WO2014063442A1 - 扩繁植物雄性不育系的方法 - Google Patents

扩繁植物雄性不育系的方法 Download PDF

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WO2014063442A1
WO2014063442A1 PCT/CN2013/001269 CN2013001269W WO2014063442A1 WO 2014063442 A1 WO2014063442 A1 WO 2014063442A1 CN 2013001269 W CN2013001269 W CN 2013001269W WO 2014063442 A1 WO2014063442 A1 WO 2014063442A1
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plant
male
seq
nucleotide sequence
gene
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PCT/CN2013/001269
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English (en)
French (fr)
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赖锦盛
赵海铭
宋伟彬
崔阳
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中国农业大学
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Priority claimed from CN201210406154.1A external-priority patent/CN102965391B/zh
Priority claimed from CN201210406155.6A external-priority patent/CN102960234B/zh
Application filed by 中国农业大学 filed Critical 中国农业大学
Priority to US14/437,633 priority Critical patent/US10246723B2/en
Publication of WO2014063442A1 publication Critical patent/WO2014063442A1/zh
Priority to US16/370,628 priority patent/US11572573B2/en

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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287—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
    • 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
    • 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
    • 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
    • A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
    • A01H1/045—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection using molecular markers
    • 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
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
    • C12N15/821—Non-antibiotic resistance markers, e.g. morphogenetic, metabolic markers
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • 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

Definitions

  • the invention relates to a new method for expanding a plant male sterile line, which utilizes a nuclear male sterility gene, a grain marker gene and a transgenic technology to expand a plant male sterile line, and belongs to the field of plant genetic breeding and seed production.
  • hybrids Due to the existence of heterosis, the biomass, resistance to pests and diseases, stress tolerance (drought, high temperature, low temperature, salinity, etc.) of hybrids are considerably higher than that of their parents, such as hybrid corn and hybrid rice. Higher than homozygous parents.
  • the commonly used methods for producing hybrids are: planting the female parent with the male parent, removing the tassel of the female parent, and retaining the tassel of the male parent, and the seed obtained by the female parent is the hybrid.
  • Self-pollination refers to the pollen of a plant, which pollinates the pistil of the same individual. In the plants of bisexual flowers, it can be divided into two-flowered pollination between the stamens and pistils of the same flower (Nymphaea) and pollination of adjacent flowers in different inflorescences (individuals), and different plants. Cross-pollination of pollination between flowers. Some plant stamens and pistils do not grow in the same flower, do not even grow on the same plant, can not self-pollinate, their pistils can only get pollen of other flowers, this is called cross-pollination. A type of crop with a natural hybridization rate higher than 50% and self-deteriorating is classified as a regular cross-pollination crop, such as corn.
  • Maize is monoecious, and male and female flowers are located in different parts of the plant. Maize can be propagated by self-pollination or by cross-pollination. Under natural conditions, when the wind blows pollen from the tassel to the filament of the ear. That is, natural pollination is completed.
  • homozygous maize inbred lines should be developed first, then two inbred lines are crossed, and the yield and stress resistance of the hybrid offspring are evaluated to determine whether they have commercial potential.
  • Each of these inbred lines may have one or more superior traits that are lacking in another inbred line, or one or more undesirable traits that complement another inbred line.
  • the first generation of seeds crossed by two inbred lines is
  • F1 generation seeds F1 generation seeds were germinated to obtain F1 generation plants, F1 generation plants were more robust than two inbred parental parents (parent), and had more biomass.
  • Hybrids can be produced by artificial emasculation of the female parent, that is, the mother of the undissolved powder (which can be planted in the field with the male parent, such as seeding 5 rows of female parents, a row of male parents). The tassel is removed, and the male tassel is kept. . Then, as long as the foreign corn pollen is isolated, the female parent can only accept the male pollen, and the obtained seed is the hybrid (F1), which can be used for agricultural production.
  • the hybrid (F1) which can be used for agricultural production.
  • the plants may be tasseled after emasculation, or emasculation is incomplete. Both of the above cases can lead to self-pollination of the female parent, resulting in the hybridization of the produced hybrids.
  • the seed of the female inbred line, the yield of the female inbred line is much lower than the yield of the hybrid.
  • Such seeds are unqualified products, which will affect the income of farmers and affect the reputation of the seed production company, which will lead to seed production.
  • the company bears the corresponding liability for compensation.
  • the machine can also be used to emasculate the female parent. The reliability of the machine emasculation and manual emasculation is basically the same, but faster and cheaper. However, most of the emasculation machines cause more damage to the plants than manual emasculation. Therefore, there is currently no satisfactory method of emasculation, and people are still looking for a more costly alternative to emasculation. .
  • Stable male sterility system provides a simple and efficient means of using nuclear-mass interaction for male sterility
  • CMS Inbred lines can be used in some genotypes to avoid heavy emasculation.
  • the means consists of three main materials, namely the sterile line: male sterile material, maintainer: can provide pollen for the sterile line, so that the offspring of the sterile line are still sterile, restorer: can restore the sterile line Fertility.
  • the hybrid line between the sterile line and the restorer line produces Fl, a hybrid used for agricultural production. More specifically, the nuclear-mass interaction is infertile, which is characterized by nuclear-mass interaction.
  • cytoplasm require a sterility gene s, but also a homozygous sterility gene (rfrf) in the nucleus, which exists at the same time, so that the plant can be expressed as male sterility.
  • rfrf homozygous sterility gene
  • the cytoplasmic gene is fertile N, then whether the nuclear gene is fertile (RfRf) or sterile (rfrf), it is male fertile.
  • the nucleus has a fertile gene (RfRf) or (Rfrf)
  • whether the cytoplasmic gene is fertile N or sterile S, it also appears to be male fertile.
  • This male-sterile line formed by nuclear-mass interaction has a genetic composition of S (rfrf) and cannot produce normal pollen, but can be used as a hybrid parent. Since the maintainer N (rfrf) can be found [using it to hybridize with the sterile line, the resulting F1 can still maintain male sterility, ⁇ : S (rfrf) ( ) XN (rfrf) ⁇ S (rfrf) (infertility) )] and can accept restorer S (RfRf) or N (RfRf) [use them to hybridize with sterile lines, the resulting F1 is fertile, BP: S (rfrf) (early) XS (RfRf) ⁇ S (Rfrf) (Fl) ( fertile), or S ( rfrf ) ( ) XN (RfRf) ⁇ S (Rfrf) (Fl) ( fertile) ] pollen, restore Fl to male fertility,
  • the male sterile line can eliminate artificial emasculation, save manpower, reduce seed costs, and ensure seed purity.
  • rice, maize, sorghum, onion, ramie, sugar beet and rapeseed have used nuclear-tolerant male sterility for hybrid seed production; nuclear-mass interaction male sterile lines for other crops are also undergoing extensive Research.
  • CMS also has its drawbacks. First, it is observed that individual CMS materials are susceptible to disease, and second, recovery systems are more difficult to find. These problems hinder the widespread application of CMS systems in seed production.
  • Fabijanski, et al. have developed a method for male sterility in plants (EP 0 89/3010153.8 Publication No. 329308 and PCT Application PCT/CA90/ published as W 90/08828). 00037).
  • the main method is to inhibit the male flower fertility of the plant by one of the following two methods.
  • One method is to transfer the promoter specifically expressed by the male tissue to the cytotoxin gene and transfer it into the plant, so that the male flower can not normally loose powder without affecting other traits;
  • genetic interference the genes of the male plants that have been cloned to control the male flowers are interfered by means of transgenic means. This makes it impossible to function properly.
  • msms nuclear recessive homozygous plants that control male sterility will behave as male sterility. Because male sterility plants are unable to self-interest, they can only hybridize with heterozygous plants (Msms). Only male sterile plants (msms) will be obtained. And male sterile seeds (msms) and fertile hybrid grains (Msms) exist simultaneously on the same ear. It is impossible to distinguish which are sterile seeds and which are fertile seeds through the grain. Can be distinguished.
  • the vector is transferred to a male sterile plant, and the vector is present in a heterozygous state in the transgenic plant, and the plant is fertile due to the restoration of the fertility gene, when it is crossed with the male sterile plant, due to the pollen containing the restorer gene (Msms) contains the lethal gene, which makes the pollen aborted. Therefore, only the pollen (ms) without the restorer gene can be crossed with the male gamete (ms) of the male sterile plant, and the offspring are all recessive homozygous individuals (msms).
  • Ms22 (mscal) 7 WEST D. R. , M. C. ALBERTSEN, 1985. Three new male-sterile genes. Maize Genet. Coop.
  • the present invention provides a method for expanding a plant male sterile line for maintaining a homozygous recessive state of a male sterile plant, the method comprising:
  • the second nucleotide sequence when it exists in a heterozygous state, can affect the grain shape or the endosperm nutrient composition, and the kernel containing the construct and the kernel not containing the construct can be distinguished by the naked eye or by an instrument;
  • the first nucleotide sequence is closely linked to the second nucleotide sequence, and the two nucleotide sequences are simultaneously present in the plant;
  • the grain shape may be a size, a length, a width, or/and a thickness, and the like; and the endosperm nutrient component may be a powdery endosperm, a starch content or/and an oil content, and the like.
  • the plant, the first plant and the second plant may each be a monocot or a dicot, such as corn, rice, sorghum, wheat, soybean, cotton or sunflower.
  • the first nucleotide sequence includes a gene for controlling male fertility, such as the wild type allele Ms45 of ms45 in Table 1, and the gene for controlling male fertility is not limited to the genes listed in Table 1.
  • Genes controlling male fertility in maize or other species may also achieve the objects of the present invention and are therefore within the scope of the present invention.
  • the first plant is a maize male sterile mutant ms45; and / or,
  • the first nucleotide sequence is an Ms45 expression element, and the Ms45 expression element expresses the protein Ms45 represented by SEQ ID No: 4 in the first plant.
  • SEQ ID No: 4 consists of 412 amino acids.
  • the Ms45 coding sequence in the Ms45 expression element is SEQ ID No: 8.
  • the nucleotide sequence of the Ms45 expression element is SEQ ID No: 1, including a promoter and a gene, wherein positions 8-254 of SEQ ID No: 1 are promoter sequences, and 142 ID2 of SEQ ID No: 1.
  • the 2972 bit is 0RF.
  • the inventors constructed a plant expression vector comprising the Ms45 expression element represented by SEQ ID No: 1, and the vector was transferred into the male sterile mutant ms45 to restore the culture of the mutant. Sex.
  • the second nucleotide sequence affects the size of the second plant kernel when present in the hybrid state in the second plant.
  • the second nucleotide sequence is a DNA fragment which interferes with the expression of the protein represented by SEQ ID No: 5.
  • SEQ ID No: 5 consists of 590 amino acid residues and is the amino acid sequence of the Mn1 protein.
  • the DNA fragment which interferes with the expression of the protein represented by SEQ ID No: 5 may be SEQ forward-X-SEQ reverse;
  • the nucleotide sequence of 850 ⁇ is position 14-276 of SEQ ID No: 2; the sequence of the SEQ is inversely complementary to the sequence of the SEQ; the X is the SEQ and the SEQ ⁇ The sequence between the spacers, in the sequence, the X is not complementary to the SEQ and the SEQ.
  • the nucleotide sequence of the SEQ forward-X-SEQ fi direction may be from positions 14-663 of SEQ ID No: 2.
  • SEQ ID No: 2 is a Mn1 interference fragment MnlRNAi which silences the Mn1 gene and is composed of 675 nucleotides.
  • the 7th to 3rd position of SEQ ID No : 2 is the BstEII recognition sequence
  • the 14th to 276th positions of SEQ ID No : 2 is the nucleotide sequence of Mn1SEQ ⁇
  • the 277 to 400th position of SEQ ID No : 2 is X
  • the intron of the hairpin structure is formed
  • the nucleotide sequence of Mnl SEQ fi is located at positions 401-463 of SEQ ID No: 2
  • the Hindlll recognition sequence is located at positions 664-669 of SEQ ID No. 2.
  • the second nucleotide sequence affects whether the kernel of the second plant is a silty endosperm when present in the second plant in a heterozygous state.
  • the second nucleotide sequence encodes the SEQ ID No: 7 MC16_KDY-gliadin.
  • SEQ ID No: 7 consists of 138 amino acids.
  • the MC16-KDY-gliadin gene in the second nucleotide sequence is at positions 1244-1780 of SEQ ID No: 6.
  • SEQ ID No: 6 consists of 1666 nucleotides, positions 91-1149 are promoter sequences, and positions 1244-1780 are coding sequences of MC16-KDY-gliadin gene, encoding MC16_KDY- of SEQ ID No: 7. Prolamin.
  • the second nucleotide sequence is specifically as shown in SEQ ID No: 6.
  • the present invention also protects the construct (DNA construct), the second plant, and the homozygous recessive male sterile plant produced by the above method.
  • the above DNA construct can restore the fertility of the male sterile mutant, while changing the shape of the grain (such as size, length, width, thickness, etc.) or simultaneously changing the nutrient composition of the grain endosperm (such as starch content, oil content, or silty) Endosperm, etc.).
