6。
在某些实施方案中，所述Wi2基因编码氨基酸序列为SEQ ID NO:9的蛋白。在某些实施方案中，所述Wi2基因的核苷酸序列为SEQ ID NO:8。
在某些实施方案中，所述第二多核苷酸还包含，与所述筛选基因的核苷酸序列可操作连接的表达调控元件，例如启动子和增强子。在某些实施方案中，所述表达调控元件选自：启动子，增强子，调控序列，可诱导元件，及其任何组合。在某些实施方案中，所述启动子选自：组成型启动子，诱导型启动子，组织优选的启动子，组织特异性启动子，生长期优选的启动子。可用于本申请的启动子并不局限于上述所列举的启动子。易于理解，在本申请的实施方案中，可以根据实际需要，使用本领域技术人员已知的任何一种启动子。
在某些实施方案中，所述雄性不育基因为隐性雄性不育基因，其在纯合状态下导致植物雄性不育。
在某些实施方案中，所述雄性不育基因选自ms1，ms2，ms3，ms4，ms5，ms6，ms7，ms8，ms9，ms10，ms11，ms12，ms13，ms14，ms15，ms16，ms17，ms18，ms19，ms20，ms21，ms22，ms23，ms24，ms25，ms26，ms27，ms28，ms29，ms30，ms31，ms32，ms33，ms34，ms35，ms36，ms37，ms38，ms43，ms45，ms47，ms48，ms49，ms50，ms52，以及其任何组合。在某些实施方案中，所述雄性不育基因为ms45。
在某些实施方案中，所述恢复基因选自Ms1，Ms2，Ms3，Ms4，Ms5，Ms6，Ms7，Ms8，Ms9，Ms10，Ms11，Ms12，Ms13，Ms14，Ms15，Ms16，Ms17，Ms18，Ms19，Ms20，Ms21，Ms22，Ms23，Ms24，Ms25，Ms26，Ms27，Ms28，Ms29，Ms30，Ms31， Ms32，Ms33，Ms34，Ms35，Ms36，Ms37，Ms38，Ms43，Ms45，Ms47，Ms48，Ms49，Ms50，Ms52，以及其任何组合。在某些实施方案中，所述恢复基因为Ms45。
易于理解，在本申请的实施方案中，所述恢复基因与所述雄性不育基因应当是相对应的，从而，其能够挽救所述雄性不育基因导致的不育性状。在本申请中，优选地，所述雄性不育基因为隐性基因，并且所述恢复基因可以是所述隐性基因的显性等位基因，能够恢复植物的雄性能育性。
在某些实施方案中，所述雄性不育基因为ms45，并且，所述恢复基因为Ms45。
在某些实施方案中，所述恢复基因编码氨基酸序列为SEQ ID NO:2的蛋白。在某些实施方案中，所述恢复基因的核苷酸序列为SEQ ID NO:1。
在某些实施方案中，所述第一多核苷酸还包含，与所述恢复基因的核苷酸序列可操作连接的表达调控元件，例如启动子和增强子。在某些实施方案中，所述表达调控元件选自：启动子，增强子，调控序列，可诱导元件，及其任何组合。在某些实施方案中，所述启动子选自：组成型启动子，诱导型启动子，组织优选的启动子，组织特异性启动子，生长期优选的启动子。可用于本申请的启动子并不局限于上述所列举的启动子。易于理解，在本申请的实施方案中，可以根据实际需要，使用本领域技术人员已知的任何一种启动子。
在某些实施方案中，所述启动子的核苷酸序列为SEQ ID NO:15。
在某些实施方案中，所述第一多核苷酸序列包含或者由SEQ ID NO:3组成。
在某些实施方案中，所述第一多核苷酸和第二多核苷酸通过或者不通过连接核苷酸而共价相连。在某些实施方案中，所述连接核苷酸的长度不超过10kb，不超过5kb，不超过1kb，不超过500bp，不超过100bp，不超过50bp，不超过10bp，不超过5bp，或者更短。在某些实施方案中，所述第一核苷酸序列与所述第二核苷酸序列是基因连锁的。
在某些实施方案中，所述分离的核酸分子还包含第三多核苷酸，所述第三多核苷酸包含选择标记基因的核苷酸序列。在某些实施方案中，所述选择标记基因为抗生素抗性基因或除草剂抗性基因，例如双丙氨磷抗性基因(bar基因)。可用于本申请实施方案的选择标记基因包括但不限于，新霉素抗性基因(例如编码新霉素磷酸转移酶的基因)，潮霉素抗性基因(例如编码潮霉素磷酸转移酶的基因)，氯霉素抗性基因，链霉素抗性基因，壮观霉素抗性基因，博来霉素抗性基因，磺酰胺抗性基因，溴苯腈抗性基因，草甘膦抗性基因，双丙氨磷抗性基因和草胺膦抗性基因。在某些实施方案中，所述选择标记基因为除草剂抗性基因。在某些实施方案中，所述除草剂抗性基因为bar基因。在某些实施方案中，所述选择标记基因的核苷酸序列为SEQ ID NO:10。
在本申请的第二方面，提供了一种载体，其包含如上所述的分离的核酸分子。
在某些实施方案中，所述载体可以是克隆载体，转移载体，或表达载体。在某些实施方案中，所述载体是质粒(例如，pCAMBAI3301)，粘粒，噬菌体等等。在某些实施方案中，所述载体能够在植物细胞(例如，玉米)中表达如上所述的分离的核酸分子。
在本申请的第三方面，提供了一种宿主细胞，其包含，如上所述的分离的核酸分子或者如上所述的载体。
在某些实施方案中，所述宿主细胞为农杆菌细胞(例如，农杆菌EHA105)或植物细胞(例如，玉米)。在某些实施方案中，所述植物细胞为单子叶植物或双子叶植物的细胞。在某些实施方案中，所述植物细胞为选自下列的植物的细胞：玉米(Zea mays)、油菜(Brassica napus)、稻(Oryza sativa)、拟南芥(Arabidopsis thaliana)、大麦(Hordeumvulgare)、小麦(Triticumaestivum)、高粱(Sorghum bicolor)、大豆(Glycine max)、苜蓿(Medicago sativa)、烟草(Nicotiana tabacum)、棉花(Gossypiumhirsutum)、向日葵(Helianthus annuus)或甘蔗(Saccharum officinarum)。
本申请还提供了所述宿主细胞的组织培养物，以及由所述组织培养物产生的原生质体。
在本申请的第四方面，提供了一种植株或植物种子，其中，所述植株或植物种子在基因组中包含如上所述的核酸分子。
在某些实施方案中，所述植株或植物种子在基因组中还包含所述雄性不育基因(例如，ms45)。在某些实施方案中，所述雄性不育基因是纯合的隐性雄性不育基因(例如，ms45ms45)。
在某些实施方案中，所述分离的核酸分子整合在所述植株或植物种子的基因组中。在某些实施方案中，所述分离的核酸分子整合在所述植株或植物种子的基因组中，并且位于与所述雄性不育基因不同的染色体上。在某些实施方案中，所述核酸分子以杂合的形式存在于所述植株或植物种子的基因组中。如本文中所使用的，术语“以杂合的形式存在”具有本领域技术人员通常理解的含义。例如，其可以是指，与所述核酸分子整合至植物基因组中的位置相对应的基因座位上不存在相同的等位基因(即，杂合)。例如，所述核酸分子仅存在于一条染色单体中，而其姊妹染色单体不含有所述核酸分子。
可以使用本领域技术人员已知的任何方法，将所述核酸分子整合在所述植株或植物种子的基因组中。此类方法包括但不限于，稳定转化方法，瞬时转化方法，病毒介导方法， 农杆菌介导方法。
在某些实施方案中，所述植株或植物种子是雄性能育的。在某些实施方案中，所述植株或植物种子能够用作包含所述雄性不育基因的雄性不育植株的保持系。
在某些实施方案中，所述植株或植物种子为单子叶植物或双子叶植物的植株或植物种子。在某些实施方案中，所述植株或植物种子为玉米、油菜、稻、拟南芥、大麦、小麦、高粱、大豆、苜蓿、烟草、棉花、向日葵或甘蔗的植株或植物种子。
在本申请的第五方面，提供了一种获得植株的方法，所述方法包括，(1)将如上所述的核酸分子或如上所述的载体导入植物细胞，以及，(2)将所述植物细胞培养成植株。
可以使用本领域技术人员已知的任何方法，将所述核酸分子或载体导入植物细胞。此类方法包括但不限于，稳定转化方法，瞬时转化方法，病毒介导方法，农杆菌介导方法。此外，可以使用本领域技术人员已知的任何方法，将所述植物细胞培养成植株。例如，可参见McCormicketal.(1986)Plant Cell Reports5：81-84中描述的方法。
在某些实施方案中，在步骤(1)中，使用农杆菌介导方法。在某些实施方案中，在步骤(1)中，使用农杆菌将所述核酸分子或载体导入植物细胞。
在某些实施方案中，所述植物细胞在其基因组中包含所述雄性不育基因，并且优选地，在导入所述核酸分子或载体之前，所述植物细胞是雄性不育的。在某些实施方案中，所述植物细胞在其基因组中包含纯合的隐性雄性不育基因。
在某些实施方案中，在步骤(1)中，所述核酸分子被整合入所述植物细胞的基因组中。在某些实施方案中，所述核酸分子在整合入所述植物细胞的基因组后，位于与所述雄性不育基因不同的染色体上。
在某些实施方案中，所述植物细胞为单子叶植物或双子叶植物的细胞。在某些实施方案中，所述植物细胞为选自下列的植物的细胞：玉米、油菜、稻、拟南芥、大麦、小麦、高粱、大豆、苜蓿、烟草、棉花、向日葵或甘蔗。
在某些实施方案中，所述植株包含纯合的隐性雄性不育基因，以及所述核酸分子或载体，并且，其是雄性能育的。在某些实施方案中，所述核酸分子或载体在所述植株中以杂合的形式存在于基因组中。在某些实施方案中，所述核酸分子整合在所述植株的基因组中，且位于与所述雄性不育基因不同的染色体上。
在某些实施方案中，所述植株能够用作包含所述雄性不育基因的雄性不育植株的保持系植株。