  • the above second plant can maintain the sterility of the male sterile plants.
  • tissue culture of the regenerated cells produced by the second plant in the above method and the protoplasts produced from the tissue culture are also within the scope of the present invention.
  • the above plants may be all or part of a plant, such as seeds, roots, stems, leaves, embryos, root tips, pollen or anthers.
  • the inventors have invented a new method for efficiently expanding a plant male sterile line by controlling a plant male fertility gene, a grain marker gene, and a transgenic technique.
  • the seed marker gene is a nucleotide that controls a particular shape (such as size, length, width, thickness, thickness, etc.) of the grain; in another embodiment of the present invention, the seed marker group Because of the nucleotides that control the main nutrient components of the endosperm (such as starch content, oil content, whether silty endosperm, etc.).
  • the present invention combines the wild-type nucleotide sequence controlling the male fertility with the nucleotide sequence of the grain shape or the seed endosperm nutrient component, and transfers it to the conventional corn, and then returns to the homozygous recessive male sterile plant. After the obtained transgenic plants are crossed with the homozygous recessive sterile lines, a large number of sterile lines and maintainer lines can be simultaneously obtained.
  • the sterile line and the maintainer line can be distinguished by the grain shape or the endosperm component by controlling the shape of the grain or the nucleotide sequence of the nutrient component of the grain endosperm.
  • the seeds with normal shape or normal endosperm composition are sterile lines (without transgenic sequences), abnormal shape (such as grain size, length, width, thickness, etc.) or abnormalities of endosperm components (starch content, oil content, whether or not powder)
  • abnormal shape such as grain size, length, width, thickness, etc.
  • abnormalities of endosperm components starch content, oil content, whether or not powder
  • the inventors constructed a plant transformation vector comprising an expression element for restoring a male fertility gene and an expression element for controlling a grain shape (e.g., size, length, width, thickness, thickness, etc.),
  • the gene controlling the shape of the grain (such as size, length, width, thickness, etc.) is a dominant gene or a sequence of interference
  • the vector is transferred into the waxy corn hybrid, and then the obtained transgenic plant is subjected to the male sterile plant.
  • the nucleotide sequence controlling the male fertility of the plant, the special shape of the grain (such as size, length, width, thickness, etc.) is introduced into the male sterile plant.
  • the plant appears to be fertile due to the presence of a restorer gene.
  • Transgenic hybrid When the plant (Msmsms) is crossed with the male sterile plant (msms), the following two offsprings are produced, one is the normal male sterile seed (the sterile line, the genotype is msms), and the sterile line can be arbitrarily One wild type plant restores fertility; the other is fertile grain (maintenance line, genotype is Msmsms) with abnormal grain status, and the maintainer line is recessive homozygous in controlling male fertility locus, which contains complementary Transgenic sequence, the plant appears to be fertile, because it also contains a nucleotide sequence that affects the shape of the grain (such as size, length, width, thickness, etc.), the grain shape (such as size, length, width, thickness, etc.) is different from wild type.
  • the inventors constructed a plant transformation vector comprising an expression element for restoring a male fertility gene and controlling major nutrient components of the endosperm (eg, starch content, oil content, or silty) The endosperm, etc.
  • major nutrient components of the endosperm eg, starch content, oil content, or silty
  • the gene that controls the main nutrient components of the endosperm (such as starch content, oil content, silty endosperm, etc.) is a dominant gene or a sequence of interference, and the vector is transferred to ⁇ ⁇ ⁇ ⁇ In the ⁇ ⁇ maize hybrid, the male sterility plants are then used to backcross the obtained transgenic plants, thereby controlling the male fertility of the plant and the main nutrient components of the endosperm (such as starch content, oil content, silty endosperm, etc.)
  • the nucleotide sequence is introduced into a male sterile plant. The plant appears to be fertile due to the presence of a restorer gene.
  • transgenic hybrid plant When a transgenic hybrid plant (Msmsms) is crossed with a male sterile plant (msms), the following two progeny are produced, one is a male sterile male embryo with normal endosperm (sterile line, genotype is msms), the infertility
  • sterile line genotype is msms
  • the infertility The fertility can be restored by any wild type plant; the other is the fertile grain of the endosperm abnormality (maintaining line, genotype is Msmsms), which is implicitly homozygous in controlling male fertility locus, due to Containing a complementary transgenic sequence, the plant appears to be fertile, and because it also contains a nucleotide sequence that affects the main nutrient components of the endosperm (such as starch content, oil content, silty endosperm, etc.), the main nutrient component of the endosperm (such as starch content, oil content, whether powder
  • Figure 1 shows the male flower phenotype of male fertility mutant ms45 and wild-type Ms45.
  • Figure 2 shows the grain phenotype of the grain size gene Mn1 mutant and the wild type.
  • Fig. 3 is a schematic view showing the structure of a plant expression vector pMs45_MnlRNAi containing a male fertility gene Ms45 expression element and a control particle size gene Mn1 interference fragment expression element.
  • Fig. 4 is a plant expression vector plant male flower and grain phenotype of the transformed male fertility gene Ms45 expression element and the control grain size gene Mnl interference fragment expression element.
  • Figure 5 is a road map for seed production using nuclear male sterility genes, genes controlling grain size, and transgenic techniques.
  • Figure 6 is a flow chart of the corn genetic transformation test.
  • Figure 7 shows the grain phenotype of the 16-KD Y-gliadin dominant mutant controlling the grain component gene.
  • Fig. 8 is a schematic view showing the structure of a plant expression vector pMs45-Mc 16-KD y -zein containing a male fertility gene Ms45 expression element and a 16-KD y - gliadin dominant allele expression element controlling the grain component gene.
  • Figure 9 shows the male flower and grain phenotype of the transformed male fertility gene Ms45 expression element and the 16-KD y-gliadin dominant allele vector plant controlling the grain component gene.
  • Figure 10 is a road map for seed production using nuclear male sterility genes, genes controlling endosperm components, and transgenic techniques.
  • Nuclear male sterility is the result of mutation, inhibition or other influences of key genes in microspore formation. These genes are collectively referred to as male sterility genes.
  • the pollen development pathway is controlled by many genes, so mutations in many genes will eventually lead to male sterility.
  • a large number of male sterile mutants have been identified in maize (as shown in Table 1), and each male sterility gene is There are specific restorer genes, ie, each male sterile mutant, which can only be restored by its wild-type allele.
  • the present invention is exemplified by a male male sterile mutant in Table 1.
  • ms45 the mutant male flower cannot be normally loose powder (as shown in Fig. 1, the left plant is a male sterile mutant, and the right plant is a wild type).
  • the wild type restorer gene is derived from the inbred line B73, and its sequence is shown in SEQ ID No: 1.
  • This sequence contains the promoter of the Ms45 gene (positions 8-542 of SEQ ID No: 1) and the sequence of 0RF (positions 1422-2972 of SEQ ID No: 1). After transferring the SEQ ID No: l to the ms45 male sterile mutant, the mutant plants showed fertility.
  • the present invention constructs plant expression interference of the endosperm-specific cel l wal l invertase (CWI-2) gene (Cheng, WH et al. 1996) specifically expressed in maize endosperm.
  • Vector the gene is silenced.
  • the mutant of this gene was named miniaturel ( mnl ), and the grain became smaller after mutation or silencing. Since the gene is specifically expressed in the endosperm, the gene does not affect other traits of the plant after silencing.
  • fertilization of corn fertilization of the gametes containing the vector affects the development of the endosperm, resulting in smaller kernels.
  • the Mnl gene encodes a cell wall invertase, which inactivates the development of the grain endosperm, resulting in smaller kernels.
  • Figure 2 shows that A is a mMn mutant grain and B is a wild-type grain), but does not affect the development of embryos and plants. Most of the Mn1 mutants obtained under natural conditions or artificial mutagenesis are recessive mutants. The inventors interfered with the wild-type Mn1 gene at the mRNA level by transgenic technology, so that the seeds containing the transformed interfering nucleotide fragments would change. Small, as shown in A of Fig. 4, therefore, when the transforming fragment is present in the grain in a heterozygous state, the grain containing the transformed fragment can be quickly distinguished by the grain size.
  • RNAi interference sequence of the Mn1 gene in the present invention is derived from B73, but is not limited to the inbred line B73, and may also be derived from any other wild type maize inbred line, or a homologous gene of other species, or a synthetic nucleotide. sequence.
  • the present invention constructs an interference fragment of the male restorer gene Ms45 and the grain size marker gene Mnl in a vector, which can restore the fertility of the male sterile mutant ms45 and make the grain containing the transgene sequence smaller.
  • the grain containing the restorer gene is labeled to distinguish between fertile seeds (maintaining lines) and sterile grains (sterile lines).
  • the present invention constructs a gene Ms45 controlling the male fertility of maize and an interference fragment of the gene Mn1 controlling the grain size in a vector, transferring the vector into the ⁇ XHillB corn hybrid, and then using the ms45 male.
  • the planted plants were backcrossed to the obtained transgenic plants, thereby simultaneously introducing the Ms45 restorer gene and the interference fragment of the Mn1 gene into the male sterile mutant ms45.
  • Transgenic plants behaved as fertile due to the presence of the wild-type Ms45 gene.
  • transgenic hybrid plant Ms45ms45ms45
  • ms45ms45 male sterile plant
  • two offspring are produced, one is a male sterile normal grain (sterile line ms45ms45) that does not contain the transgene sequence, and the sterile line can Fertility is restored by any wild type plant (Ms45Ms45), which can be used as a sterile line in the seed production process; the other is a fertile grain with a small grain size (maintaining line M S 45ms45ms45), which maintains the male in control
  • the fertility locus is recessive homozygous, and the plant appears to be fertile due to the inclusion of complementary transgenic sequences, which become smaller due to the nucleotide sequence that also affects the size of the kernel.
  • the present invention uses a dominant allele that controls the kernel component gene 16-KD Y-gliadin, and the 16-KD Y-gliadin gene encodes a prolamin, a mutant Mucronate (Mc) has a 38-base deletion due to the 16-KD ⁇ -gliadin gene 438_476bp, which changes the coding frame of the gene, making the translated protein significantly different from the wild type. This in turn affects the development of the grain endosperm, resulting in opaque endosperm ( Figure A, MC is a MC16-KD Y-gliadin mutant, B is wild type), but does not affect the development of embryos and plants.
  • Mc Mucronate
  • Mc is a dominant mutant
  • the grain will appear as an opaque endosperm as long as the grain contains a dominant allele of Mc.
  • the present invention transfers the Mcdominant allele into the wild type by transgenic technology, and ensures that the gene is normally expressed in the plant, and the opaque phenotype of the endosperm is shown, as shown in A in Fig. 9, under such conditions,
  • the transformed fragments are present in a heterozygous state in a single kernel individual and can be resolved quickly by the naked eye or by means of an instrument.
  • the same method can be used to perform the same operation on other dominant genes affecting the development of the endosperm component, and the transgenic kernels which are easy to distinguish can be obtained.
  • the nucleotide sequence of the 16-KD Y-gliadin gene of the present invention is derived from a Mc mutant, and the specific sequence is shown in SEQ ID No: 6, which comprises a promoter sequence and a coding sequence of the gene, and the above nucleotides
  • the sequence is not limited to the Mc mutant and may be derived from any other maize endosperm mutant, or a homologous gene of another species, or a synthetic nucleotide sequence.
  • the present invention constructs a vector for controlling the 16-KD Y-gliadin gene (CheolSoo Kim et al. 2006) of the corn endosperm component.
  • the vector affects the development of the endosperm, making the corn a silty endosperm, and the silty endosperm appears opaque.
  • the mutant of this gene is Mucrone (Mc). This gene silence does not affect other traits of the plant.
  • fertilization of corn fertilization of gametes containing the carrier affects the development of the endosperm, resulting in opaque endosperm.
  • the invention constructs a male restorer gene Ms45 and a Mcl6-KD y-gliadin endosperm marker gene in a vector, which can restore the fertility of the male sterile mutant ms45, and at the same time make the grain endosperm containing the transgenic sequence opaque, ie Seeds containing the restorer gene are labeled to distinguish between fertile seeds (maintainer lines) and sterile seeds (sterile lines).
  • the invention constructs a gene Ms45 which controls the male fertility of corn and a Mc 16-KD Y-gliadin gene which controls the endosperm component, and transfers the vector into the waxy corn hybrid, and then uses the ms45 male
  • the transgenic plants were backcrossed to the obtained transgenic plants, thereby simultaneously introducing the Ms45 restorer gene and the Mc16-KD Y-gliadin gene into the male sterile mutant ms45.
  • Transgenic plants behaved as fertile due to the presence of the wild-type Ms45 gene.
  • transgenic hybrid plant Ms45ms45ms45
  • ms45ms45 male sterile plant
  • two offspring are produced, one is a male sterile normal grain (sterile line ms45ms45) that does not contain the transgene sequence, and the sterile line can It is restored to fertility by any wild type plant (Ms45Ms45), and can be used as a sterile line in the seed production process; the other is a fertile seed that is opaque to the endosperm (maintaining line M S 45ms45ms45), which maintains male fertility.