在某些实施方案中，所述方法还包括：
(3)用步骤(2)的植株对包含所述雄性不育基因的雄性不育植株授粉，产生后代种子或植株；和
(4)筛选显示所述筛选基因调控的外在性状的后代种子或植株。
在本申请的第六方面，提供了一种获得雄性不育系植株和保持系植株的后代种子或植株的方法，所述方法包括，将如上所述的植株或通过如上所述的方法获得的植株作为父本与作为母本的包含所述雄性不育基因的雄性不育植株杂交，并产生后代种子或植株。
在某些实施方案中，所述方法包括：
(1)提供作为母本的包含所述雄性不育基因的雄性不育植株；优选地，所述雄性不育基因是纯合的隐性雄性不育基因(例如，ms45ms45)；
(2)提供作为父本的如上所述的植株或通过如上所述的方法获得的植株；
(3)用步骤(2)的植株对步骤(1)的植株授粉，以产生后代种子；
(4)任选地，将所述后代种子培养成后代植株；
其中，显示所述筛选基因调控的外在性状的后代种子或植株是雄性能育的，能够用作保持系；且，不显示所述筛选基因调控的外在性状的后代种子或植株是雄性不育的，能够用作雄性不育系。
在本申请中，两个或更多个外在性状的同时使用在某些情况下是优选和有利的。例如，在某些实施方案中，步骤(2)中作为父本的植株可以包含：(a)能够调控种子的外在性状(例如种子颜色或种子尺寸)的第一筛选基因，以及，(b)能够调控植株的外在性状(例如植株颜色或植株萎蔫程度)的第二筛选基因。由此，在将步骤(2)的植株与步骤(1)的植株杂交后，可以利用种子的外在性状(例如种子颜色或种子尺寸)与植株的外在性状(例如植株颜色或植株萎蔫程度)来对杂交后代进行筛选，从而确保杂交后代的纯度。
因此，在某些实施方案中，所述方法包括：
(1)提供作为母本的包含所述雄性不育基因的雄性不育植株；优选地，所述雄性不育基因是纯合的隐性雄性不育基因；
(2)提供作为父本的如上所述的植株或通过如上所述的方法获得的植株；其中，所述第二多核苷酸包括：(a)能够调控种子的外在性状(例如种子颜色或种子尺寸)的第一筛选基因，以及，(b)能够调控植株的外在性状(例如植株颜色或植株萎蔫程度)的第二筛选基因；
(3)用步骤(2)的植株对步骤(1)的植株授粉，产生两种后代种子；其中，第一种 后代种子显示所述第一筛选基因调控的种子的外在性状；且，第二种后代种子不显示所述第一筛选基因调控的种子的外在性状；
(4)分离所述第一种和第二种后代种子，且任选地，将它们分别培养成第一种和第二种后代植株。
任选地，所述方法还包括下述步骤：
(5)从第一种后代植株中去除这样的植株，其不显示所述第二筛选基因调控的植株的外在性状，由此，剩余的第一种后代植株为雄性能育的，能够用作保持系植株；和/或
从第二种后代植株中去除这样的植株，其显示所述第二筛选基因调控的植株的外在性状，由此，剩余的第二种后代植株为雄性不育的，能够用作雄性不育系植株；
(6)用所述剩余的第一种后代植株对所述剩余的第二种后代植株授粉，并产生进一步的后代种子。
在又一个方面，本申请提供了一种制备杂交种子的方法，所述方法包括：
(1)提供通过如上所述的方法获得的雄性不育系植株的后代种子，其不显示所述第一筛选基因调控的种子的外在性状；并且，提供目标品系植株的种子；
(2)将所述雄性不育系植株的后代种子与所述目标品系植株的种子田间播种，以获得雄性不育系植株和目标品系植株；
(3)从所述雄性不育系植株中去除这样的植株，其显示所述第二筛选基因调控的植株的外在性状；
(4)用所述目标品系植株对剩余的雄性不育系植株授粉；
(5)从雄性不育系植株上收获种子，即为杂交种子。
本申请的方法可以利用两种或更多种外在性状来对雄性不育系植株后代和保持系植株后代进行筛选和区分，这避免了育种过程中可能因异雄核受精、基因突变、基因丢失、染色体重组、染色体易位等而导致的“假阳性”或“假阴性”性状，从而进一步提高了雄性不育系植株后代和保持系植株后代各自的纯度，提高了育种效率和所产生的杂交种子的质量和纯度。
在某些实施方案中，所述第一筛选基因和第二筛选基因是相同的。在此类实施方案中，所述第二多核苷酸含有能够控制种子的外在性状与植株的外在性状的一种筛选基因。例如，在某些实施方案中，所述筛选基因为Lc基因，其能够控制种子的颜色和植株的颜色。在某些实施方案中，所述Lc基因编码氨基酸序列为SEQ ID NO:5的蛋白。在某些实施方案中，所述Lc基因的核苷酸序列为SEQ ID NO:4。
在某些实施方案中，所述第一筛选基因和第二筛选基因是不同的。例如，在某些实施方案中，所述第二多核苷酸包括：
(1)能够调控种子颜色的第一筛选基因和能够调控植株颜色的第二筛选基因，
(2)能够调控种子颜色的第一筛选基因和能够调控植株萎蔫程度的第二筛选基因，
(3)能够调控种子尺寸的第一筛选基因和能够调控植株颜色的第二筛选基因，或，
(4)能够调控种子尺寸的第一筛选基因和能够调控植株萎蔫程度的第二筛选基因。
在某些实施方案中，所述第二多核苷酸包括：Lc基因和Wi2基因。在某些实施方案中，所述第二多核苷酸包括：编码CWI-2基因的干扰RNA的核苷酸和Lc基因。在某些实施方案中，所述第二多核苷酸包括：编码CWI-2基因的干扰RNA的核苷酸和Oy1基因。在某些实施方案中，所述第二多核苷酸包括：编码CWI-2基因的干扰RNA的核苷酸和Wi2基因。
在某些实施方案中，所述编码CWI-2基因的干扰RNA的核苷酸具有如SEQ ID NO:18所示的核苷酸序列。
在某些实施方案中，所述Oy1基因编码氨基酸序列为SEQ ID NO:7的蛋白。在某些实施方案中，所述Oy1基因的核苷酸序列为SEQ ID NO:6。
在某些实施方案中，所述Wi2基因编码氨基酸序列为SEQ ID NO:9的蛋白。在某些实施方案中，所述Wi2基因的核苷酸序列为SEQ ID NO:8。
在本申请的第七方面，提供了一种制品，其由如上所述的植株或其部分制成。在某些实施方案中，所述制品为食品，并且其由如上所述的植株的可食用部分(例如种子)制成。
在本申请的第八方面，提供了如上所述的核酸分子或如上所述的载体或如上所述的宿主细胞(例如植物细胞)用于产生保持系植株的用途。在本申请的第九方面，提供了通过如上所述的方法获得的植株或后代种子或植株或如上所述的植株用于产生杂交子代(例如植株或种子)的用途。在本申请的第十方面，提供了一种组织培养物或由其产生的原生质体，其中，所述组织培养物包含如上所述的宿主细胞(例如植物细胞)或通过如上所述的方法获得的植株的细胞或通过如上所述的方法获得的后代种子或植株的细胞。
在某些实施方案中，所述植株为单子叶植物或双子叶植物的植株。在某些实施方案中，所述植株为选自下列的植物：玉米、油菜、稻、拟南芥、大麦、小麦、高粱、大豆、苜蓿、烟草、棉花、向日葵或甘蔗。
此外，本申请还公开了如下技术方案：
本发明的一个目的是提供一种方法。本发明提供的方法，用于保持雄性不育植株的纯合隐性状态，所述方法包括：
(a)提供第一植株，其包含使植物雄性不育的纯合隐性等位基因(例如，纯合隐性等位基因具体为ms45ms45)；
(b)提供第二植株，该植株包含同所述第一植株相同使植物雄性不育的纯合隐性等位基因，并且含有下述构建体，所述构建体(例如，转基因元件Ms45-Lc或Ms45-Oy1或Ms45-Wi2)在所述第二植株中以杂合状态存在(杂合Ms45-Lc/-或Ms45-Oy1/-或Ms45-Wi2/-插入片段仅存在于一条染色单体中，其姊妹染色单体不含有转基因元件)，所述构建体包含：
i.第一核苷酸序列，当其在所述第一植株中表达时将恢复所述第一植株雄性生育力；
ii.第二核苷酸序列，当其以杂合状态存在时即可影响植株颜色(如Lc基因使植株和/或籽粒变为紫色，或，Oy1基因使植株变为黄色)或影响植株形态(如Wi2基因使植株顶部叶片萎蔫)，通过肉眼或仪器可以分辨含有该构建体的植株或籽粒和不含有该构建体的植株或籽粒；所述第一核苷酸序列(例如，SEQ ID NO:3所示的Ms45基因表达元件)与所述第二核苷酸序列(例如，SEQ ID NO:4所示的Lc基因或SEQ ID NO:6所示的Oy1基因或SEQ ID NO:8所示的Wi2基因)紧密相连，这两个核苷酸序列在植株中同时存在(例如，实施例中的转基因元件Ms45-Lc、Ms45-Oy1或Ms45-Wi2)；
(c)用所述第二植株的雄性配子与所述第一植株的雌性配子受精，以产生保持了所述第一植株纯合隐性状态的后代。
上述方法为扩繁植物雄性不育系的方法；
和/或，所述植物、所述第一植株和所述第二植株均为双子叶植物或者单子叶植物；
和/或，所述植物、所述第一植株和所述第二植株均不仅可为玉米(Zea mays)，同样可为水稻(Oryza sativa)、高粱(Sorghum bicolor)、小麦(Triticumaestivum)、大豆(Glycine max)、棉花(Gossypiumhirsutum)、向日葵(Helianthus annuus)等作物。
上述方法中，所述第一核苷酸序列包括控制雄性生育能力的恢复基因，如表1中的ms45的野生型等位基因Ms45。这个控制雄性生育能力的恢复基因不限于表1中列出的基因，玉米中或其他物种中控制雄性生育能力的恢复基因也可达到本发明的目的，因此也在本发明的保护范围内。
上述方法中，在本发明的实施例中，所述第一植株为玉米雄性不育突变体ms45，具体为ms45纯合隐性自交系郑58(郑58(ms45ms45))，其源自ms45纯合隐性突变体(Maize Genetics Cooperation Stock Center，905I)和郑58(zheng58)的回交后代。