  • the ability site is recessive homozygous, and the plant appears to be fertile due to the inclusion of a complementary transgene sequence, which is opaque due to the nucleotide sequence that also affects the endosperm component.
  • the present invention provides an efficient seed labeling method which is applicable not only to maize (Zea mays) but also to rice (Oryza sativa), sorghum bicolor, wheat (Triticumaestivum), soybean (Glycine max), cotton. (Gossypiumhirsutum), sunflower (Helianthus annuus) and other crops.
  • Example 1 Construction of a DNA fragment containing a control maize male fertility and control of corn kernel size (DNA construct) Plant transformation vector pMs45_MnlRNAi
  • the plant transformation vector P Ms45-MnlRNAi shown in Fig. 3 contains a DNA fragment (DNA construct) and a selectable marker gene for controlling male fertility in maize and controlling corn kernel size.
  • the DNA fragment which controls the male fertility of corn and controls the size of corn kernel is Ms45_MnlRNAi, which is a DNA fragment between LB and RB of pMs45-MnlRNAi.
  • Ms45_MnlRNAi comprises the Ms45 expression element (first nucleotide sequence) of SEQ ID No: 1 and the expression element (second nucleotide sequence) of the silenced Mn1 gene.
  • the expression element for silencing the Mn1 gene is ligated by the promoter of Mn1 of SEQ ID No: 3 (Mnl promoter), the Mn1 interference fragment MnlRNAi and a terminator.
  • Mnl promoter Mn1 of SEQ ID No: 3
  • the Ms45 expression element is tightly linked to the expression element of the silenced Mnl gene, and when pMs45-MnlRNAi is transferred into a plant, the two expression elements are simultaneously present in the plant.
  • the construction method of pMs45_MnlRNAi is as follows:
  • the present invention exemplifies the embodiment by taking the ms45 male sterile mutant in Table 1 as an example.
  • the wild type allele Ms45 of ms45 was amplified and the gene was derived from inbred line B73, and its sequence is shown in SEQ ID No: 1.
  • genomic DNA of maize inbred line B73 as a template, reference B73 genome sequence
  • Tcccgg gGGTTGCGCATGAAATAGGGGT 3' The EcoRI recognition site was added to the 5' end of the upstream amplification primer, and the Smal recognition site was added to the 5' end of the downstream amplification primer.
  • the amplification reaction system was: template DNA 2 L, primer Ms45F 0.5 L, primer Ms45R 0.5 L , dNTP 1.6 ⁇ , 10 X Buffer 2 L, Gao Bao True taq enzyme 0.3 ⁇ , ddH20 13. L.
  • the reaction conditions were pre-denaturation at 95 ° C for 5 min, denaturation at 95 ° C for 45 s, annealing at 59 ° C for 45 s, extension at 72 ° C for 3 min, 32 cycles, and extension at 72 ° C for 10 min.
  • the amplified target band was 3518 bp in length.
  • the sequence was ligated into the T-easy sequencing vector, and the positive clone was sequenced.
  • the sequencing result showed that the 3518 bp DNA contained the Ms45 gene expression element represented by SEQ ID No: 1. , EcoRI recognition site and Smal recognition site.
  • position 8-542 is the promoter
  • position 1422-2972 is the coding sequence of the Ms45 gene, which encodes the Ms45 protein of SEQ ID No: 4.
  • the Mn1 gene encodes a cell wall invertase protein (the amino acid sequence of which is SEQ ID No: 5, which inactivates the endosperm, resulting in smaller kernels (shown in Figure 2), but does not affect embryos and Plant development.
  • the invention silences the Mn1 gene by RNAi technology, the interference fragment name is MnlRNAi, the structure is MnlSEQ forward-X-MnlSEQ reverse, and the reverse sequence of MnlSEQ is reverse complementary to the forward sequence of MnlSEQ, X is an intron forming a hairpin structure.
  • the nucleotide sequence of MnlRNAi is shown in SEQ ID No: 2, the 7th to 3rd position of SEQ ID No: 2 is the BstEII recognition sequence, and the 14th of SEQ ID No: 2 276 is the nucleotide sequence of Mn1SEQ ⁇ , 277-400 of SEQ ID No: 2 is X is an intron forming a hairpin structure, and positions 401-663 of SEQ ID No: 2 are nucleotides of MnlSEQ The sequence, positions 664-669 of SEQ ID No: 2, is the Hindlll recognition sequence.
  • the present invention activates the Mn1 interference fragment MnlRNAi using a promoter controlling the grain size gene Mn1 (Mnl promoter), and the Mn1 gene is endosperm-specific expression, and thus the mRNA which is initiated by the promoter is present only in the endosperm cells.
  • the promoter is derived from the genomic DNA of maize inbred line B73. The specific sequence is shown in SEQ ID No: 3, and the maize inbred line B73 genomic DNA is used as a template, and the B73 genome sequence (www. maizesequence.org) is used to design primers.
  • the promoter of the gene was amplified, and the amplification primers were as follows: Mnlpro bF: 5' at ccc ggGCTCGCATGAGAGAACAACCA 3', Mnlpro bR: 5' gcaagcttGGGGGTGCTATTTGTACTGTGC 3'.
  • the Smal recognition site was added to the 5' end of the upstream amplification primer, and the Hindlll recognition site was added to the 5' end of the downstream amplification primer.
  • the amplification reaction system was: template DNA 2 ⁇ , primer Mnlpro bF 0.5 ⁇ primer Mnlpro bRO.5 L, dNTP 1.6 L, 10 X Buffer 2 L, high fidelity taq enzyme 0.3 ⁇ , ddH20 13. L.
  • the reaction conditions were pre-denaturation at 95 °C for 5 min, denaturation at 95 °C for 45 s, annealing at 59 °C for 45 s, extension at 72 °C for 2 min, 32 cycles, and extension at 72 °C for 10 min.
  • the amplified target band was 2422 bp in length. After amplification, the sequence was ligated into the T-easy sequencing vector, and the positive clone was sequenced.
  • the sequencing result showed that the 2422 bp DNA was the promoter fragment of Mnl gene, and the promoter fragment of Mn1 gene was amplified.
  • the Mnl gene promoter, Smal recognition site and Hindlll recognition site of SEQ ID No: 3 were included.
  • the plant transformation vector pMs45_MnlRNAi shown in Fig. 3 was constructed by assembling the DNA components of the above steps 1, 2 and 3.
  • the plasmid PCAMBIA3301 International Agricultural Molecular Biology Application Center CAMBIA, Australia
  • constructing the male fertility gene Ms45, Mnl interference fragment expression elements and selection markers Plant transformation vector pMs45_MnlRNAi of the gene bar expression element.
  • the Mn1 interference fragments MnlRNAi and pCAMBAI3301 of step 2 were digested with BstEII and Hindlll, and the large fragments of Mnl interference fragment MnlRNAi and pCAMBAI3301 were ligated to detect positive clones, and then the positive clones were double-digested with EcoRI and Smal and the 3518 bp DNA of step 1.
  • the target band is recovered, the two fragments are ligated, the positive clone is detected, and finally the positive clone and the Mnl gene promoter of step 3 are digested by Smal and Hindlll, the target band is recovered, and the two fragments are ligated.
  • a positive clone was detected, and a plant transformation vector pMs45_MnlRNAi containing the male fertility gene Ms45, the Mn1 interference fragment expression element, and the selectable marker gene bar expression element was obtained (Fig. 3).
  • pMs45_MnlRNAi will be
  • the present invention obtains a transgenic plant by a method in which Agrobacterium infects maize immature embryos.
  • the plant transformation vector pMs45-MnlRNAi of Example 1 was transformed into Agrobacterium tumefaciens EHA105, and the maize embryos were infested with the Agrobacterium containing the gene of interest.
  • the specific transgenic method was as follows:
  • the receptor used in the transgenic process of the laboratory is the hybrid F1 generation of inbred lines ⁇ and ⁇ .
  • Maize Genetics Co., Ltd. and Hill B Arrow 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).
  • the pMs45_MnlRNAi was introduced into the immature embryos of the recipient plants by Agrobacterium tumefaciens EHA105 infection, and the transgenic plants were obtained after screening by the herbicide dipropylamine.
  • the specific method is:
  • Agrobacterium tumefaciens EHA105 should be cultured one week in advance on YEP (containing 33mg/L kanamycin and 100m g /L rifampicin) medium, and stored in a refrigerator at 4°C for about one month. -80 ° C glycerol preservation.
  • Agrobacterium tumefaciens EHA105 was cultured on YEP medium at 19 °C for 3 days, while kanamycin was added to a concentration of 33 mg/L and rifampicin to a concentration of 50 mg/L.
  • Agrobacterium tumefaciens EHA105 into a 50 ml centrifuge tube containing 5 mL of infestation medium, and add AS (inf+AS) (solute as shown in Table 2, solvent is water) at room temperature (25 °C) Shake the bacteria for 2-4 hours at 75rpm.
  • the young embryos were transferred to a resting medium (solutes as shown in Table 2, the solvent was water), and the culture dishes were sealed with a parafilm, and cultured at 28 ° C for 7 days in the dark.
  • the whole medium was transferred to a selection medium (solute as shown in Table 2, the solvent was water) (35 immature embryos per dish), cultured for two weeks, the selection medium containing dipropylamine 1. 5 mg / L The concentration of the sub-cultured dipropylamine can be increased to 3 mg/L after two weeks.
  • MS salt was purchased from phyto Technology Laboratories under the product number M524.
  • the ms45 homozygous recessive mutant (Maize Genetics Cooperation Stock Center, 9051) was used as the female parent, and hybridized with different inbred lines (such as Zheng 58 (zheng58)), and the obtained F1 continued with the maize inbred line Zheng 58 (Henan Autumn Le Seed Industry Technology Co., Ltd.
  • the ms45 homozygous recessive inbred line female was crossed with the TO-generation pMs45_MnlRNAi transformed maize plant ( father) obtained in step one, and then the ms45 homozygous recessive inbred line was used as the recurrent parent for multi-generation backcrossing.
  • the TO-generation pMs45_MnlRNAi transformed maize plant obtained in step 1 is converted into containing
  • Ms45-MnlRNAi and the ms45 site is a homozygous recessive and Ms45_MnlRNAi heterozygous inbred line, which is a second plant homozygous for Ms45_MnlRNAi and homozygous for ms45.
  • the 50 strains of TO-derived pMs45_MnlRNAi transformed into maize plants (parent) obtained in step one were hybridized with the ms45 homozygous recessive inbred line Zheng 58 (Zheng 58 (ms45ms45)) (mother) obtained in step two, respectively. Seeds selected from the hybrid progeny were sown in the field and sprayed with 200 mM of dipropylamine. The surviving plants were backed up with the ms45 homozygous recessive inbred line Zheng 58 (Zheng 58 (ms45ms45)) obtained in step two.
  • the molecular marker was used to screen the transgenic site (Ms45-MnlRNAi) for heterozygosity, the Ms45 site for recessive homozygosity, and the other sites were single plants with Zheng 58 background.
  • Ms45_Mn lRNAi A second plant heterozygous for Ms45_Mn lRNAi and homozygous for ms45, the second plant was named Zheng 58 (Ms45ms45ms45) second plant.
  • the above-mentioned Zheng 58 (Ms45ms45ms45) second plant was used as the male parent, and the ms45 homozygous recessive inbred line Zheng 58 (Zheng 58 (ms45ms45)) (mother) was obtained in step 2.
  • the resulting offspring not only had male sterility.
  • step 1 obtained by the generation of pMs45-MnlRNAi transformed maize plants
  • the T0 generation pMs45_MnlRNAi transformed maize plants and their progeny obtained from step 1 were evaluated for the overall morphology of the plants, and the pollen and grain phenotypes were analyzed. Except for the kernel, no other morphological differences were observed between the T0 generation pMs45-MnlRNAi transformed maize plants and the non-transgenic control plants.
  • the T0 generation pMs45_MnlRNAi transformed maize plant was crossed with the ms45 male sterile material, the plants containing Ms45_MnlRNAi in the progeny of the hybrid showed fertility, as shown by C in Fig. 4, and the hybrid progeny containing no Ms45_MnlRNAi showed complete infertility.
  • Ms45 gene complements the recessive homozygous ms45 male sterility.
  • the grain containing Ms45_MnlRNAi is smaller than the normal grain containing no Ms45-MnlRNAi, and the phenotype is the same as the mnl mutant, as shown in A in Fig. 4.
  • the Mnl gene interference fragment can function normally in transgenic plants.
  • the inventors sow the small grain and the normal grain identified by the grain phenotype into the field, and the seeds can germinate normally, and the sprouting rate is not significantly different from the normal grain.
  • Example 3 using the male sterile maintainer line in Example 2 to carry out large-scale expansion of the ms45 male sterile inbred line
  • the male sterile line Zheng 58 (ms45ms45) in Example 2 and the male sterile line Zheng 58 (Ms45ms45ms45) in Example 2 were sown into the field, and the two materials were planted separately. For each row of sowing, the corresponding line of 5 lines of sterile lines is planted to ensure that no other corn is planted within 300 meters of the breeding area, and the sterile lines are maintained in the field.