所述第一核苷酸序列在本发明的实施例中为Ms45基因表达元件，所述Ms45基因表达元件在所述第一植株中表达蛋白质Ms45。
所述蛋白质Ms45为如下a)或b)：
a)SEQ ID NO:2所示的氨基酸残基组成的蛋白；
b)经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与SEQ ID NO:2具有相同功能的蛋白质。
上述方法中，所述Ms45基因表达元件包括Ms45基因启动子、Ms45基因5’UTR、Ms45基因外显子、Ms45基因内含子、Ms45基因3’UTR和Ms45基因终止子；
所述Ms45基因表达元件为如下1)或2)或3)所示的DNA分子：
1)编码区包括SEQ ID NO:1的DNA分子；
2)编码区为SEQ ID NO:1的DNA分子；
3)将1)或2)经过一个或几个核苷酸的取代和/或缺失和/或添加且与1)或2)具有相同功能的DNA分子。
上述方法中，所述第二核苷酸序列包括控制植株颜色或控制植物籽粒颜色或控制植株萎蔫程度的基因；
上述方法中，所述第二核苷酸序列在所述第二植株中以杂合状态存在时影响所述第二植株颜色或籽粒颜色或植株萎蔫程度：
本发明的一个实施例中，上述控制植株颜色或控制植物籽粒颜色的基因为花青素合成的关键基因Lc；上述花青素合成关键基因Lc在所述第二植株中表达蛋白质LC。所述第二核苷酸序列在所述第二植株中以杂合状态存在时影响所述第二植株的籽粒或植株颜色是否紫色；含有第二核苷酸序列，则籽粒或植株颜色为紫色，不含有，则籽粒或植株颜色为非紫色；
本发明的另一个实施例中，上述控制植株颜色的基因为叶绿素合成相关基因Oy1；上述Oy1基因在所述第二植株中表达蛋白质Oy1。所述第二核苷酸序列在所述第二植株中以杂合状态存在时影响所述第二植株的植株颜色是否黄色；含有第二核苷酸序列，则植株颜色为黄色，不含有，则植株颜色为非黄色；
本发明的另一个实施例中，上述控制植株萎蔫程度的基因在本发明的实施例中为Wi2基因；上述Wi2基因在所述第二植株中表达蛋白质Wi2。所述第二核苷酸序列在所述第二植株中以杂合状态存在时影响所述第二植株的植株是否萎蔫；含有第二核苷酸序列，则植株萎蔫，不含有，则植株为非萎蔫。
上述蛋白质LC为如下a)或b)：
a)SEQ ID NO:5所示的氨基酸残基组成的蛋白；
b)经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与SEQ ID NO:5具有相同功能的蛋白质；
或，所述蛋白质Oy1为如下a)或b)：
a)SEQ ID NO:7所示的氨基酸残基组成的蛋白；
b)经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与SEQ ID NO:7具有相同功能的蛋白质；
或，所述蛋白质Wi2为如下a)或b)：
a)SEQ ID NO:9所示的氨基酸残基组成的蛋白；
b)经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与SEQ ID NO:9具有相同功能的蛋白质。
所述基因Lc为如下1)或2)或3)所示的DNA分子：
1)编码区包括SEQ ID NO:4的DNA分子；
2)编码区为SEQ ID NO:4的DNA分子；
3)将1)或2)经过一个或几个核苷酸的取代和/或缺失和/或添加且与1)或2)具有相同功能的DNA分子。
所述基因Oy1为如下1)或2)或3)所示的DNA分子：
1)编码区包括SEQ ID NO:6的DNA分子；
2)编码区为SEQ ID NO:6的DNA分子；
3)将1)或2)经过一个或几个核苷酸的取代和/或缺失和/或添加且与1)或2)具有相同功能的DNA分子。
所述基因Wi2为如下1)或2)或3)所示的DNA分子：
1)编码区包括SEQ ID NO:8的DNA分子；
2)编码区为SEQ ID NO:8的DNA分子；
3)将1)或2)经过一个或几个核苷酸的取代和/或缺失和/或添加且与1)或2)具有相同功能的DNA分子。
本发明涉及控制雄性生育能力、控制植株或种子颜色和控制植株萎焉的核苷酸序列，及利用这些核苷酸序列和转基因技术开发的扩繁植物雄性不育的方法。
本发明的上述方法可以用于如下目的：
一个目的在于提供一种高效的种子标记方法，利用该方法可以扩繁植物雄性不育种子，为杂交制种节约人力，降低成本，保证种子纯度。
另一目的是提供一种通过植株或种子颜色或植株萎蔫程度高效分辨可育籽粒及不育籽粒的方法。
另一目的是提供一种转基因植株，该植株可以保持雄性不育植株的不育性。
本发明另一个目的是提供一种DNA构建物。
本发明提供的构建物如上述方法中所述的构建体。
上述DNA构建物可以恢复雄性不育突变体的育性，同时改变植株颜色或改变植株籽粒颜色或改变植株萎蔫程度。
上述方法中所述的构建体如下：
本发明构建了含有SEQ ID NO:3所示的Ms45基因表达元件的植物表达载体，将该载体转入雄性不育突变体ms45中，可以恢复该突变体的育性。
本发明构建了控制玉米花青素合成基因Lc(Ludwig et al.1993)的载体。该载体会影响花青素合成，过表达该基因使植株或籽粒表现为紫色。
本发明构建了调控叶绿体合成的Oy1基因的载体。该载体会影响叶绿体合成，过表达该基因使植株变为黄色。
本发明构建了调控根木栓质合成相关基因Wi2的载体。该载体会影响水分运输，过表达该基因使植株萎蔫(顶部叶片萎蔫)。
本发明第3个目的是提供如下任一种的物质。
本发明提供的如下物质：
1)一种植物，该植物如上述方法中所述的第二植株；
2)由1)所述的植物产生的再生细胞的组织培养物；
3)由2)所述的组织培养物产生的原生质体；
4)一种植物，所述植物是利用上述方法产生的纯合隐性的雄性不育植株。
上述第二植株可以保持雄性不育植株的不育性。
上述植株可为植株的全部或部分，如根、茎、叶、胚、根尖、花粉或花药等。
利用上述方法产生的纯合隐性雄性不育植株在生产杂交种中的用途也是本发明保护的范围。
本发明的一个关键是将雄性恢复基因Ms45、控制植株颜色或控制种子颜色或调控植株萎焉等控制植株形态的基因构建在一个载体中，该载体可以恢复雄性不育突变体ms45的育性，同时使含有转基因序列的植株颜色为所需颜色或所需植株萎焉状态，即对含有恢复基因的植株或籽粒进行标记，以便区分可育个体(保持系)和不育个体(不育系)。
以Lc为例，本发明的一个实施例中，发明人利用控制植物雄性生育能力、过表达花青素合成相关基因Lc的核苷酸序列及转基因技术，发明了一种高效扩繁植物雄性不育系的新方法。控制植物雄性生育能力的基因包括表1中所列出的雄性不育基因及其他雄性不育基因以及其他物种的雄性不育基因。发明人首先构建了一个植物转化载体，该载体中包含恢复雄性生育能力基因的表达元件和过表达花青素合成相关基因Lc的表达元件，将该载体转入HiIIA×HiIIB玉米杂交种中，该杂交种正常条件下，植株为绿色。然后利用雄性不育植株对获得的转基因植株进行回交，从而将控制植物雄性生育能力Ms45恢复基因、控制植株或种子颜色(Lc基因)的核苷酸序列导入雄性不育植株ms45中。由于存在恢复基因Ms45，转基因植株表现为可育。当转基因杂合体植株(Msmsms)与雄性不育植株(msms)杂交时，会产生以下两种后代，一种为绿色植株和黄色种子的雄性不育个体(不育系，基因型为msms)，不含有转基因序列的雄性不育正常籽粒(不育系ms45ms45)，该不育系可以被任意一种野生型植株恢复育性，在制种过程中可作为不育系；另一种为紫色植株或种子的可育籽粒(保持系，基因型为Msmsms)，该保持系在控制雄性生育能力位点为隐性纯合，由于含有可以互补的转基因序列，该植株表现为可育，由于同时含有控制植株或种子颜色(花青素合成基因)的核苷酸序列，该植株或种子颜色不同于野生型。
以Oy1为例，本发明的一个实施例中，发明人利用控制植物雄性生育能力、过表达花青素合成相关基因Oy1的核苷酸序列及转基因技术，发明了一种高效扩繁植物雄性不育系的新方法。控制植物雄性生育能力的基因包括表1中所列出的雄性不育基因及其他雄性不育基因以及其他物种的雄性不育基因。发明人首先构建了一个植物转化载体，该载体中包含恢复雄性生育能力基因的表达元件和过表达叶绿素合成相关基因Oy1的表达元件，将该载体转入HiIIA×HiIIB玉米杂交种中,该杂交种正常条件下，植株为绿色。然后利用雄性不育植株对获得的转基因植株进行回交，从而将控制植物雄性生育能力Ms45恢复基因、控制植株颜色(Oy1基因)的核苷酸序列导入雄性不育植株ms45中。由于存在恢复基因Ms45， 转基因植株表现为可育。当转基因杂合体植株(Msmsms)与雄性不育植株(msms)杂交时，会产生以下两种后代，一种为绿色植株的雄性不育个体(不育系，基因型为msms)，不含有转基因序列的雄性不育正常籽粒(不育系ms45ms45)，该不育系可以被任意一种野生型植株恢复育性，在制种过程中可作为不育系；另一种为黄色植株的可育籽粒(保持系，基因型为Msmsms)，该保持系在控制雄性生育能力位点为隐性纯合，由于含有可以互补的转基因序列，该植株表现为可育，由于同时含有控制植株或种子颜色(控叶绿体合成相关基因)的核苷酸序列，该植株颜色不同于野生型。