  • the maintainer can only accept its own pollen, and the resulting offspring are discarded because the seeds containing the homozygous transgenic component (Ms45_MnlRNAi) are indistinguishable from the heterozygous kernels, and the normal size of the kernel (large grain) can be used as a sterile system.
  • the male sterile line Zheng 58 (ms45ms45) received the pollen of the male sterile line Zheng 58 (Ms45ms45ms45).
  • the normal size of the offspring was a sterile line without transgenic components, while the small grain was kept with genetically modified ingredients. system.
  • the maintainer line is used for the expansion of the sterile line and the maintainer line in the next year. Most of the sterile line is used for the producer variety, and the remaining small part is used for the expansion of the sterile line and the maintainer line in the next year. The flow is shown in Figure 5.
  • Example 4 Large-scale production of hybrids using the male sterile line of Example 3
  • the sterile line produced in Example 3 is a nuclear-controlled recessive homozygous sterile line which can be restored to fertility by any wild type plant (Ms45Ms45). So just choose one and male infertility
  • Inbred lines such as male infertility Zheng 58 (ms45ms45) Inbred lines with high combining ability, such as Chang 7-2, can produce hybrids with excellent agronomic traits.
  • the inventors sown the male sterile inbred lines and the wild-type inbred lines in the field to ensure that no other corn was planted within 300 meters of the breeding, and the ears of the sterile lines could only accept wild-type inbred lines. Pollen, while wild-type inbreds can only be self-sufficient.
  • the seeds produced on the ears of the sterile line are hybrids.
  • the plant transformation vector pMs45_Mc 16-KD y -zein shown in Fig. 8 contains a DNA fragment (DNA construct) and a selectable marker gene which control the male male fertility and control whether the corn seed endosperm is transparent.
  • the name of the spiritual fragment controlling the male fertility of corn and controlling the size of corn kernel is Ms45-Mc 16-KD y -zein, which is the DNA fragment between LB and RB of pMs45_Mc 16-KD y -zein.
  • Ms45_Mc 16-KD y -zein comprises the Ms45 expression element of SEQ ID No: 1 (first nucleotide sequence) and the MC16-KD Y-gliadin gene expression element of SEQ ID No: 6 (second nucleotide sequence) ).
  • the Ms45 expression element is tightly linked to the MC16-KD Y-gliadin gene expression element, and when pMs45-Mc 16-KD y -zein is transferred into a plant, the two expression elements are simultaneously present in the plant.
  • the construction method of pMs45_Mc 16-KD ⁇ -zein is as follows:
  • positions 9-1149 are promoter sequences, and positions 1244-1780 are coding sequences of the MC16-KD Y-gliadin gene, and Mcl6-KD Y-alcohol encoding SEQ ID No: 7 protein.
  • PCAMBIA3301 International Agricultural Molecular Biology Application Center CAMBIA, Australia
  • Mc 16-KD Y-Gliadin expression element and PCAMBAI3301 large fragment were ligated, positive clones were detected, and even fragments were ligated, positive clones were detected, and then EcoRI was used.
  • the Smal double-cleavage-positive clone and the Ms45 wild-type allele of step 1 the target band was recovered, the two fragments were ligated, and the positive clone was detected to obtain the male fertility gene Ms45 and Mc 16-KD y-glycoprotein.
  • pMs45_Mc 16-KD y -zein The expression vector and the plant transformation vector pMs45_Mc 16-KD y -zein of the selection marker gene bar, and the constructed vector are as shown in Fig. 8 .
  • pMs45-Mc 16-KD y -zein is a fragment of the EcoRI and Smal recognition site of pCAMBAI3301 replaced with the Ms45 gene expression element of SEQ ID No: 1 and a fragment between the BstEII and Hindlll recognition sites of pCAMBAI3301
  • Example 6 Preparation of Ms45_Mc 16-KD ⁇ -zein heterozygous and ms45 homozygous second plant I. Transformation of maize with the plant transformation vector pMs45_Mc 16-KD y -zein of Example 5 The present invention infects corn by Agrobacterium The method of embryos obtains transgenic plants.
  • the plant transformation vector pMs45_Mc 16-KD ⁇ -zein of Example 5 was transformed into Agrobacterium tumefaciens EHA105, and the hybrid F1 generation maize immature embryos of the inbred lines ⁇ and ⁇ were infested with Agrobacterium containing the gene of interest, and Agrobacterium tumefaciens The infected immature embryos were screened on a selection medium for multiple times to obtain a resistant callus, and the resistant callus was regenerated to produce a transgenic T0 generation plant.
  • the pollen of the T0 transgenic plants was used to hybridize some of the female parent and the Ms45 male sterile material, and the phenotype was observed.
  • the specific experimental method is the same as the first step of the second embodiment.
  • the ms45 homozygous recessive mutant (Maize Genetics Cooperation Stock Center, 9051) was used as the female parent, and the maize inbred line Zheng 58 (zheng58) (Henan Qiuliao Seed Industry Technology Co., Ltd.) was crossed, and the obtained F1 continued with corn.
  • Inbred line Zheng 58 backcross genotype analysis of the obtained BC1 population, identification of plants with heterozygous Ms45 locus continued to cross back with Zheng 58, so after 5-6 generations of backcrossing, screening Ms45 locus using molecular markers For heterozygosity, other sites are self-crossing of Zheng 58, thus obtaining ms45 homozygous recessive inbred line Zheng 58 (Zheng 58 (ms45ms45)), which can be used as a sterile line. It is called the first plant.
  • the specific experimental method is the same as that in the second step of the second embodiment. 3.
  • Ms45_Mc 16-KD y-zein heterozygous and ms45 homozygous second plant transformed ms45 homozygous recessive inbred line (mother) with TO-generation pMs45_Mc 16-KD y-zein obtained in step one into maize plants ( Parental) Hybridization and then multi-generation backcrossing with the ms45 homozygous recessive inbred line as the recurrent parent, transforming the TO-generation pMs45-Mc 16-KD y -zein transformed maize plant obtained in step one into Ms45_Mc 16-KD
  • the ⁇ -zein and ms45 loci are homozygous recessive and Ms45_Mc 16-KD y -zein heterozygous inbred lines, which are Ms45_Mc 16-KD ⁇ -zein heterozygous and ms45 homozygous second plant .
  • the 50 strains of TO-derived pMs45_Mc 16-KD ⁇ -zein obtained from step 1 were transformed into maize plants ( father) and the ms45 homozygous recessive inbred lines obtained in step 2 were Zheng 58 (Zheng 58 (ms45ms45) (mother) hybridization, selecting the opaque seeds of the endosperm from the hybrid progeny, sowing the field, spraying 200 mM of dipropylamine, and continuing the same with the ms45 homozygous recessive inbred line obtained by the second step.
  • Zheng 58 Zheng Zheng 58 ( ms45ms45 )
  • the molecular marker was used to screen the transgenic locus (Ms45_Mc 16-KD y -zein ) as heterozygous, Ms45 locus as recessive homozygous, and other loci were positive.
  • a single plant of 58 background the single plant is a second plant heterozygous for Ms45_Mc 16-KD ⁇ -zein and homozygous for ms45, and the second plant is named Zheng 58 (Ms45ms45ms45) second plant.
  • the above-mentioned Zheng 58 (Ms45ms45ms45) second plant was used as the male parent, and the ms45 homozygous recessive inbred line Zheng 58 (Zheng 58 (ms45ms45)) (mother) was obtained in step 2.
  • the resulting offspring not only had male sterility.
  • Department Zheng 58 (ms45ms45) and there is a male infertility Zheng 58's maintenance system Zheng 58
  • the TO-generation pMs45-Mc 16-KD y-zein transformed maize plants obtained in step 1 were evaluated for the overall morphology of the plant.
  • the T0 generation pMs45_Mc 16-KD y-zein transformed maize plants and their progeny from step 1 were evaluated for pollen. And the grain phenotype is focused on analysis. Except for the kernel, no other morphological differences were observed between the T0 generation pMs45-Mc 16-KD ⁇ -zein transformed maize plants and the non-transgenic control plants.
  • Example 7 The male sterile line in Example 6 was used for large-scale expansion of the ms45 male sterile inbred line.
  • the male sterile line Zheng 58 (ms45ms45) in Example 6 and the male sterile line Zheng 58 (Ms45ms45ms45) in Example 6 were sown into the field, and the two materials were planted separately.
  • the corresponding line of 5 lines of sterile lines is planted to ensure that no other corn is planted within 300 meters of the breeding area, and the sterile lines are maintained in the field.
  • the maintainer can only accept its own pollen, and the resulting offspring are discarded because the seeds containing the homozygous genetically modified ingredients are indistinguishable from the heterozygous kernels, and the normal kernels can be used as sterile lines.
  • the sterile material receives the pollen of the maintainer line, and the normal kernel of the progeny is a sterile line containing no transgenic components, and the endosperm opaque kernel is a maintainer containing the transgenic component.
  • the maintainer line is used for the expansion of the sterile line and the maintainer line in the next year. Most of the sterile line is used for the producer variety, and the remaining small part is used for the expansion of the sterile line and the maintainer line in the next year. The flow is shown in Figure 10.
  • Example 8 Large-scale production of hybrids using the male sterile line of Example 7
  • the sterile line produced in Example 7 is a nuclear-controlled recessive homozygous sterile line which can be restored to fertility by any wild type plant (Ms45Ms45). Therefore, just choose an inbred line with male infertility (ms45ms45), such as male infertility Zheng 58 (ms45ms45) with high combining ability, such as Chang
  • Hybridization can be carried out in 7-2 to produce hybrids with excellent agronomic traits.
  • the male sterile inbred line and the wild type inbred line are sown in the field, ensuring that no other corn is planted within 300 meters of the breeding area, and the ear of the sterile line can only accept the pollen of the wild type inbred line. Wild-type inbred lines can only be self-interested.
  • the seeds produced on the ears of the sterile line are hybrids.
  • the method for expanding the male sterile line of the invention adopts an efficient seed labeling method, which can expand the male sterile seed of the plant, save manpower for the hybrid seed production, reduce the cost and ensure the purity of the seed.
  • the expanded plant male sterile line method of the invention utilizes the shape of the grain (such as size, length, width, thickness, etc.) or the main nutrient components of the endosperm (such as starch content, oil content, whether powdery endosperm, etc.) Nucleotides and wild-type alleles and transgenic techniques of a nuclear male sterility gene, either through grain shape (eg size, length, width, thickness, etc.) or endosperm nutrient components (eg starch content, oil content, Whether silty endosperm, etc.) efficiently distinguishes fertile seeds and sterile seeds in transgenic grains. Homozygous recessive male sterile plants produced by the methods of the invention can be used to produce hybrids.

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Abstract

提供了扩繁植物雄性不育系的方法。方法包括（a）提供包含使植物雄性不育的纯合隐性等位基因的第一植株；（b）提供包含所述纯合隐性等位基因并且含有下述构建体的第二植株，所述构建体在第二植株中以杂合状态存在，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的雄性生育力；ii）第二核苷酸序列，当其以杂合状态存在时即可影响籽粒形状或胚乳营养物质成分，通过肉眼或仪器可以分辨含有该构建体的籽粒和不含有该构建体的籽粒；所述第一核苷酸序列和第二核苷酸序列紧密相连，在这两个核苷酸序列在植株中同时存在；（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。

Description

扩繁植物雄性不育系的方法 技术领域
本发明涉及扩繁植物雄性不育系的新方法， 该方法利用细胞核雄性不育基 因、 籽粒标记基因及转基因技术扩繁植物雄性不育系， 属于植物遗传育种及种 子生产领域。
背景技术
由于杂种优势的存在， 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， 如杂交玉米、 杂交水稻的 产量远远高于纯合的双亲。 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， 将雌性亲本的雄穗去除， 而保留雄性亲本的雄穗， 雌性亲本收 获的种子即为杂交种。
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 粉指一株植物的花粉， 对同一个体的雌蕊进行授粉的现象。 在两性花的植物中， 又可分为同一花的雄蕊与雌蕊间进行授粉的同花授粉 （菜豆属） 和在一个花序 (个体） 中不同花间进行授粉的邻花授粉， 以及同株不同花间进行授粉的同株 异花授粉。 有的植物雄蕊和雌蕊不长在同一朵花里， 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， 如玉米。
玉米为雌雄同株， 并且雌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， 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。
在玉米育种中首先应开发出纯合的玉米自交系， 然后将两个自交系进行杂 交， 对杂交的后代进行产量、 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， 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。
在生产杂交种的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， 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， 即会影响农民收入又会影响制种公司信誉， 严重的将导致 制种公司承担相应的赔偿责任。 也可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， 目前没有令人完全满意的去雄手段， 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手段， 通过使用核-质互作雄性不育
(CMS) 自交系， 可以在一些基因型中得以避免繁重的去雄工作。 该手段包括三 个主要材料， 即不育系： 雄性不育材料， 保持系： 可以为不育系提供花粉， 使 不育系的后代仍为不育系， 恢复系： 可以恢复不育系的育性。 不育系与恢复系 杂交产生 Fl， 即用于农业生产的杂交种。 更具体的说， 核-质互作不育型， 表现 为核-质互作遗传。 不但需要细胞质有不育基因 s， 而且需要细胞核里有纯合的 不育基因 （rfrf) ， 二者同时存在， 方能使植株表现为雄性不育。 如胞质基因 为可育 N， 则不论核基因是可育 （RfRf) 还是不育 （rfrf) ， 都表现为雄性可 育。 同样， 如核里具有可育基因 （RfRf) 或 （Rfrf) ， 则不论胞质基因是可育 N还是不育 S， 也都表现为雄性可育。 这种由核-质互作形成的雄性不育系， 其 遗传组成为 S (rfrf) ， 不能产生正常的花粉， 但可作为杂交母本。 由于能找 到保持系 N (rfrf) [用它与不育系杂交， 所产生的 F1仍能保持雄性不育， 艮卩： S (rfrf) ( ) XN (rfrf) →S (rfrf) (不育） ]并能接受恢复系 S (RfRf) 或 N (RfRf) [用它们与不育系杂交， 所产生的 F1都是可育的， BP: S (rfrf) (早） XS (RfRf) →S (Rfrf) (Fl) (可育） ， 或 S ( rfrf ) ( ) XN (RfRf) →S (Rfrf) (Fl) (可育） ]的花粉， 使 Fl 恢复为雄性可育， F1 植株自交产 生 F2， 所以在农业生产上可以广泛应用。 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进行杂交种子的生产； 对 其他作物的核-质互作雄性不育系， 也正在进行广泛的研究。
CMS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。
Brar et al的美国专利 4654465和 4727219中公开了一种类型的遗传不育 性。 但是， 这种类型的遗传不育需要在基因组内的多个不同位点保持相应的基 因型， 需要每代对这些位点进行分子标记跟踪检测。 Patterson 还描述了一种 可能有用的染色体易位基因系统， 但该系统更为复杂 （见美国专利 No.3861709 和 3710511) 。
人们一直在尝试对雄性不育系统进行优化， 例如， Fabijanski, et al.开 发了使植物雄性不育的方法 （EP0 89/3010153.8 公开号 329308 和作为 W 90/08828公开的 PCT申请 PCT/CA90/00037) 。 主要是通过以下两种途径抑制植 株的雄花育性， 一种方法是将雄性组织特意表达的启动子与细胞毒素基因相连 转入植株中， 使雄花不能正常散粉同时不影响其他性状； 另一种是通过基因干 扰手段， 将已经克隆的控制植株雄花育性的基因通过转基因的手段将其干扰， 从而使其不能正常行使功能。 还有通过一些基因调控元件来抑制基因表达， 从 而影响植株育性的手段 （W090/08829 ) 。
在多数情况下， 只有控制雄性不育的核基因隐性纯合 （msms ) 植株才会表 现为雄性不育， 由于雄性不育植株无法自交， 因此只能通过杂合植株 (Msms)与 其杂交，才会得到雄性不育植株（msms)。并且在同一果穗上雄性不育籽粒（msms) 与可育的杂合籽粒 (Msms)同时存在， 通过籽粒无法分辨哪些是不育籽粒， 哪些 是可育籽粒， 只能通过播种后， 植株散粉时才可分辨。
近年来， 也有利用转基因的手段来保持雄性不育植株的不育性 ( US6743968 ) 。 该方法将花粉致死基因与雄性生育能力恢复基因构建在一个载 体中， 导入雄性不育植株中， 转基因后代表现为可育， 但只能产生不含恢复基 因的花粉。 当这样的植株与雄性不育植株杂交， 便保持了隐性不育植株的纯合 隐性状态。 其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， 同时该载体还含有一个恢复植株育性的显性基因。将该载体转入雄性不育植株， 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使得植 株可育， 当其与雄性不育植株杂交， 由于含有恢复基因的花粉 （Msms ) 同时含 有致死基因， 使得花粉败育， 因此只有不含恢复基因的花粉 （ms ) 才能与雄性 不育植株雌配子 （ms ) 进行杂交， 后代均为隐性纯合个体（msms)。
如前文所述， 采用雄性不育系统制种的很多工作的一个重要问题在于如何 利用雄性不育基因及如何分辨雄性不育种子及可育种子， 同时还需要考虑如何 将不育个体的不育性保持下来。
在玉米中已经鉴定了多种雄性不育突变体 （Skibbe et al. 2005 ) ， 具体见 下表：
表 1.由核基区引起的雄性不育突变体
突变体 （等位 染色体 参考文献
突变）
ms l 6 Singleton W. R and Jonnes D. F. 1930. Heritable characters of maize. XXXV. Male steri le. J Hered 21 : 266-268
ms2 9 Eyster W. H. 1931. J Hered 22 : 99-102 ; ALBERTSEN
M. C. , R. L. PHILLIPS, 1981. Developmental cytology of 13 genetic male steri le loci in maize. Can. J. Genet. Cytol. 23： 195-208 ms3 3 Eyster W. H. 1931. J Hered 22 : 99-102 ms4 (pol) BEADLE G. W. , 1932. GENES IN MAIZE FOR POLLEN
STERILITY. GENETICS17 : 413-431
ms5 5 BEADLE G. W. , 1932. GENES IN MAIZE FOR POLLEN
STERILITY. GENETICS17 : 413-431 ; ALBERTSEN M. C. , R. L. PHILLIPS, 1981. Developmental
cytology of 13 genetic male steri le loci in maize. Can. J. Genet. Cytol. 23： 195-208 ms6 (pol) BEADLE G. W. , 1932. GENES IN MAIZE FOR POLLEN
STERILITY. GENETICS17 : 413-431 ; ALBERTSEN M. C. , R. L. PHILLIPS, 1981. Developmental cytology of 13 genetic male steri le loci in maize. Can. J. Genet. Cytol. 23： 195-208 ms7 7 BEADLE G. W. , 1932. GENES IN MAIZE FOR POLLEN
STERILITY. GENETICS17 : 413-431 ; ALBERTSEN M. C. , R. L. PHILLIPS, 1981. Developmental cytology of 13 genetic male steri le loci in maize. Can. J. Genet. Cytol. 23： 195-208 ms8 8 BEADLE G. W. , 1932. GENES IN MAIZE FOR POLLEN
STERILITY. GENETICS17 : 413-431 ; ALBERTSEN M. C. , R. L. PHILLIPS, 1981. Developmental cytology of 13 genetic male steri le loci in maize. Can. J. Genet. Cytol. 23： 195-208 ms9 1 BEADLE G. W. , 1932. GENES IN MAIZE FOR POLLEN
STERILITY. GENETICS17 : 413-431 ; ALBERTSEN M. C. , R. L. PHILLIPS, 1981. Developmental cytology of 13 genetic male steri le loci in maize. Can. J. Genet. Cytol. 23： 195-208 ms lO 10 BEADLE G. W. , 1932. GENES IN MAIZE FOR POLLEN
STERILITY. GENETICS17 : 413-431 ; ALBERTSEN M. C. , R. L. PHILLIPS, 1981. Developmental cytology of 13 genetic male steri le loci in maize. Can. J. Genet. Cytol. 23： 195-208 ms l l 10 BEADLE G. W. , 1932. GENES IN MAIZE FOR POLLEN
STERILITY. GENETICS17 : 413-431 ; ALBERTSEN M. C. , R. L. PHILLIPS, 1981. Developmental cytology of 13 genetic male steri le loci in maize. Can. J. Genet. Cytol. 23： 195-208 ms l2 1 BEADLE G. W. , 1932. GENES IN MAIZE FOR POLLEN
STERILITY. GENETICS17 : 413-431 ; ALBERTSEN M. C. , R. L. PHILLIPS, 1981. Developmental cytology of 13 genetic male steri le loci in maize. Can. J. Genet. Cytol. 23： 195-208 msl3 5 BEADLE G. W. , 1932. GENES IN MAIZE FOR POLLEN
STERILITY. GENETICS17: 413-431; ALBERTSEN M. C. , R. L. PHILLIPS, 1981. Developmental cytology of 13 genetic male sterile loci in maize. Can. J. Genet. Cytol. 23： 195-208 msl4 1 BEADLE G. W. , 1932. GENES IN MAIZE FOR POLLEN
STERILITY. GENETICS17: 413-431; ALBERTSEN M. C. , R. L. PHILLIPS, 1981. Developmental cytology of 13 genetic male sterile loci in maize. Can. J. Genet. Cytol. 23： 195-208 msl5 BEADLE G. W. , 1932. GENES IN MAIZE FOR POLLEN
STERILITY. GENETICS17: 413-431
msl6 BEADLE G. W. , 1932. GENES IN MAIZE FOR POLLEN
STERILITY. GENETICS17: 413-431
msl7 1 EMERSON R. A. , 1932. A recessive zygotic lethal resulting in 2:1 ratios for normal vs.
male-sterile and colored vs. colorless pericarp in F2 of certain maize inbreds. Science 75: 566; ALBERTSEN M. C. ,
R. L. PHILLIPS, 1981. Developmental cytology of 13 genetic male sterile loci in maize. Can. J. Genet. Cytol. 23:195-208
msl8 1 EYSTER W. H. , 1934. Genetics of Zea mays.
Bibliogr. Genet. 11:187-392
msl9 9 EYSTER W. H. , 1934. Genetics of Zea mays.
Bibliogr. Genet. 11:187-392
ms20 EYSTER W. H. , 1934. Genetics of Zea mays.
Bibliogr. Genet. 11:187-392
Ms21 6 SCHWARTZ D. , 1951 . The interaction of
nuclear and cy t op lasmicf actors in the inheritance of male sterility in maize.
Genetics36： 676 - 696
ms22 (mscal) 7 WEST D. R. , M. C. ALBERTSEN, 1985. Three new male-sterile genes. Maize Genet. Coop.
Newsletter 59: 87; TRIMNELL M. R. , T. W. FOX, M. C. ALBERTSEN, 2001 New male-sterilemutant allele of Ms22. Maize Genet. Coop.
Newsletter 75:31; CHAUBAL R. , J. R. ANDERSON, M. R. TRIMNELL, T. W. FOX, M. C. ALBERTSEN, P. BEDINGER, 2003 . The transformation of anthers in themscal mutant of maize. Planta 216： 778-788
ms23 WEST D. R. , M. C. ALBERTSEN, 1985. Three new male-sterile genes. Maize Genet. Coop.
Newsletter 59: 87; CHAUBAL R. , C. ZANELLA, M. R. TRIMNELL, T. W. FOX, M. C. ALBERTSEN, P.
BEDINGER, 2000. Two male-sterile mutants of Zea mays (Poaceae) with an extra cell division in the anther wall. Am. J. Bot. 87: 1193-1201
ms24 10 WEST D. R. , M. C. ALBERTSEN, 1985. Three new male-sterile genes. Maize Genet. Coop.
Newsletter 59: 87; FOX T. W. , M. R. TRIMNELL, M. C. ALBERTSEN, 2002. Male-sterile mutant ms24 mapped to chromosome 10. Maize
Genet. Coop. Newsletter 76: 37
ms25 9 LOUKIDES C. A. , A. H. BROADWATER, P. A. BEDINGER,
1995. Two newmale-sterile mutants of Zea mays (Poaceae) with abnormal tapetal cell morphology. Am. J. Bot. 82: 1017-1023 ms26 1 LOUKIDES C. A. , A. H. BROADWATER, P. A. BEDINGER,
1995. Two newmale-sterile mutants of Zea mays (Poaceae) with abnormal tapetal cell morphology. Am. J. Bot. 82: 1017-1023 ms27 ALBERTSEN M. C. , 1996. Ms-gene designations.
Maize Genet. Coop. Newsletter 70: 30-31 ms28 1 GOLUBOVSKAYA I. N. , D. V. SITNIK0VA, 1980.
Three meiotic mutations of maize, causing irregular segregation of chromosomes in the first division of meiosis. Genetika 16: 656-666
ms29 10 TRIMNELL M. R. , T. W. FOX, M. C.
ALBERTSEN, 1998. New chromosome 10S male-sterile mutant： ms29. Maize Genet.
Coop. Newsletter 72: 37-38
ms30 (msx) 4 TRIMNELL M. R. , T. W. FOX, M. C.
ALBERTSEN, 1998. New chromosome 2L male-sterile mutants ms30 and ms31. Maize Genet. Coop. Newsletter 72: 38
ms31 2 TRIMNELL M. R. , T. W. FOX, M. C.
ALBERTSEN, 1998. New chromosome 2L male-sterile mutants ms30 and ms31. Maize Genet. Coop. Newsletter 72: 38
ms32 2 CHAUBAL R. , C. ZANELLA, M. R. TRIMNELL, T. W.
FOX, M. C. ALBERTSEN, P. BEDINGER, 2000. Two male-sterile mutants of Zea
mays (Poaceae) with an extra cell division in the anther wall. Am. J. Bot. 87: 1193-1201 ms33 2 TRIMNELL M. R. , E. PATTERSON, T. W. FOX,
P. BEDINGER, M. C. ALBERTSEN, 1999. New chromosome 2L male-sterile mutantms33 and alleles. Maize Genet. Coop. Newsletter 73: 48-49
ms34 7 TRIMNELL M. R. , E. PATTERSON, M. C.