以Wi2为例，本发明的一个实施例中，发明人利用控制植物雄性生育能力、过表达调控根木栓质合成相关的基因Wi2的核苷酸序列及转基因技术，发明了一种高效扩繁植物雄性不育系的新方法。控制植物雄性生育能力的基因包括表1中所列出的雄性不育基因及其他雄性不育基因以及其他物种的雄性不育基因。发明人首先构建了一个植物转化载体，该载体中包含恢复雄性生育能力基因的表达元件和过表达调控根木栓质合成相关的基因Wi2的表达元件，将该载体转入HiIIA×HiIIB玉米杂交种中，该杂交种正常条件下，植株为非萎蔫状态。然后利用雄性不育植株对获得的转基因植株进行回交，从而将控制植物雄性生育能力Ms45恢复基因、调控根木栓质合成(Wi2的基因)的核苷酸序列导入雄性不育植株ms45中。由于存在恢复基因Ms45，转基因植株表现为可育。当转基因杂合体植株(Msmsms)与雄性不育植株(msms)杂交时，会产生以下两种后代，一种为非萎蔫状态(或者萎蔫程度低于保持系)的雄性不育个体(不育系，基因型为msms)，不含有转基因序列的雄性不育正常籽粒(不育系ms45ms45)，该不育系可以被任意一种野生型植株恢复育性，在制种过程中可作为不育系；另一种为植株叶片萎蔫的可育籽粒(保持系，基因型为Msmsms)，该保持系在控制雄性生育能力位点为隐性纯合，由于含有可以互补的转基因序列，该植株表现为可育，由于同时含有控制植株或种子颜色(花青素合成基因)的核苷酸序列，该植株萎蔫程度不同于野生型。
本发明的其他目的在下文说明书和权利要求书中将是显而易见的。
本发明的实验证明，本发明提供方法可以扩繁植物核雄性不育种子，为杂交制种带来便利。该体系主要是利用一个可以分辨植株或种子颜色或调控植物萎蔫的核苷酸序列和一种细胞核雄性不育基因的野生型等位基因及转基因技术。转基因的籽粒可以通过颜色分辨，转基因植物或可以通过颜色区分植株，或可以通过萎蔫程度区分植株。
有益效果
本申请提供了一种扩繁雄性不育植株的方法，以及用于扩繁雄性不育植株的保持系植株。本申请所提供的保持系植株在与雄性不育植株杂交后，能够同时收获并且有效区分雄性不育植株和保持系植株的后代。由此，本申请的所提供的方法和保持系植株能够实现雄性不育植株的高效扩繁，提高了育种效率。此外，通过种子的外在性状与植株的外在性状的联合使用，本申请的所提供的方法能够对杂交后代进行两次或更多次的筛选，这进一步提高了雄性不育系植株后代和保持系植株后代各自的纯度，提高了育种效率和所产生的杂交种子的质量和纯度。
在以往的技术中，仅对种子或仅对植株进行标记、筛选，然而，这些方法具备以下不足：(1)仅对种子进行筛选，由于异雄核受精等原因，通过对胚乳表型进行筛选获得的部分种子中，胚乳中不含转基因成分，胚中却含有转基因成分，这些种子发育成的植株是可育的，并且这些可育种子难以与不育种子区分开，只能等到植株散粉时才能确定，但散粉时再去除可育的植株将会对繁殖的杂交种纯度产生影响，不能完全满足实际生产的需要；(2)仅对植株进行筛选，则无法在种植时将不育种子与可育种子区分开，耗费大量人力物力。而本申请的方法结合种子和植株的标记和筛选，并且能够在植株播种后、散粉前(例如苗期)确定不育株和可育株，及时去除混杂的可育株，显著提高了育种效率和所产生的杂交种子的质量和纯度。
序列信息
本申请所涉及的序列的信息如下面的表2所示。
表2.序列信息
Figure PCTCN2020095082-appb-000003
Figure PCTCN2020095082-appb-000004
Figure PCTCN2020095082-appb-000005
Figure PCTCN2020095082-appb-000006
Figure PCTCN2020095082-appb-000007
Figure PCTCN2020095082-appb-000008
Figure PCTCN2020095082-appb-000009
Figure PCTCN2020095082-appb-000010
Figure PCTCN2020095082-appb-000011
Figure PCTCN2020095082-appb-000012
Figure PCTCN2020095082-appb-000013
附图说明
图1显示了雄性不育玉米植株ms45(A)及野生型玉米植株Ms45(B)的雄花表型。
图2显示了不含有转基因元件Ms45-Lc的杂交后代籽粒(A，籽粒颜色为黄色)以及含有转基因元件Ms45-Lc的杂交后代籽粒(B，籽粒颜色为紫色)。
图3显示了不含有转基因元件Ms45-Lc的杂交后代幼苗(其为非紫色)以及含有转基因元件Ms45-Lc的杂交后代幼苗(其为紫色)。
图4显示了pMs45-Lc载体的结构示意图。
图5显示了含有转基因元件Ms45-Lc的杂交后代植株(A，植株表现为紫色，且是雄性能育的)以及不含有转基因元件Ms45-Lc杂交后代植株(B，植株表现为非紫色，且是雄性不育的)。
图6示意性显示了保持系植株、不育系植株及其杂种后代的选育过程。
图7显示了含有转基因元件Ms45-Oy1的杂交后代植株(A，植株表现为黄色)以及不含有转基因元件Ms45-Oy1的杂交后代植株(B，植株表现为绿色)。
图8显示了含有转基因元件Ms45-Wi2的杂交后代植株(A，植株表现为萎蔫)以及不含有转基因元件Ms45-Wi2杂交后代的植株(B，植株表现为正常)。
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。
实施例1：载体的构建
本实施例选用表1中的雄性不育基因ms45及其恢复基因Ms45进行实验。
1、雄性不育恢复基因Ms45的扩增
雄性不育恢复基因Ms45来源于玉米品种B73，其核苷酸序列如SEQ ID NO:1所示，其编码的Ms45蛋白的氨基酸序列如SEQ ID NO:2所示。以B73的基因组DNA为模板，参考B73基因组序列(www.maizesequence.org)，设计扩增引物，以扩增恢复基因Ms45。所设计的引物如下：
Ms45F:5'tgaattcTGCTGAGTTCTCCTTGGGTTATCC 3'(SEQ ID NO:11)，
Ms45R:5'tcccgggGGTTGCGCATGAAATAGGGGT 3'(SEQ ID NO:12)。
所设计的上游引物Ms45F在5’端含有EcoRI酶切位点，下游引物Ms45R在5’端含有SmaI酶切位点，以此进行扩增。扩增的反应体系为：模板DNA 2μL，引物Ms45F 0.5μL，引物Ms45R 0.5μL，dNTP 1.6μL，10×Buffer 2μL，高保真taq酶0.3μL，ddH 2O 13.1μL。反应条件为：95℃变性5min；32个循环的(95℃变性45s，59℃退火45s，72℃延伸3min)；72℃延伸10min。
实验结果：扩增得到的目标产物全长约为3500bp。将其回收并连接至T-easy测序载体，然后用于转化并测序。测序结果证实，扩增产物是由EcoRI酶切位点、SEQ ID NO:3所示序列和SmaI酶切位点组成的3518bp的DNA片段，其用作Ms45基因构建体。SEQ ID NO:3所示序列含有Ms45基因的启动子(SEQ ID NO:15)和编码区核苷酸序列。
2、调控种子颜色、植株颜色和植株萎蔫的相关基因的扩增
1)Lc基因的扩增
Lc基因的核苷酸序列如SEQ ID NO:4所示，其编码的Lc蛋白的氨基酸序列如SEQ ID NO:5所示。在SEQ ID NO:4的5’端添加NcoI酶切位点和保护碱基，3’端添加BstEII酶切位点和保护碱基，并将此序列进行人工合成，用作Lc基因构建体。
2)Oy1基因的扩增
Oy1基因的核苷酸序列如SEQ ID NO:6所示，其编码的Oy1蛋白氨基酸序列如SEQ ID NO:7所示。在SEQ ID NO:6的5’端添加NcoI酶切位点和保护碱基，3’端添加BstEII酶切位点和保护碱基，并将此序列进行人工合成，用作Oy1基因构建体。
3)合成调控植株萎蔫的Wi2基因
Wi2基因能够调控根木栓质，其表达可以影响植株的水分运输从而使植株更容易萎蔫。Wi2基因的核苷酸序列如SEQ ID NO:8所示，其编码的Wi2蛋白氨基酸序列如SEQ ID NO:9所示。在SEQ ID NO:8的5’端添加NcoI酶切位点和保护碱基，3’端添加BstEII酶切位点和保护碱基，并将此序列进行人工合成，用作Wi2基因构建体。
3、构建重组农杆菌
使用质粒pCAMBAI3301(国际农业分子生物学应用中心CAMBIA,Australia)来构建下述重组表达载体，该质粒上含有选择标记基因bar(其核苷酸序列如SEQ ID NO:10所示)。
1)载体pMs45-Lc
通过双酶切基因构建体和质粒，将Lc基因构建体克隆至pCAMBAI3301质粒的BstEII和NcoI酶切位点之间，并且，将Ms45基因构建体克隆至pCAMBAI3301质粒的EcoRI和SmaI酶切位点之间。所构建的载体命名为pMs45-Lc。该载体的结构示意图如图4所示，该载体包含Ms45基因、Lc基因和载体本身的选择标记基因bar。
2)载体pMs45-Oy1
通过双酶切基因构建体和质粒，将Oy1基因构建体克隆至pCAMBAI3301质粒的BstEII和NcoI酶切位点之间，并且，将Ms45基因构建体克隆至pCAMBAI3301质粒的EcoRI和SmaI酶切位点之间。所构建的载体命名为pMs45-Oy1。该载体包括Ms45基因、Oy1基因和载体本身的选择标记基因bar。