ALBERTSEN, 1999. New chromosome 7L male-sterile mutant ms34. Maize Genet. Coop. Newsletter 73: 49
ms35 (ms23) TRIMNELL M. R. , E. PATTERSON, T. W. FOX,
M. C. ALBERTSEN, 1999. New chromosome 9L male-sterile mutants ms35 and ms36. Maize Genet. Coop. Newsletter 73: 49-50; TRIMNELL M. R. , T. W. FOX, M. C. ALBERTSEN, 2002. We made a mistake！ ms35 is allelic to ms23, but what is the correct map location? Maize Genet. Coop. Newsletter 76: 37-38; ALBERTSEN M. C. , T. W. FOX, M. R. TRIMNELL, 1999 . Changing a duplicated designation for two different male-sterile mutationsMaize Genet. Coop. Newsletter 73: 48
ms36 9 TRIMNELL M. R. , E. PATTERSON, T. W. FOX, M. C. ALBERTSEN, 1999. New chromosome 9L male-sterile mutants ms35 and ms36. Maize Genet. Coop. Newsletter 73: 49-5
ms37 3 TRIMNELL M. R. , T. W. FOX, M. C.
ALBERTSEN, 1999. New chromosome 3L male-sterile mutant ms37. Maize Genet. Coop. Newsletter 73: 48
ms38 2 TRIMNELL M. R. , T. W. FOX, M. C.
(ms*WL89A) ALBERTSEN, 1998a New chromosome 10S male-sterile mutant： ms29. Maize Genet. Coop. Newsletter 72: 37-38; ALBERTSEN M. C. , T. W. FOX, M. R. TRIMNELL, 1999. Changing a duplicated designation for two different male-sterile mutationsMaize Genet. Coop. Newsletter 73: 48
Ms41 4 NEUFFER M. G. , 1987. Location of dominant male sterile on chromosome 4L. Maize Genet. Coop. Newsletter 61： 51
Ms42 5 ALBERTSEN M. C. , T. W. FOX, M. R.
TRIMNELL, M. G. NEUFFER, 1993. Interval mapping a new dominant male-sterile mutant, Ms42. Maize Genet. Coop. Newsletter 67: 64 ms43 8 GOLUBOVSKAYA I.N.， 1979 . Genetical control of meiosis. Int. Rev. Cytol. 58: 247-290
Ms44 4 ALBERTSEN M. C. , L. M. SELLNER, 1988. An independent, EMS-induced dominant male sterile that maps similar to
Ms41. Maize Genet. Coop. Newsletter 62: 70; ALBERTSEN M. C. , M. G. NEUFFER, 1990.
Dominant male sterile inmaize. Maize Genet. Coop. Newsletter 64: 52
ms45 9 ALBERTSEN M. C. , M. R. TRIMNELL, T. W. FOX, 1993.
Tagging, cloningand characterizing a male fertility gene in maize. Am. J. Bot.80： 16 ms47 10 TRIMNELL M. R. , T. W. FOX, M. C.
ALBERTSEN, 2002. New chromosome 10 male-sterile mutant： ms47. Maize Genet. Coop. News letter 76 : 38
ms48 9 TRIMNELL M. R. , T. W. FOX, M. C.
ALBERTSEN, 2002. New chromosome 9L male-steri le mutant： ms48. Maize Genet. Coop. News letter 76 : 38
ms49 10 TRIMNELL M. R. , T. W. FOX, M. C. ALBERTSEN,
2002. New chromosome 10 male-steri le mutant： ms49. Maize Genet. Coop. News letter 76 : 38-39
ms50 6 TRIMNELL M. R. , T. W. FOX, M. C.
ALBERTSEN, 2002e New chromosome 6L male-steri le mutant： ms50. Maize Genet. Coop. News letter 76 : 39
ms52 10 Skibbe DS, Schnable PS : Male steri l ity in maize. Maydica 2005, 50 : 367-376
以上这些基因已经陆续被克隆， 如 ms45 (Albertsen et al. 1993)和 ms26 (PTC/US2006/024273) , 同时， 水稻中也有一些雄性不育基因被陆续克隆， 如 dpw ( Jing Shi et al. 2011 ) 以及拟南芥中鉴定到的一些雄性不育基因， 如 (Aarts, et al. 1993 ) 。
发明公开
本发明提供了一种扩繁植物雄性不育系的方法， 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；
(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， 所述构建体在所述第二植株中以杂合状 态存在， 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；
ϋ.第二核苷酸序列， 当其以杂合状态存在时即可影响籽粒形状或胚乳营养 物质成分， 通过肉眼或仪器可以分辨含有该构建体的籽粒和不含有该构建体的 籽粒；
所述第一核苷酸序列与所述第二核苷酸序列紧密相连， 这两个核苷酸序列 在植株中同时存在；
(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。
上述方法中， 所述籽粒形状可为大小、 长短、 宽窄或 /和薄厚等； 所述胚乳 营养物质成分为可为是否粉质胚乳、 淀粉含量或 /和油份含量等。 上述方法中， 所述植物、 所述第一植株和所述第二植株均可为单子叶植物 或双子叶植物， 如玉米、 水稻、 高粱、 小麦、 大豆、 棉花或向日葵。
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基因， 如表一 中的 ms45的野生型等位基因 Ms45， 这个控制雄性生育能力的基因不限于表一 中列出的基因， 玉米中或其他物种中控制雄性生育能力的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。
在本发明的一个实施例中， 所述第一植株为玉米雄性不育突变体 ms45; 和 / 或，
所述第一核苷酸序列为 Ms45表达元件， 所述 Ms45表达元件在所述第一植 株中表达 SEQ ID No:4所示的蛋白质 Ms45。
其中， SEQ ID No :4由 412个氨基酸组成。
在本发明的一个实施例中，所述 Ms45表达元件中的 Ms45编码序列是 SEQ ID No:8。 所述 Ms45表达元件的核苷酸序列是 SEQ ID No:l, 包括启动子和基因， 其中， SEQ ID No:l的第 8-542位为启动子序列， SEQ ID No:l的第 1422-2972 位为 0RF。
在本发明的一个实施例中， 发明人构建了含有 SEQ ID No:l所示的 Ms45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。
在本发明的一个实施方式中， 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籽粒的大小。
在本发明的一个实施例中， 所述第二核苷酸序列为干扰 SEQ ID No:5所示 的蛋白质表达的 DNA片段。
其中， SEQ ID No: 5由 590个氨基酸残基组成， 是 Mnl蛋白质的氨基酸序列。 在本发明的一个实施例中， 所述干扰 SEQ ID No:5所示的蛋白质表达的 DNA 片段可为 SEQ正向- X - SEQ反向；
所述 850^的核苷酸序列为 SEQ ID No :2的第 14-276位； 所述 SEQ^的序 列与所述 SEQ 的序列反向互补； 所述 X是所述 SEQ 与所述 SEQ ^之间的间 隔序列， 在序列上， 所述 X与所述 SEQ 及所述 SEQ^均不互补。
所述 SEQ正向- X - SEQfi向的核苷酸序列可为 SEQ ID No :2的第 14-663位。 其中， SEQ ID No:2是沉默 Mnl基因的 Mnl干扰片段 MnlRNAi, 由 675个核 苷酸组成。 SEQ ID No :2的第 7-13位为 BstEII识别序列， SEQ ID No :2的第 14-276 位为 MnlSEQ ^的核苷酸序列， SEQ ID No :2的第 277-400位为 X为形成发卡结 构的内含子， SEQ IDNo:2的第 401-663位为 MnlSEQfi 的核苷酸序列， SEQ IDNo:2 的第 664-669位为 Hindlll识别序列。
在本发明的另一个实施方式中， 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的籽粒是否粉质胚乳。
在本发明的一个实施例中， 所述第二核苷酸序列编码 SEQ ID No: 7所示的 MC16_KDY-醇溶蛋白。 其中， SEQ ID No: 7由 138个氨基酸组成。
所述第二核苷酸序列中的 MC16-KDY-醇溶蛋白基因为 SEQ ID No:6的第 1244-1780位。
其中， SEQ ID No:6由 1666个核苷酸组成， 第 9_1149位为启动子序列， 第 1244-1780位为 MC16-KDY-醇溶蛋白基因的编码序列， 编码 SEQ ID No:7的 MC16_KDY-醇溶蛋白。
所述第二核苷酸序列具体如 SEQ ID No:6所示。
本发明还保护上述方法中的构建体 （DNA构建物） 、 第二植株、 利用上述 方法产生的纯合隐性的雄性不育植株。
上述 DNA构建物可以恢复雄性不育突变体的育性， 同时改变籽粒形状 （如 大小、 长短、 宽窄、 薄厚等） 或同时改变籽粒胚乳营养物质成分 （如淀粉含量、 油份含量、 是否粉质胚乳等） 。
上述第二植株可以保持雄性不育植株的不育性。
由上述方法中的第二植株产生的再生细胞的组织培养物以及由该组织培养 物产生的原生质体也属于本发明的保护范围。
上述植株可为植株的全部或部分， 如种子、 根、 茎、 叶、 胚、 根尖、 花粉 或花药等。
本发明中， 发明人利用控制植物雄性生育能力基因、 籽粒标记基因及转基 因技术， 发明了一种高效扩繁植物雄性不育系的新方法。 在本发明的一个实施 方式中， 所述种子标记基因为控制籽粒特殊形状 （如大小、 长短、 宽窄、 薄厚 等） 的核苷酸； 在本发明的另一个实施方式中， 所述种子标记基因为控制胚乳 主要营养物质成分 （如淀粉含量、 油份含量、 是否粉质胚乳等） 的核苷酸。
本发明将控制雄性生育能力的野生型核苷酸序列和籽粒形状或籽粒胚乳营 养物质成分的核苷酸序列连在一起转入常规玉米， 然后回交到纯合隐性雄性不 育系植株， 利用获得的转基因植株与纯合隐性不育系杂交后， 可以同时得到大 量的不育系和保持系种子。 由于控制籽粒形状或籽粒胚乳营养物质成分核苷酸 序列的作用， 可以通过籽粒形状或胚乳成分区分不育系和保持系。 其中形状正 常或胚乳成分正常的种子为不育系 （不含转基因序列） ， 形状异常 （如籽粒大 小、 长短、 宽窄、 薄厚等发生变化） 或胚乳成分异常 （淀粉含量、 油份含量、 是否粉质胚乳等发生变化） 的种子为保持系。
在本发明的一个实施方式中， 发明人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控制籽粒形状 （如大小、 长短、 宽窄、 薄厚等） 基因的表达元件， 控制籽粒形状 （如大小、 长短、 宽窄、 薄厚等） 的 基因为一种显性基因或一段干扰序列， 将该载体转入 ΗΠΙΑΧΗΠΙΒ玉米杂交种 中， 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、 籽粒特殊形状 （如大小、 长短、 宽窄、 薄厚等） 的核苷酸序列导 入雄性不育植株中。 由于存在恢复基因， 该植株表现为可育。 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 籽粒正常的雄性不育籽粒 （不育系， 基因型为 msms ) ， 该不育系可以被任意一 种野生型植株恢复育性； 另一种为籽粒异常的可育籽粒 （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， 由于同时含有影响籽粒形状 （如大小、 长 短、 宽窄、 薄厚等） 的核苷酸序列， 该籽粒形状 （如大小、 长短、 宽窄、 薄厚 等） 不同于野生型。