3)载体pMs45-Wi2
通过双酶切基因构建体和质粒，将Wi2基因构建体克隆至pCAMBAI3301质粒的BstEII和NcoI酶切位点之间，并且，将Ms45基因构建体克隆至pCAMBAI3301质粒的EcoRI和SmaI酶切位点之间。所构建的载体命名为pMs45-Wi2。该载体包括Ms45基因、Wi2基因和载体本身的选择标记基因bar。
将上述获得的载体pMs45-Lc、pMs45-Oy1和pMs45-Wi2分别转化农杆菌EHA105，得到重组农杆菌EHA105/pMs45-Lc、EHA105/pMs45-Oy1和EHA105/pMs45-Wi2。
另外，通过与上述类似的方法，制备了载体pMs45-Oy1-CWI-2(其携带Ms45基因、Oy1基因、编码CWI-2基因的干扰RNA的核苷酸(SEQ ID NO:18)以及载体本身的选择标记基因bar)和pMs45-Lc-CWI-2(其携带Ms45基因、Lc基因、编码CWI-2基因的干扰RNA的核苷酸(SEQ ID NO:18)以及载体本身的选择标记基因bar)，并制备了重组农杆菌EHA105/pMs45-Oy1-CWI-2和EHA105/pMs45-Lc-CWI-2。
实施例2：转基因玉米的获得
在田间种植玉米品种HiIIA和HiIIB(Armstrong C L,Green C E and Phillips R L.Development and availability of germplasm with high Type II culture formation response.Maize Genetics Cooperation News Letter,1991,65:92-93)，到散粉时将其分别套袋、授粉， 进行杂交。使用两种杂交方式：HiIIA作母本，HiIIB作父本；或者，HiIIA作父本，HiIIB作母本。授粉后9-11天，取授粉果穗籽粒上的未成熟的杂交幼胚，用获得的重组农杆菌EHA105/pMs45-Lc、EHA105/pMs45-Lc和EHA105/pMs45-Wi2分别对玉米幼胚进行侵染。将侵染后的幼胚放在选择培养基上进行多次筛选，获得抗性愈伤组织，将抗性愈伤组织再生成苗，得到转基因T0代植株。获得转基因T0代以后，用T0代转基因植株的花粉对制种母本HiIIA和HiIIB及ms45雄性不育材料(获自Maize Genetics Cooperation Stock Center，905I)授粉，并观察表型。
具体的实施步骤如下所示：
1、玉米幼胚的获得
1)将HiIIA和HiIIB杂交F1代的果穗顶端约1cm左右切除，用镊子从顶端插入果穗。然后把果穗放入含有消毒液的烧杯中，根据实际需要，可以在同一个烧杯里放4-6个果穗。
2)向烧杯里加入约700ml的消毒液(50％的漂白剂或5.25％的次氯酸钠，并加入一滴Tween 20)用来浸泡果穗。在消毒20分钟的过程中，不时旋转果穗同时轻轻拍打烧杯以驱除籽粒表面的气泡，从而达到最佳的消毒效果。消毒结束后，取出果穗并放入盛满灭菌水的烧杯里，在水里洗3次，然后准备剥胚。
3)把消毒过的果穗放在一个大的培养皿上，用大的手术刀削掉籽粒的顶部(1.5-1.8mm)。
4)用剥胚刀的刀尖插在胚和胚乳之间，然后轻轻向上撬出幼胚，用小的手术刀尖轻轻托起幼胚，确保幼胚不受到任何的损伤，把幼胚的胚轴面紧贴放有滤纸的N6E培养基，胚的放置密度大约是2x2cm(30个/皿)。
5)用封口膜封住培养皿，28℃暗培养2-3天。
2、农杆菌的浸染
1)将重组农杆菌EHA105/pMs45-Lc、EHA105/pMs45-Oy1和EHA105/pMs45-Wi2分别在YEP(含Kana33mg/L和Str100mg/L抗生素)培养基上提前一周培养。
2)将上述培养的重组农杆菌转移至新鲜的YEP(含Kana33mg/L，Str50mg/L)培养基上，19℃培养3天。
3)3天以后，挑取重组农杆菌放入含有5mL浸染培养基的50ml离心管中，同时加入100uM的AS(inf+AS)，在室温(25℃)转速75rpm下培养2-4个小时。
4)浸染幼胚，把刚剥离的幼胚放入含有inf+AS液体培养基(2ml)的离心管中，每管约放置20-100个幼胚，用这样的培养基洗涤2次，然后加入1-1.5ml特定浓度(OD550＝0.3-0.4) 的农杆菌，轻轻颠倒离心管20次，然后直立放置在暗箱里5分钟，确保幼胚全部浸泡在农杆菌液体里，整个过程避免旋涡振荡。
3、共培养
1)浸染后，把浸染过的玉米幼胚转移到共培养培养基(溶质如表3，溶剂为水)，使幼胚的胚轴接触培养基表面，同时去除培养基表面多余的农杆菌。
2)用封口膜封住培养皿，在20℃条件下暗培养3天。
4、静息培养
1)共培养3天后，把幼胚转移到静息培养基(溶质如表3，溶剂为水)上，同时用封口膜封住培养皿，在28℃条件下暗培养7天。
5、选择
1)7天后，把所有的幼胚转移到选择培养基(溶质如表3，溶剂为水)上(35个幼胚/每皿)培养两周，选择培养基含有双丙胺磷1.5mg/L。两周后再进行亚培养，双丙胺磷的浓度可以上升到3mg/L。
2)浸染大约5周左右，含有转化子的细胞会长成可以看的见的Ⅱ型愈伤组织。
6、转基因植株的再生
1)在再生培养基I(溶质如表3，溶剂为水)上生长3周，然后在再生培养基II(溶质如表3，溶剂为水)上面发芽(置于光照培养室中)，得到T0代转基因pMs45-Lc玉米、pMs45-Oy1玉米和pMs45-Wi2玉米。
2)待生长出3-4片叶时，将其分别转移到温室，待其生长至吐丝散粉期时，对其分别进行授粉。
7、分析获得的转基因植株
针对植株整体形态(例如，花粉、植株和籽粒表型)对获得的T0代转基因pMs45-Lc玉米、pMs45-Oy1玉米和pMs45-Wi2玉米及其后代进行评估。将上述转基因玉米分别与ms45纯合隐性的雄性不育材料(获自Maize Genetics Cooperation Stock Center，905I)杂交，得到杂交后代。
1)杂交后代的基因型检测
通过检测bar基因以确定杂交后代是否含有转基因元件Ms45-Lc、Ms45-Oy1或Ms45-Wi2。bar基因检测方法为：用如下引物Bar669F(SEQ ID NO:13)和Bar669R(SEQ ID NO:14)对杂交后代的基因组进行PCR扩增，若扩增产物含有大小约为669bp的目的 片段，则杂交后代为含有转基因元件的杂交后代；若扩增产物不含有大小669bp目的片段，则杂交后代为不含有转基因元件杂交后代。
上述引物Bar669F和Bar669R序列如下：
Bar669F:5'TCTCGGTGACGGGCAGGAC 3'(SEQ ID NO:13)；
Bar669R:5'TGACGCACAATCCCACTATCCTT 3'(SEQ ID NO:14)。
经PCR和测序验证，分别得到含有转基因元件Ms45-Lc的杂交后代、含有转基因元件Ms45-Oy1的杂交后代和含有转基因元件Ms45-Wi2的杂交后代。
2)杂交后代表型的检测
(1)含有转基因元件Ms45-Lc的杂交后代的检测
观察大田中栽培的杂交后代的表型，杂交后代的植株颜色如图5所示，其中，含有转基因元件Ms45-Lc的杂交后代如图5A所示，植株表现为紫色(检测可育)；而不含有转基因元件Ms45-Lc杂交后代如图5B所示，植株表现为非紫色(检测不育)。杂交后代的籽粒颜色结果如图2所示，图2B为含有转基因元件Ms45-Lc的杂交后代籽粒，籽粒颜色为紫色(检测可育)，图2A为不含有转基因元件Ms45-Lc的杂交后代籽粒，籽粒颜色为黄色(非紫色，检测不育)。杂交后代的幼苗颜色结果如图3所示，其中，含有转基因元件Ms45-Lc的杂交后代幼苗为紫色(检测可育)，不含有转基因元件Ms45-Lc的杂交后代幼苗为非紫色(检测不育)。
上述检测结果表明，Ms45基因在植株中的表达拯救了隐性纯合ms45基因导致的雄性不育表型。同时，含有Ms45-Lc的转基因植株或籽粒表现紫色。这表明Lc基因在转基因植株中均能正常行使功能。将上述获得的紫色籽粒及非紫色(黄色)籽粒播种到田间，这些籽粒均能够正常发芽，且出芽率没有显著差异。并且，在双丙胺磷的筛选下，紫色种子、植株仍能够正常生长。以上结果表明，选择标记基因bar、Ms45基因及Lc基因均能正常行使功能，并且这三个基因连锁遗传。在T0代转基因植株与雄性不育系植株ms45杂交产生的后代中，正常籽粒:紫色籽粒为1:1，紫色籽粒后代均为紫色幼苗。
(2)含有转基因元件Ms45-Oy1的杂交后代的检测
观察大田中栽培的杂交后代的表型，杂交后代植株颜色如图7所示，其中，含有转基因元件Ms45-Oy1的杂交后代如图7A所示，植株表现为黄色(检测可育)；而不含有转基因元件Ms45-Oy1杂交后代如图7B所示，植株表现为绿色(检测不育)。
(3)含有转基因元件Ms45-Wi2的杂交后代的检测
观察大田中栽培的杂交后代的表型，杂交后代植株萎蔫程度如图8所示，其中，含有转基因元件Ms45-Wi2的杂交后代如图8A所示，植株表现为萎蔫(检测可育)；而不含有转基因元件Ms45-Wi2杂交后代的植株如图8B所示，植株表现为正常(检测不育)。
表3.培养基的溶质及浓度
Figure PCTCN2020095082-appb-000014
上表中，MS盐购自phyto Technology Laboratories公司，货号为M524。
另外，通过与上述类似的方法，分别利用重组农杆菌EHA105/pMs45-Oy1-CWI-2和EHA105/pMs45-Lc-CWI-2，制备了转基因pMs45-Oy1-CWI-2玉米(其含有转基因元件Ms45-Oy1-CWI-2)和转基因pMs45-Lc-CWI-2玉米(其含有转基因元件Ms45-Lc-CWI-2)。
实施例3.利用雄性不育保持系对雄性不育系进行大规模扩繁
1、雄性不育系的制备
以ms45纯合隐性突变体(Maize Genetics Cooperation Stock Center，905I)为母本，与郑58(河南省农科院粮作所)杂交，获得的F1继续与玉米自交系郑58回交。对获得的BC1(回交一代)群体进行基因型分析，筛选其中Ms45位点为杂合的植株继续与郑58回 交。如此回交5-6代后，利用分子标记筛选Ms45位点为杂合，农艺性状表型与郑58接近的单株进行自交，从而获得ms45纯合隐性自交系郑58(ms45ms45)，该自交系即可作为雄性不育系植株。
上述筛选Ms45位点的基因型的方法如下：用如下引物Ms45F1(SEQ ID NO:16)和Ms45R1(SEQ ID NO:17)对植物基因组进行PCR扩增，并对扩增结果进行测序。Ms45目的片段大小为859bp，ms45目的片段大小为811bp。若扩增产物同时包含859bp和811bp目的片段，则该位点的基因型为杂合的Ms45/ms45；若扩增产物不含811bp片段，则该位点的基因型为Ms45/Ms45显性纯合；若扩增产物不含859bp目的片段，则该位点的基因型为ms45/ms45隐性纯合。
Ms45F1:5'CTTGAGCGACAGCGGGAACT 3'(SEQ ID NO:16)；
Ms45R1:5'TGTTGTTTCTTGGCAAAGGTCAG 3'(SEQ ID NO:17)。
2、保持系的制备
(1)制备Ms45-Lc杂合且ms45纯合的保持系植株
将实施例2中的T0代转pMs45-Lc玉米作为父本，与上述得到的作为母本的郑58(ms45ms45)杂交，从杂交后代中挑选紫色籽粒播种到田间后喷施200mM的双丙胺磷。将存活的植株继续与母本郑58回交。在回交过程中一直选择紫色籽粒及植株与母本杂交，如此回交5-6代后，后代中紫色籽粒及植株的转基因位点(Ms45-Lc)均为杂合。利用紫色籽粒及植株的花粉给母本授粉，如果得到的正常籽粒(黄色籽粒)或正常植株(绿色植株)均为不育，则提供花粉的植株转基因位点Ms45-lc为杂合且ms45位点为隐形纯合，即为保持系植株。
(2)制备Ms45-Oy1杂合且ms45纯合的保持系植株
将实施例2中的T0代转pMs45-Oy1玉米作为父本，与上述得到的作为母本的郑58(ms45ms45)杂交，从杂交后代中挑选黄色植株播种到田间后喷施200mM的双丙胺磷。将存活的植株继续与母本郑58回交。在回交过程中一直选择黄色植株与母本杂交，如此回交5-6代后，后代中黄色植株转基因位点(Ms45-Oy1)均为杂合。利用黄色植株的花粉给母本授粉，如果得到的正常植株(绿色植株)均为不育，则提供花粉的植株转基因位点Ms45-Oy1为杂合且ms45位点为隐形纯合，即为保持系植株。
(3)制备Ms45-Wi2杂合且ms45纯合的保持系植株
将实施例2中的T0代转pMs45-Wi2玉米作为父本，与上述得到的作为母本的郑58(ms45ms45)杂交，从杂交后代中挑选萎蔫植株(萎蔫程度如图8A所示，主要表现为心 叶卷曲)播种到田间后喷施200mM的双丙胺磷。将存活的植株继续与母本回交。在回交过程中一直选择萎蔫植株与母本杂交，如此回交5-6代后，代中萎蔫植株转基因位点(Ms45-Wi2)均为杂合。利用萎蔫植株的花粉给母本授粉，如果得到的正常植株(绿色植株)均为不育，则提供花粉的植株转基因位点Ms45-Wi2为杂合且ms45位点为隐形纯合，即为保持系植株。
3、保持系的获得
将获得的保持系植株(Ms45-Lc杂合且ms45ms45)作为父本，与作为母本的郑58进行杂交，产生的后代不但有雄性不育系(ms45ms45)，而且还有保持系(Ms45-Lc杂合且ms45ms45)。并且雄性不育系(ms45ms45)的籽粒显现正常，而其保持系(Ms45-Lc杂合且ms45ms45)籽粒为紫色(图2中B所示籽粒)。具体选育过程见附图6。
将获得的保持系植株(Ms45-Oy1杂合且ms45ms45)作为父本，与作为母本的郑58进行杂交，产生的后代不但有雄性不育系(ms45ms45)，而且还有保持系(Ms45-Oy1杂合且ms45ms45)。并且雄性不育系植株(ms45ms45)正常，显示绿色(图7B所示)，而其保持系(Ms45-Oy1杂合且ms45ms45)植株为黄色。
将获得的保持系植株(Ms45-Wi2杂合且ms45纯合)作为父本，与作为母本的郑58进行杂交，产生的后代不但有雄性不育系(ms45ms45)，而且还有保持系(Ms45-Wi2杂合且ms45ms45)。并且雄性不育植株(ms45ms45)正常(图8B所示)，而其保持系(Ms45-Wi2杂合且ms45ms45)植株显示萎蔫状态。
4、使用雄性不育保持系对雄性不育系进行大规模扩繁
以郑58自交系为例，将得到的雄性不育系郑58(ms45ms45)分别和上述获得的保持系(Ms45-Lc杂合且ms45ms45、Ms45-Oy1杂合且ms45ms45以及Ms45-Wi2杂合且ms45ms45)进行播种。两个材料相隔播种，每播种1行保持系相应的播种5行不育系，并确保繁种周边300米内无其他玉米播种，让不育系与保持系田间自然授粉。
保持系只能接受自己的花粉，会产生两种后代。一种为表现出转基因元件外在性状的后代(例如，紫色籽粒和紫色植株、黄色植株、萎蔫植株)，这些后代的转基因元件可能是纯合的，可能是杂合的，较难辨别。因此，这些籽粒或植株予以丢弃。而第二种外在性状正常的后代，不含有转基因元件，可以作为不育系后代并保留。
不育系材料接受了保持系的花粉，会产生两种后代。一种为表现出转基因元件外在性状的后代(例如，紫色籽粒和紫色植株、黄色植株、萎蔫植株)，这些后代的转基因元件是杂合的，可以作为保持系后代并保留。另一种为外在性状正常的后代(例如，黄色籽粒， 绿色植株，不萎蔫植株)，不含有转基因元件，可以作为不育系后代并保留。保持系可用于下一年继续扩繁不育系和保持系，而不育系中大部分用于生产商品种，剩余的小部分用于下一年继续扩繁不育系和保持系，具体生产流程如附图6所示。
另外，通过与上述类似的方法，获得了Ms45-Oy1-CWI-2杂合且ms45纯合的保持系植株，以及，Ms45-Lc-CWI-2杂合且ms45纯合的保持系植株，它们都可用于对雄性不育系(ms45ms45)进行大规模扩繁。
实施例4.利用雄性不育系大规模生产杂交种
在实施例3中生产的不育系为细胞核控制的隐性纯合不育系，该不育系可以被任意的野生型植株(Ms45Ms45)恢复育性。因此只要选择一个与雄性不育系(例如，郑58)配合力高的自交系(例如，昌7-2)进行杂交，便可以生产出农艺性状优良的杂交种。
为了达到以上目的，将郑58与昌7-2隔行播种于田间，并确保繁种周边300米内无其他玉米播种。使得不育系的果穗只能接受野生型自交系的花粉，而野生型自交系只能自交。如此，不育系果穗上所产生的种子既为优势的杂交种。
实施例5：不育系繁育杂交种的质量评估
之前，本申请发明人已利用同时含有Ms45和mn1RNAi的载体(中国专利ZL201210406155.6)，创建了以籽粒大小标记的雄性不育保持系。然而，由于异雄核受精现象(双受精过程中同一个种子的胚和胚乳由2个不同的雄配子体形成的精子受精获得)的存在，利用此类雄性不育保持系生产的雄性不育系子代群体无法实现100％的不育性。具体来说，我们利用中国专利ZL201210406155.6中描述的方法，扩繁制备了大量不育系玉米种子；随后，将其播种于田间，并在散粉期观测记录散粉植株的数量。结果显示，10万株中共观测到320株散粉植株(此类植株含有源自保持系的转基因成分)。这表明，所获得的不育系种子中，混杂了少量具有雄性可育的个体(约占所生产的不育系后代群体的3.2‰)。进一步，将此类混有雄性可育个体(即保持系个体)的不育系植株群体与目标玉米品系(如昌7-2)植株进行杂交，制备杂交种。然后，将所生产的杂交种播种于田间，随机挑选了10000株进行基因检测。结果显示，含有源自保持系的转基因成分的杂交种有210株，占比达到2.1％。该结果表明，所述生产的杂交种的种子纯度仍然有待提高，不能完全满足生产要求。这对杂交种市场化带来了不良影响和潜在风险。
在本实施例中，利用Lc基因对植株进行了双重标记(种子颜色和植株颜色)，解决了异雄核受精所引发的问题，显著提高了所生产的杂交种的种子纯度(达到100％)。
简言之，将上述实施例获得的保持系植株(转基因pMs45-Lc/ms45ms45玉米)和不育系植株(ms45ms45玉米)相隔播种，每播种1行保持系相应的播种5行不育系，确保繁种周边300米内无其他玉米播种，让不育系植株与保持系植株田间自然授粉。收集不育系植株的后代种子，并根据种子的颜色进行首次的筛选，即，区分不育系子代(种子为黄色)和保持系子代(种子为紫色)。
然后，将获得的不育系种子(作为母本)和目标玉米品系种子(作为父本，昌7-2自交系)相隔播种，每播种1行目标玉米品系种子，相应的播种5行不育系种子，并且，确保繁种周边300米内无其他玉米播种。在玉米生长的苗期，观察不育系植株的外在性状，并且，从不育系植株中去除显示紫色植株颜色的植株。经统计，10万株中不育系植株中，有290株紫色苗被去除，即，扩繁的不育系的纯度为99.71％。