在本发明的另一个实施方式中， 发明人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控制胚乳主要营养物质成分 （如淀 粉含量、 油份含量、 是否粉质胚乳等） 基因的表达元件， 控制胚乳主要营养物 质成分 （如淀粉含量、 油份含量、 是否粉质胚乳等） 的基因为一种显性基因或 一段干扰序列， 将该载体转入 ΗΠ ΙΑ Χ ΗΠ ΙΒ玉米杂交种中， 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、 胚乳主要 营养物质成分 （如淀粉含量、 油份含量、 是否粉质胚乳等） 的核苷酸序列导入 雄性不育植株中。 由于存在恢复基因， 该植株表现为可育。 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， 一种为胚 乳正常的雄性不育籽粒 （不育系， 基因型为 msms ) ， 该不育系可以被任意一种 野生型植株恢复育性； 另一种为胚乳异常的可育籽粒 （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， 由于同时含有影响胚乳主要营养物质成分 (如淀粉含量、 油份含量、 是否粉质胚乳等） 的核苷酸序列， 该胚乳主要营养 物质成分 （如淀粉含量、 油份含量、 是否粉质胚乳等） 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。
附图说明
图 1为雄性生育力突变体 ms45及野生型 Ms45的雄花表型。
图 2为控制籽粒大小基因 Mnl突变体及野生型的籽粒表型。
图 3为含有雄性生育力基因 Ms45表达元件及控制籽粒大小基因 Mnl干扰片 段表达元件的植物表达载体 pMs45_MnlRNAi的结构示意图。
图 4为转化雄性生育力基因 Ms45表达元件及控制籽粒大小基因 Mnl干扰片 段表达元件的植物表达载体植株雄花及籽粒表型。
图 5为利用细胞核雄性不育基因、 控制籽粒大小的基因及转基因技术制种 的路线图。
图 6为玉米遗传转化试验流程图。
图 7为控制籽粒成分基因 16-KD Y -醇溶蛋白显性突变体籽粒表型。
图 8为含有雄性生育力基因 Ms45表达元件及控制籽粒成分基因 16-KD y - 醇溶蛋白显性等位基因表达元件的植物表达载体 pMs45-Mc 16-KD y -zein的结 构示意图。 图 9为转化雄性生育力基因 Ms45表达元件及控制籽粒成分基因 16-KD y - 醇溶蛋白显性等位基因载体植株的雄花及籽粒表型。
图 10为利用细胞核雄性不育基因、控制胚乳成分的基因及转基因技术制种 的路线图。
实施发明的最佳方式
本文所用的所有技术和科学术语都具有本发明所属领域普通技术人员通常 所理解的相同的含义， 除非特殊说明， 本文所使用的或提到的技术是本领域普 通技术人员公认的标准技术， 材料， 方法和例子仅作阐述， 不加以限制。
细胞核雄性不育是由于小孢子形成过程中的关键基因被突变、 抑制或受到 其他影响的结果， 这些基因被统称为雄性不育基因。 花粉发育途径中受到很多 基因的控制， 因此很多基因的突变最终都会导致雄性不育， 目前在玉米中鉴定 了大量的雄性不育突变体 （如表一所示） ， 每个雄性不育基因都有其特定的恢 复基因， 即每种雄性不育突变体， 只能被其野生型等位基因恢复。
本发明以表一中的一个玉米雄性不育突变体为例， 如 ms45， 突变体雄花不 能正常散粉 （图 1所示， 左侧植株为雄性不育突变体， 右侧植株为野生型） ， 该突变体育性可以被野生型植株恢复。 本发明中野生型恢复基因来源于自交系 B73, 其序列见 SEQ ID No : l。 该序列包含 Ms45基因的启动子 （SEQ ID No : l的 第 8-542位） 和 0RF序列 ( SEQ ID No : l的第 1422-2972位） 。 将 SEQ ID No : l 所示的靈转入 ms45雄性不育突变体后， 突变体植株表现为可育。
在本发明的一个实施方式中， 本发明构建了玉米胚乳特异表达的细胞壁转 化酉每 ( endosperm-specific cel l wal l invertase, CWI- 2)基因 ( Cheng, WH et al. 1996)的植物表达干扰载体， 将该基因沉默。 该基因的突变体命名为 miniaturel ( mnl ) ， 该基因突变或沉默后籽粒会变小。 由于该基因特异性的在 胚乳中表达， 因此该基因沉默后不会影响植株的其他性状。 在玉米受精过程中， 含有该载体的配子受精后会影响胚乳的发育， 从而导致籽粒变小。 Mnl基因编 码一种细胞壁转化酶， 该基因失活后会影响到籽粒胚乳的发育， 导致籽粒变小
(图 2所示， A为 mnl突变体籽粒， B为野生型籽粒） ， 但并不影响胚和植株的 发育。 自然条件下或人工诱变获得的 Mnl突变体大多为隐性突变体， 发明人通 过转基因技术对野生型的 Mnl基因在 mRNA水平进行干扰， 使得只要含有转化干 扰核苷酸片段的籽粒就会变小， 图 4中 A所示， 因此， 转化片段在籽粒中以杂 合状态存在时， 就可以通过籽粒大小快速分辨含有转化片段的籽粒。 同理， 也 可以通过相同方法对影响籽粒形状 （如大小、 长短、 宽窄、 薄厚等） 发育的其 他基因进行相同的操作，获得便于分辨的转基因籽粒。本发明中的 Mnl基因 RNAi 干扰序列来源于 B73， 但不限于自交系 B73，， 同样可以来源于其他任何野生型 玉米自交系， 或其他物种的同源基因， 或人工合成的核苷酸序列。 本发明将雄 性恢复基因 Ms45及籽粒大小标记基因 Mnl的干扰片段构建在一个载体中， 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籽粒变小， 即对含有恢复基因的籽粒进行标记， 以便区分可育籽粒 （保持系） 和不育籽粒 (不育系） 。 在下述实施例中， 本发明将控制玉米雄性生育能力的基因 Ms45与 控制籽粒大小的基因 Mnl的干扰片段构建在一个载体中， 将该载体转入 ΗΠΙΑ XHillB玉米杂交种中， 然后利用 ms45雄性不育植株对获得的转基因植株进行 回交， 从而将 Ms45恢复基因和 Mnl基因的干扰片段同时导入雄性不育突变体 ms45中。 由于野生型 Ms45基因的存在， 转基因植株表现为可育。 当转基因杂 合体植株 （Ms45ms45ms45) 与雄性不育植株（ms45ms45)杂交时， 会产生两种后 代， 一种为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 （Ms45Ms45) 恢复育性， 在制种过程中可作为 不育系； 另一种为籽粒变小的可育籽粒 （保持系 MS45ms45ms45) ， 该保持系在 控制雄性生育能力位点为隐性纯合， 由于含有互补的转基因序列， 该植株表现 为可育， 由于同时含有影响籽粒大小的核苷酸序列， 该籽粒变小。
在本发明的另一个实施方式中， 本发明使用控制籽粒成分基因 16-KD Y-醇 溶蛋白的显性等位基因， 16-KD Y -醇溶蛋白基因编码一种醇溶蛋白， 突变体 Mucronate (Mc)由于 16-KD γ -醇溶蛋白基因 438_476bp有 38个碱基的缺失， 该 38bp的缺失改变了基因的编码框，从而使得翻译的蛋白较野生型有较大的差别。 进而影响到籽粒胚乳的发育， 导致胚乳不透明 （图 7所示， A为 MC16-KD Y-醇 溶蛋白突变体， B为野生型） ， 但并不影响胚和植株的发育。 由于 Mc为显性突 变体， 因此只要籽粒中含有 Mc的显性等位基因该籽粒就会表现为不透明胚乳。 本发明通过转基因技术将 Mc显性等位基因转入野生型中， 并确保该基因在植株 中正常表达， 就会表现出胚乳不透明的表型， 图 9中 A所示， 这种条件下， 转 化片段在单个籽粒个体中以杂合状态存在， 就可以通过肉眼或借助仪器进行快 速分辨。 同理， 也可以通过相同方法对影响胚乳成分发育的其他显性基因进行 相同的操作， 获得便于分辨的转基因籽粒。 本发明中的 16-KD Y-醇溶蛋白基因 核苷酸序列来源于 Mc突变体， 具体序列见 SEQ ID No:6所示， 其中包含启动子 序列以及基因的编码框序列， 以上核苷酸序列不限于 Mc突变体， 同样可以来源 于其他任何玉米胚乳突变体， 或其他物种的同源基因， 或人工合成的核苷酸序 列。 本发明构建了控制玉米胚乳成分的 16-KD Y-醇溶蛋白基因 （CheolSoo Kim et al. 2006)的载体。 该载体会影响胚乳的发育， 使玉米为粉质胚乳， 粉质胚 乳表现为不透明， 该基因的突变体为 Mucronate (Mc)。 该基因沉默后不会影响 植株的其他性状。 在玉米受精过程中， 含有该载体的配子受精后会影响胚乳的 发育， 从而导致胚乳不透明。本发明将雄性恢复基因 Ms45及 Mcl6-KD y -醇溶蛋 白胚乳标记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籽粒胚乳不透明， 即对含有恢复基因的籽粒进行 标记， 以便区分可育籽粒 （保持系） 和不育籽粒 （不育系） 。 本发明将控制玉 米雄性生育能力的基因 Ms45与控制胚乳成分的 Mc 16-KD Y -醇溶蛋白基因构建 在一个载体中， 将该载体转入 ΗΠΙΑΧΗΠΙΒ玉米杂交种中， 然后利用 ms45雄 性不育植株对获得的转基因植株进行回交， 从而将 Ms45 恢复基因和 Mc 16-KD Y -醇溶蛋白基因同时导入雄性不育突变体 ms45中。由于野生型 Ms45基因的存 在， 转基因植株表现为可育。 当转基因杂合体植株 （Ms45ms45ms45) 与雄性不 育植株（ms45ms45)杂交时， 会产生两种后代， 一种为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 (Ms45Ms45) 恢复育性， 在制种过程中可作为不育系； 另一种为胚乳不透明的 可育籽粒 （保持系 MS45ms45ms45) ， 该保持系在控制雄性生育能力位点为隐性 纯合， 由于含有互补的转基因序列， 该植株表现为可育， 由于同时含有影响胚 乳成分的核苷酸序列， 该籽粒胚乳不透明。
本发明提供了一种高效的种子标记方法， 该方法不仅适用于玉米 （Zea mays) ， 同样适用于水稻 (Oryza sativa) 、 高粱 (Sorghum bicolor) 、 小麦 (Triticumaestivum) 、 大豆 (Glycine max) 、 棉花 (Gossypiumhirsutum) 、 向日葵 （Helianthus annuus) 等作物。
下文通过说明和阐述提供了更为详细的描述， 这并非意欲对本发明的范围 加以限制。
实施例 1、构建含有控制玉米雄性生育能力和控制玉米籽粒大小的謹片段 (DNA构建体） 植物转化载体 pMs45_MnlRNAi
图 3所示的植物转化载体 PMs45-MnlRNAi中含有控制玉米雄性生育能力和 控制玉米籽粒大小的 DNA片段 （DNA构建体） 和选择标记基因。 其中， 控制玉米 雄性生育能力和控制玉米籽粒大小的 DNA片段名称为 Ms45_MnlRNAi， 为 pMs45-MnlRNAi的 LB和 RB之间的 DNA片段。 Ms45_MnlRNAi包含 SEQ ID No: 1 的 Ms45表达元件 （第一核苷酸序列） 和沉默 Mnl基因的表达元件 （第二核苷酸 序列）。沉默 Mnl基因的表达元件由 SEQ IDNo:3的 Mnl的启动子（Mnl promoter), Mnl干扰片段 MnlRNAi和终止子连接而成。 Ms45表达元件和沉默 Mnl基因的表 达元件紧密相连， 当将 pMs45-MnlRNAi转入植物时， 这两个表达元件在植物中 同时存在。 pMs45_MnlRNAi的构建方法如下：
1、 恢复玉米雄性不育突变体 ms45雄性生育能力的 Ms45野生型等位基因 (Ms45表达元件） 的扩增
本发明以表一中的 ms45雄性不育突变体为例， 具体阐述实施方案。 首先扩 展扩增 ms45的野生型等位基因 Ms45，该基因来源于自交系 B73,其序列见 SEQ ID No:l。 以玉米自交系 B73的基因组 DNA为模板， 参考 B73基因组序列
(www.maizesequence.org) ， 设计引物对该基因的整个表达元件（Ms45基因的 启动子和编码框序列） 进行扩增， 扩增引物如下： Ms45F: 5'
tgaattcTGCTGAGTTCTCCTTGGGTTATCC 3' , Ms45R: 5'
t c c c g g gGGTTGCGCATGAAATAGGGGT 3'。上游扩增引物的 5' 端添加了 EcoRI识别 位点， 下游扩增引物的 5' 端添加了 Smal识别位点， 扩增反应体系为： 模板 DNA 2 L，引物 Ms45F 0.5 L， 引物 Ms45R 0.5 L， dNTP 1.6μΐ, 10 X Buffer 2 L，高保 真 taq酶 0.3μΐ, ddH20 13. L。 反应条件为 95°C预变性 5min, 95°C变性 45s， 59°C退火 45s， 72°C延伸 3min， 32个循环， 72°C后延伸 10min。 扩增的目标条 带全长为 3518bp，扩增后将该序列连接 T-easy测序载体，将阳性克隆进行测序， 测序结果表明该 3518bp 的 DNA含有 SEQ ID No： 1所示的 Ms45基因表达元件、 EcoRI识别位点和 Smal识别位点。 SEQ ID No:l中， 第 8-542位为启动子， 第 1422-2972位为 Ms45基因的编码序列， 编码 SEQ ID No： 4的 Ms45蛋白质。