筛选后，让不育系植株与目标玉米品系植株田间自然授粉。收集不育系植株上结出的杂交种子。然后，将所生产的杂交种子播种于田间，并随机挑选10000株进行基因检测，以确定杂交种子中含有源自保持系的转基因成分(pMs45-Lc)的种子比例，评估杂交种子的质量(纯度)。实验结果显示，在利用种子颜色和植株颜色进行双重筛选后，所产生的杂交种子的纯度达到100％，即，所有杂交种子均不含有源自保持系的转基因成分(pMs45-Lc)。
基于这一结果可以确定：通过利用本发明的雄性不育保持系，借助于双重筛选(种子筛选和苗期筛选)，可在苗期实现子代不育系植株和子代保持系植株各自100％的纯度。本发明的雄性不育保持系以及种子繁育方法可用于产生高纯度的不育系后代种子以及高纯度的杂交种子。
实施例6：不育系繁育杂交种的质量评估
在本实施例中，本发明人利用籽粒大小(CWI-2基因的干扰RNA)和植株颜色(Oy1基因)两个性状对植株进行标记，由此，在制种过程中，在苗期即可将母本不育系地块中由于异雄核受精产生的具有育性的保持系(即，黄色苗；约3‰)去除，从而在散粉期确保所有母本100％的雄性不育，进而改善和确保了生产的杂交种的纯度，很好的满足了生产的需求。
简言之，根据上述实施例中描述的实验方法，将Ms45基因(SEQ ID NO:1)、Oy1基因(SEQ ID NO:6)和编码CWI-2基因的干扰RNA的核苷酸(SEQ ID NO:18)构建至 载体中，并制备获得了保持系植株，即转基因玉米植株pMs45-Oy1-CWI-2/ms45ms45，其能够表达Ms45蛋白，Oy1蛋白，以及抑制CWI-2基因的干扰RNA。将获得的保持系植株(pMs45-Oy1-CWI-2/ms45ms45)和不育系植株(ms45ms45玉米)相隔播种，每播种1行保持系相应的播种5行不育系，确保繁种周边300米内无其他玉米播种，让不育系植株与保持系植株田间自然授粉。收集不育系植株的后代种子，并根据种子的大小进行首次的筛选，即，区分不育系子代(种子大小为正常)和保持系子代(种子大小为小粒)。
然后，将获得的不育系种子(作为母本，种子大小为正常)和目标玉米品系种子(作为父本，昌7-2自交系，种子大小为正常)相隔播种，每播种1行目标玉米品系种子，相应的播种5行不育系种子，并且，确保繁种周边300米内无其他玉米播种。在玉米生长的苗期，观察不育系植株的外在性状，并且，从不育系植株中去除显示黄色植株颜色的植株。经统计，10万株不育系植株中有310株黄色苗被去除，即，扩繁的不育系的纯度为99.69％。
筛选后，让不育系植株与目标玉米品系植株田间自然授粉。收集不育系植株上结出的杂交种子。然后，将所生产的杂交种子播种于田间，并随机挑选10000株进行基因检测，以确定杂交种子中含有源自保持系的转基因成分(pMs45-Oy1-CWI-2)的种子比例，评估杂交种子的质量(纯度)。实验结果显示，在利用种子颜色和植株颜色进行双重筛选后，所产生的杂交种子的纯度达到100％，即，所有杂交种子均不含有源自保持系的转基因成分(pMs45-Oy1-CWI-2)。
基于这一结果可以确定：通过利用本发明的雄性不育保持系，借助于双重筛选(种子筛选和苗期筛选)，可在苗期实现子代不育系植株和子代保持系植株各自100％的纯度。本发明的雄性不育保持系以及种子繁育方法可用于产生高纯度的不育系后代种子以及高纯度的杂交种子。
实施例7：不育系繁育杂交种的质量评估
在本实施例中，本发明人利用籽粒大小(CWI-2基因的干扰RNA)和植株颜色(Lc基因)两个性状对植株进行标记，由此，在制种过程中，在苗期即可将母本不育系地块中由于异雄核受精产生的具有育性的保持系(即，紫色苗；约3‰)去除，从而在散粉期确保所有母本100％的雄性不育，进而改善和确保了生产的杂交种的纯度，很好的满足了生产的需求。
简言之，根据上述实施例中描述的实验方法，将Ms45基因(SEQ ID NO:1)、Lc基因(SEQ ID NO:4)和编码CWI-2基因的干扰RNA的核苷酸(SEQ ID NO:18)构建至载 体中，并制备获得了保持系植株，即转基因玉米植株pMs45-Lc-CWI-2/ms45ms45玉米，其能够表达Ms45蛋白，Lc蛋白，以及抑制CWI-2基因的干扰RNA。将获得的保持系植株(pMs45-Lc-CWI-2/ms45ms45玉米)和不育系植株(ms45ms45玉米)相隔播种，每播种1行保持系相应的播种5行不育系，确保繁种周边300米内无其他玉米播种，让不育系植株与保持系植株田间自然授粉。收集不育系植株的后代种子，并根据种子的大小进行首次的筛选，即，区分不育系子代(种子大小为正常)和保持系子代(种子大小为小粒)。
然后，将获得的不育系种子(作为母本，种子大小为正常)和目标玉米品系种子(作为父本，昌7-2自交系，种子大小为正常)相隔播种，每播种1行目标玉米品系种子，相应的播种5行不育系种子，并且，确保繁种周边300米内无其他玉米播种。在玉米生长的苗期，观察不育系植株的外在性状，并且，从不育系植株中去除显示紫色植株颜色的植株。经统计，10万株不育系植株中有305株紫色苗被去除，即，扩繁的不育系的纯度为99.695％。
筛选后，让不育系植株与目标玉米品系植株田间自然授粉。收集不育系植株上结出的杂交种子。然后，将所生产的杂交种子播种于田间，并随机挑选10000株进行基因检测，以确定杂交种子中含有源自保持系的转基因成分(pMs45-Lc-CWI-2)的种子比例，评估杂交种子的质量(纯度)。实验结果显示，在利用种子颜色和植株颜色进行双重筛选后，所产生的杂交种子的纯度达到100％，即，所有杂交种子均不含有源自保持系的转基因成分(pMs45-Lc-CWI-2)。
基于这一结果可以确定：通过利用本发明的雄性不育保持系，借助于双重筛选(种子筛选和苗期筛选)，可在苗期实现子代不育系植株和子代保持系植株各自100％的纯度。本发明的雄性不育保持系以及种子繁育方法可用于产生高纯度的不育系后代种子以及高纯度的杂交种子。

Claims (12)

  1. 一种分离的核酸分子，其包含第一多核苷酸和第二多核苷酸，所述第一多核苷酸包含恢复基因的核苷酸序列，所述恢复基因能够恢复因雄性不育基因导致雄性不育的植物的雄性能育性；所述第二多核苷酸包含能够调控种子和/或植株的外在性状的筛选基因的核苷酸序列，所述外在性状选自种子颜色，种子尺寸，植株颜色，植株萎蔫程度，茎形态，叶形态，以及其任何组合；
    优选地，所述外在性状选自种子颜色，植株颜色，植株萎蔫程度，以及其任何组合；
    优选地，所述第二多核苷酸包括：(a)能够调控种子的外在性状(例如种子颜色或种子尺寸)的第一筛选基因，以及，(b)能够调控植株的外在性状(例如植株颜色或植株萎蔫程度)的第二筛选基因；
    优选地，所述第一筛选基因和第二筛选基因是相同的或者不同的；
    优选地，所述第二多核苷酸含有能够控制种子的外在性状与植株的外在性状的一种筛选基因，例如Lc基因；
    优选地，所述第二多核苷酸包括：
    (1)能够调控种子颜色的第一筛选基因和能够调控植株颜色的第二筛选基因，
    (2)能够调控种子颜色的第一筛选基因和能够调控植株萎蔫程度的第二筛选基因，
    (3)能够调控种子尺寸的第一筛选基因和能够调控植株颜色的第二筛选基因，或，
    (4)能够调控种子尺寸的第一筛选基因和能够调控植株萎蔫程度的第二筛选基因；
    优选地，所述第二多核苷酸包括：Lc基因和Wi2基因；或者，编码CWI-2基因的干扰RNA的核苷酸和Lc基因；或者，编码CWI-2基因的干扰RNA的核苷酸和Oy1基因；或者，编码CWI-2基因的干扰RNA的核苷酸和Wi2基因；
    优选地，所述Lc基因编码氨基酸序列为SEQ ID NO:5的蛋白；优选地，所述Lc基因的核苷酸序列为SEQ ID NO:4；
    优选地，所述编码CWI-2基因的干扰RNA的核苷酸具有如SEQ ID NO:18所示的核苷酸序列；
    优选地，所述Oy1基因编码氨基酸序列为SEQ ID NO:7的蛋白；优选地，所述Oy1基因的核苷酸序列为SEQ ID NO:6；
    优选地，所述Wi2基因编码氨基酸序列为SEQ ID NO:9的蛋白；优选地，所述Wi2基因的核苷酸序列为SEQ ID NO:8；
    优选地，所述第二多核苷酸还包含，与所述筛选基因的核苷酸序列可操作连接的表达调控元件，例如启动子和增强子；
    优选地，所述启动子选自：组成型启动子，诱导型启动子，组织优选的启动子，组织特异性启动子，生长期优选的启动子。
  2. 权利要求1所述的核酸分子，其中，所述雄性不育基因为隐性雄性不育基因，其在纯合状态下导致植物雄性不育；
    优选地，所述雄性不育基因选自ms1，ms2，ms3，ms4，ms5，ms6，ms7，ms8，ms9，ms10，ms11，ms12，ms13，ms14，ms15，ms16，ms17，ms18，ms19，ms20，ms21，ms22，ms23，ms24，ms25，ms26，ms27，ms28，ms29，ms30，ms31，ms32，ms33，ms34，ms35，ms36，ms37，ms38，ms43，ms45，ms47，ms48，ms49，ms50，ms52，以及其任何组合；优选地，所述雄性不育基因为ms45；优选地，所述恢复基因选自Ms1，Ms2，Ms3，Ms4，Ms5，Ms6，Ms7，Ms8，Ms9，Ms10，Ms11，Ms12，Ms13，Ms14，Ms15，Ms16，Ms17，Ms18，Ms19，Ms20，Ms21，Ms22，Ms23，Ms24，Ms25，Ms26，Ms27，Ms28，Ms29，Ms30，Ms31，Ms32，Ms33，Ms34，Ms35，Ms36，Ms37，Ms38，Ms43，Ms45，Ms47，Ms48，Ms49，Ms50，Ms52，以及其任何组合；优选地，所述恢复基因为Ms45；
    优选地，所述雄性不育基因为ms45，并且，所述恢复基因为Ms45；
    优选地，所述恢复基因编码氨基酸序列为SEQ ID NO:2的蛋白；优选地，所述恢复基因的核苷酸序列为SEQ ID NO:1；
    优选地，所述第一多核苷酸还包含，与所述恢复基因的核苷酸序列可操作连接的表达调控元件，例如启动子和增强子；
    优选地，所述启动子选自：组成型启动子，诱导型启动子，组织优选的启动子，组织特异性启动子，生长期优选的启动子；
    优选地，所述启动子的核苷酸序列为SEQ ID NO:15；
    优选地，所述第一多核苷酸序列包含或者由SEQ ID NO:3组成；
    优选地，所述第一多核苷酸和第二多核苷酸通过或者不通过连接核苷酸而共价相连；
    优选地，所述连接核苷酸的长度不超过10kb，不超过5kb，不超过1kb，不超过500bp，不超过100bp，不超过50bp，不超过10bp，不超过5bp，或者更短；
    优选地，所述第一核苷酸序列与所述第二核苷酸序列是基因连锁的；
    优选地，所述分离的核酸分子还包含第三多核苷酸，所述第三多核苷酸包含选择标记基因的核苷酸序列；
    优选地，所述选择标记基因为抗生素抗性基因或除草剂抗性基因，例如双丙氨磷抗性基因(bar基因)；
    优选地，所述选择标记基因的核苷酸序列为SEQ ID NO:10。
  3. 一种载体，其包含权利要求1或2所述的分离的核酸分子。
  4. 一种宿主细胞，其包含，权利要求1或2所述的分离的核酸分子或者权利要求3的载体；
    优选地，所述宿主细胞为农杆菌细胞或植物细胞；
    优选地，所述植物细胞为单子叶植物或双子叶植物的细胞；
    优选地，所述植物细胞为选自下列的植物的细胞：玉米、油菜、稻、拟南芥、大麦、小麦、高粱、大豆、苜蓿、烟草、棉花、向日葵或甘蔗。
  5. 一种植株或植物种子，其中，所述植株或植物种子在基因组中包含权利要求1或2所述的核酸分子；
    优选地，所述植株或植物种子在基因组中还包含所述雄性不育基因；优选地，所述雄性不育基因是纯合的隐性雄性不育基因；
    优选地，所述分离的核酸分子整合在所述植株或植物种子的基因组中；优选地，所述核酸分子整合在所述植株或植物种子的基因组中，且位于与所述雄性不育基因不同的染色体上；优选地，所述核酸分子以杂合的形式存在于所述植株或植物种子的基因组中；
    优选地，所述植株或植物种子是雄性能育的；
    优选地，所述植株或植物种子能够用作包含所述雄性不育基因的雄性不育植株的保持系；
    优选地，所述植株或植物种子为单子叶植物或双子叶植物的植株或植物种子；
    优选地，所述植株或植物种子为玉米、油菜、稻、拟南芥、大麦、小麦、高粱、大豆、苜蓿、烟草、棉花、向日葵或甘蔗的植株或植物种子。
  6. 一种获得植株的方法，所述方法包括，(1)将权利要求1或2所述的核酸分子或权 利要求3所述的载体导入植物细胞，以及，(2)将所述植物细胞培养成植株；
    优选地，在步骤(1)中，使用农杆菌将所述核酸分子或载体导入植物细胞；
    优选地，所述植物细胞在其基因组中包含所述雄性不育基因，并且在导入所述核酸分子或载体之前是雄性不育的；优选地，所述植物细胞在其基因组中包含纯合的隐性雄性不育基因；
    优选地，在步骤(1)中，所述核酸分子被整合入所述植物细胞的基因组中；优选地，所述核酸分子在整合入所述植物细胞的基因组后，位于与所述雄性不育基因不同的染色体上；
    优选地，所述植物细胞为单子叶植物或双子叶植物的细胞；
    优选地，所述植物细胞为选自下列的植物的细胞：玉米、油菜、稻、拟南芥、大麦、小麦、高粱、大豆、苜蓿、烟草、棉花、向日葵或甘蔗；
    优选地，所述植株包含纯合的隐性雄性不育基因以及所述核酸分子或载体，并且，其是雄性能育的；优选地，所述核酸分子或载体在所述植株中以杂合的形式存在于基因组中；优选地，所述核酸分子整合在所述植株的基因组中，且位于与所述雄性不育基因不同的染色体上；
    优选地，所述植株能够用作包含所述雄性不育基因的雄性不育植株的保持系植株；
    优选地，所述方法还包括：
    (3)用步骤(2)的植株对包含所述雄性不育基因的雄性不育植株授粉，产生后代种子或植株；和
    (4)筛选显示所述筛选基因调控的外在性状的后代种子或植株。
  7. 一种获得雄性不育系植株和保持系植株的后代种子或植株的方法，所述方法包括，将权利要求5所述的植株或通过权利要求6的方法获得的植株作为父本与作为母本的包含所述雄性不育基因的雄性不育植株杂交，并产生后代种子或植株；
    优选地，所述方法包括：
    (1)提供作为母本的包含所述雄性不育基因的雄性不育植株；优选地，所述雄性不育基因是纯合的隐性雄性不育基因；
    (2)提供作为父本的权利要求5所述的植株或通过权利要求6的方法获得的植株；
    (3)用步骤(2)的植株对步骤(1)的植株授粉，以产生后代种子；
    (4)任选地，将所述后代种子培养成后代植株；
    其中，显示所述筛选基因调控的外在性状的后代种子或植株是雄性能育的，能够用作保持系；且，不显示所述筛选基因调控的外在性状的后代种子或植株是雄性不育的，能够用作雄性不育系；
    优选地，所述方法包括：
    (1)提供作为母本的包含所述雄性不育基因的雄性不育植株；优选地，所述雄性不育基因是纯合的隐性雄性不育基因；
    (2)提供作为父本的权利要求5所述的植株或通过权利要求6的方法获得的植株；其中，所述第二多核苷酸包括：(a)能够调控种子的外在性状(例如种子颜色或种子尺寸)的第一筛选基因，以及，(b)能够调控植株的外在性状(例如植株颜色或植株萎蔫程度)的第二筛选基因；
    (3)用步骤(2)的植株对步骤(1)的植株授粉，产生两种后代种子；其中，第一种后代种子显示所述第一筛选基因调控的种子的外在性状；且，第二种后代种子不显示所述第一筛选基因调控的种子的外在性状；
    (4)分离所述第一种和第二种后代种子，且任选地，将它们分别培养成第一种和第二种后代植株；
    任选地，所述方法还包括下述步骤：
    (5)从第一种后代植株中去除这样的植株，其不显示所述第二筛选基因调控的植株的外在性状，由此，剩余的第一种后代植株为雄性能育的，能够用作保持系植株；和/或
    从第二种后代植株中去除这样的植株，其显示所述第二筛选基因调控的植株的外在性状，由此，剩余的第二种后代植株为雄性不育的，能够用作雄性不育系植株；
    (6)用所述剩余的第一种后代植株对所述剩余的第二种后代植株授粉，并产生进一步的后代种子。
  8. 一种制品，其由权利要求5所述的植株或其部分制成；
    优选地，所述制品为食品，并且其由权利要求5所述的植株的可食用部分(例如种子)制成。
  9. 权利要求1或2所述的核酸分子或权利要求3所述的载体或权利要求4所述的宿主细胞(例如植物细胞)用于产生保持系植株的用途；
    优选地，所述植株为单子叶植物或双子叶植物的植株；
    优选地，所述植株为选自下列的植物：玉米、油菜、稻、拟南芥、大麦、小麦、高粱、大豆、苜蓿、烟草、棉花、向日葵或甘蔗。
  10. 通过权利要求6所述的方法获得的植株或通过权利要求7所述的方法获得的后代种子或植株或权利要求5所述的植株用于产生杂交子代的用途；
    优选地，所述植株为单子叶植物或双子叶植物的植株；
    优选地，所述植株为选自下列的植物：玉米、油菜、稻、拟南芥、大麦、小麦、高粱、大豆、苜蓿、烟草、棉花、向日葵或甘蔗。
  11. 一种组织培养物或由其产生的原生质体，其中，所述组织培养物包含权利要求4所述的宿主细胞(例如植物细胞)或者通过权利要求6所述的方法获得的植株的细胞或通过权利要求7所述的方法获得的后代种子或植株的细胞；
    优选地，所述植株为单子叶植物或双子叶植物的植株；
    优选地，所述植株为选自下列的植物：玉米、油菜、稻、拟南芥、大麦、小麦、高粱、大豆、苜蓿、烟草、棉花、向日葵或甘蔗。
  12. 一种制备杂交种子的方法，所述方法包括：
    (1)提供通过权利要求7的方法获得的雄性不育系植株的后代种子，其不显示所述第一筛选基因调控的种子的外在性状；并且，提供目标品系植株的种子；
    (2)将所述雄性不育系植株的后代种子与所述目标品系植株的种子田间播种，以获得雄性不育系植株和目标品系植株；
    (3)从所述雄性不育系植株中去除这样的植株，其显示所述第二筛选基因调控的植株的外在性状；
    (4)用所述目标品系植株对剩余的雄性不育系植株授粉；
    (5)从雄性不育系植株上收获种子，即为杂交种子。
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