2、 沉默 Mnl基因的 Mnl干扰片段 MnlRNAi的制备
Mnl基因编码一种细胞壁转化酶的蛋白 （其氨基酸序列如 SEQ ID No :5, 该 基因失活后会影响到胚乳的发育， 导致籽粒变小 （图 2 所示） ， 但并不影响胚 和植株的发育。 本发明通过 RNAi技术将 Mnl基因沉默， 所用的干扰片段名称为 MnlRNAi, 其结构为 MnlSEQ正向- X - MnlSEQ反向， MnlSEQ反向的序列与 MnlSEQ正向 的序列反向互补， X为形成发卡结构的内含子。 MnlRNAi的核苷酸序列如 SEQ ID No :2所示， SEQ ID No :2 的第 7-13位为 BstEII识别序列， SEQ ID No :2的第 14-276位为 MnlSEQ ^的核苷酸序列， SEQ ID No： 2的第 277-400位为 X为形成 发卡结构的内含子， SEQ ID No:2的第 401-663位为 MnlSEQ 的核苷酸序列， SEQ ID No :2的第 664-669位为 Hindlll识别序列。
3、 Mnl基因启动子 （Mnl promoter) 的克隆
本发明使用控制籽粒大小基因 Mnl的启动子 （Mnl promoter) 启动 Mnl干 扰片段 MnlRNAi, Mnl基因为胚乳特异表达， 因此利用该启动子起始转录的 mRNA 只存在于胚乳细胞中。 该启动子来源于玉米自交系 B73的基因组 DNA， 具体序列 见 SEQ ID No :3所示， 以玉米自交系 B73 基因组 DNA为模板， 参考 B73基因组 序列 （www. maizesequence. org) ， 设计引物对该基因的启动子进行扩增， 扩增 引物如下： Mnlpro bF:5' at c c c ggGCTCGCATGAGAGAACAACCA 3' , Mnlpro bR:5' gcaagcttGGGGGTGCTATTTGTACTGTGC 3'。 上游扩增引物的 5' 端添加了 Smal识 别位点， 下游扩增引物的 5' 端添加了 Hindlll识别位点， 扩增反应体系为： 模 板 DNA 2μΐ,引物 Mnlpro bF 0.5μ 引物 Mnlpro bRO.5 L， dNTP 1.6 L， 10 X Buffer 2 L,高保真 taq酶 0.3μΐ, ddH20 13. L。 反应条件为 95°C预变性 5min, 95 °C 变性 45s， 59°C退火 45s， 72°C延伸 2min， 32个循环， 72°C后延伸 10min。 扩 增的目标条带全长为 2422bp，扩增后将该序列连接 T-easy测序载体， 将阳性克 隆进行测序， 测序结果表明该 2422bp的 DNA为 Mnl基因启动子片段， 该 Mnl基 因启动子片段含有 SEQ ID No:3的 Mnl基因启动子、 Smal识别位点和 Hindlll 识别位点。
4、 pMs45-MnlRNAi的构建
通过装配上述步骤 1、 2和 3的 DNA组件， 构建图 3所示的植物转化载体 pMs45_MnlRNAi。
以质粒 PCAMBIA3301 (国际农业分子生物学应用中心 CAMBIA, Australia) 为骨架 DNA， 构建包含雄性生育力基因 Ms45、 Mnl干扰片段表达元件及选择标记 基因 bar表达元件的植物转化载体 pMs45_MnlRNAi。首先利用 BstEII和 Hindlll 消化步骤 2的 Mnl干扰片段 MnlRNAi和 pCAMBAI3301， 将 Mnl干扰片段 MnlRNAi 和 pCAMBAI3301大片段进行连接， 检测阳性克隆， 然后再利用 EcoRI和 Smal双 酶切阳性克隆和步骤 1的 3518bp 的 DNA ( Ms45表达元件） ， 回收目标条带， 将两片段进行连接， 检测阳性克隆， 最后再利用 Smal和 Hindlll消化阳性克隆 和步骤 3的 Mnl基因启动子， 回收目标条带， 将两片段进行连接， 检测阳性克 隆， 获得包含雄性生育力基因 Ms45、 Mnl干扰片段表达元件及选择标记基因 bar 表达元件的植物转化载体 pMs45_MnlRNAi (图 3) 。 pMs45_MnlRNAi是将
PCAMBAI3301的 EcoRI和 Smal识别位点间的片段替换为 SEQ ID No: 1所示的 Ms45 基因表达元件、将 pCAMBAI3301的 Smal和 Hindlll识别位点间的片段替换为 SEQ ID No :3所示的 Mnl基因启动子， 且将 pCAMBAI3301的 BstEII和 Hindlll识别 位点间的片段替换为 SEQ ID No :2所示的 MnlRNAi得到的重组表达载体。
实施例 2、 制备 Ms45_MnlRNAi杂合且 ms45纯合的第二植株
一、 用实施例 1的植物转化载体 pMs45_MnlRNAi转化玉米
本发明通过农杆菌侵染玉米幼胚的方法获得转基因植株。 将实施例 1的植 物转化载体 pMs45-MnlRNAi转化根癌农杆菌 EHA105, 再用含有目的基因的农杆 菌侵染玉米幼胚， 具体的转基因方法如下：
实验室在转基因过程中所用受体为自交系 ΗΠΙΑ和 ΗΠΙΒ的杂交 F1代。 玉 米自交系 ΗΠΙΑ和 HillB ( 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) 。 首先在田间种植玉米自交系 Hi I IA和 Hi I IB, 到自交系散粉时分别 套袋； 然后准备授粉， 有两种授粉方式： ΗΠΙΑ作母本， ΗΠΙΒ作父本； ΗΠΙΑ 作父本， ΗΠΙΒ作母本， 授粉后 9-11天， 取授粉果穗籽粒上的未成熟幼胚， 然 后在室内进行根癌农杆菌 EHA105侵染， 将被根癌农杆菌 EHA105侵袭的幼胚放 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 TO代植株。 获得转基因 TO代以后， 用 TO代转基因植株的花粉对一 些制种母本及 Ms45雄性不育材料进行杂交， 并观察表型。 具体试验流程如图 6。
采用根癌农杆菌 EHA105侵染法将 pMs45_MnlRNAi导入受体植株的幼胚， 经 除草剂双丙胺磷筛选后获得转基因植株。 具体方法为：
(一） 剥离幼胚
1、 去除苞叶。 切除果穗顶端约 lcm左右， 用镊子从顶端插入果穗， 这样可 以以镊子当作把手， 有利于操作， 然后把果穗放入含有消毒液的烧杯里， 根据 实际需要， 可以在同一个烧杯里放 4-6个果穗。
2、 向烧杯里加约 700ml的消毒液（50%的漂白剂或 5.25%的次氯酸钠， 并加 入一滴 Tween20) 用来浸泡果穗， 在消毒 20分钟过程当中， 不时的旋转果穗同 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、 把消毒过果穗的一端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) ， 在这过程当中， 要勤消毒所用的工具， 如： 手术刀片、 培 养皿、 剥胚刀等。
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，胚的密度大约是 2 X 2cm (30个 /皿）。
5、 用封口膜封住培养皿， 28度暗培养 2-3天。
(二） 农杆菌浸染
1、 根癌农杆菌 EHA105要在 YEP (含 33mg/L卡那霉素和 100mg/L利福平） 培养基上提前一周培养， 并在 4°C冰箱保存一个月左右， 长期保存要在 -80°C甘 油保存。
2、 根癌农杆菌 EHA105要在 YEP培养基上在 19°C培养 3天， 同时加卡那霉 素至浓度为 33mg/L,加利福平至浓度为 50mg/L。
3、 3天以后， 挑取根癌农杆菌 EHA105放入含有 5mL侵染培养基的 50ml离 心管中，同时加 AS (inf+AS) (溶质如表 2，溶剂为水），在室温（25 °C )转速 75rpm 摇菌 2-4个小时。
4、 侵染幼胚， 把刚剥离的幼胚放入含有 AS (inf+AS)液体培养基 （2ml ) 的 离心管中，每管约 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 （0D550=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。
(三） 共培养
1、 侵染以后， 把侵染过的幼胚转移到共培养培养基 （溶质如表 2， 溶剂为 水） ，使幼胚的胚轴接触培养基表面， 同时驱除培养基表面多余的农杆菌。
2、 用封口膜封住培养皿， 在 20°C条件下暗培养 3天。
(四） 静息培养
共培养 3天后， 把幼胚转移到静息培养基 （溶质如表 2， 溶剂为水） 上面， 同时用封口膜封住培养皿， 放在 28 °C条件下暗培养 7天。
(五） 选择
7天后， 把所有的幼胚转移到选择培养基（溶质如表 2， 溶剂为水）上面（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、侵染大约 5周左右，含有转化子的细胞会长成可以看见的 II型愈伤组织。
(六） 转基因植株的再生
1、 在再生培养基 I (溶质如表 2， 溶剂为水） 上面长 3周， 然后在再生培 养基 I I (溶质如表 2， 溶剂为水） 上面发芽 （在光照培养室） ， 得到 100株 TO 代 pMs45_MnlRNAi转化玉米植株。 2、 待再生苗生长出 3-4片叶时， 将其转移到温室， 待其生长至吐丝散粉期 时， 对其进行授粉。
表 2.培养基的溶质及含量
Figure imgf000020_0001
表 2中， MS 盐购自 phyto Technology Laboratories公司， 货号为 M524。 二、 将 Ms45Ms45野生型自交系转变为 ms45ms45纯合隐性自交系
以 ms45纯合隐性突变体 (Maize Genetics Cooperation Stock Center, 9051) 为母本， 与不同自交系 （如郑 58 (zheng58) ) 杂交， 获得的 F1继续与 玉米自交系郑 58 (河南秋乐种业科技股份有限公司） 回交， 对获得的 BC1群体 进行基因型分析， 鉴定 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， 其他位点均为郑 58的单株进行自 交， 从而获得 ms45纯合隐性自交系郑 58 (郑 58 (ms45ms45) ) ， 该自交系即 可作为不育系， 将其称为第一植株。 三、 制备 Ms45_MnlRNAi杂合且 ms45纯合的第二植株
将 ms45纯合隐性自交系 （母本） 与步骤一获得的 TO代 pMs45_MnlRNAi转 化玉米植株 （父本） 杂交然后再以 ms45纯合隐性自交系为轮回亲本进行多代回 交， 将步骤一获得的 TO代 pMs45_MnlRNAi转化玉米植株转变为含有
Ms45-MnlRNAi且 ms45位点为纯合隐性且 Ms45_MnlRNAi杂合的自交系， 该自交 系即为 Ms45_MnlRNAi杂合且 ms45纯合的第二植株。
为达到以上目的， 将步骤一获得的 50株 TO代 pMs45_MnlRNAi转化玉米植 株（父本）分别与步骤二获得的 ms45纯合隐性自交系郑 58 (郑 58 ( ms45ms45 ) ) (母本） 杂交， 从杂交后代中挑选小粒的籽粒播种到田间后喷施 200mM的双丙 胺磷， 对存活的植株继续与步骤二获得的 ms45纯合隐性自交系郑 58 (郑 58 ( ms45ms45 ) ) 回交， 如此回交 5_6代后， 利用分子标记筛选转基因位点 (Ms45-MnlRNAi ) 为杂合， Ms45位点为隐性纯合， 其他位点均为郑 58背景的单 株， 该单株即为 Ms45_Mn lRNAi杂合且 ms45纯合的第二植株， 将该第二植株命 名为郑 58 ( Ms45ms45ms45 ) 第二植株。
以上述郑 58 ( Ms45ms45ms45 )第二植株为父本， 与步骤二获得的 ms45纯合 隐性自交系郑 58 (郑 58 ( ms45ms45 ) ) (母本） 杂交， 产生的后代不但有雄性 不育系郑 58 ( ms45ms45 ) ， 而且还有雄性不育郑 58 ( ms45ms45 ) 的保持系郑 58 ( Ms45ms45ms45 ) ， 并且郑 58 ( ms45ms45 ) 的籽粒大小正常（图 4中 B所示） ， 而其保持系郑 58 ( Ms45ms45ms45 ) 籽粒为小粒 （图 4中 A所示籽粒） 。 具体选 育过程见附图 5。
四、 分析步骤一获得的 TO代 pMs45-MnlRNAi转化玉米植株
针对植株整体形态对来自步骤一获得的 T0代 pMs45_MnlRNAi转化玉米植株 及其后代加以评估， 针对花粉和籽粒表型着重进行分析。 除了籽粒之外， 在 T0 代 pMs45-MnlRNAi转化玉米植株与非转基因对照植株之间没有观察到其他形态 的不同。 当将 T0代 pMs45_MnlRNAi转化玉米植株与 ms45雄性不育材料进行杂 交时， 杂交后代中含有 Ms45_MnlRNAi的植株表现为可育， 图 4中 C所示， 而不 含有 Ms45_MnlRNAi的杂交后代表现为完全不育。 这表明 Ms45基因的表达互补 了隐性纯合 ms45雄性不育表现， 同时， 含有 Ms45_MnlRNAi的籽粒较不含有 Ms45-MnlRNAi的正常籽粒小， 表型同 mnl突变体， 图 4中 A所示。 这表明 Mnl 基因干扰片段在转基因植株中均能正常行使功能。 同时发明人对通过籽粒表型 鉴定的小粒籽粒及正常籽粒播种到田间， 这些籽粒均能够正常发芽， 出芽率与 正常籽粒没有显著差异。 当植株生长至 4-5片叶时， 喷施 200mM的双丙胺磷， 小粒籽粒均能正常成活， 并且生长不受到抑制。 而正常籽粒出的苗全部死亡。 这表明 Ms45雄性生育力基因及 mnl干扰片段及选择标记基因 bar均能正常行使 功能，并且这三个基因连锁遗传。转 pMs45_MnlRNAi植株与雄性不育突变体 ms45 杂交产生的后代中， 正常籽粒： 小粒籽粒为 1 : 1。
图 4的具体实验方法同步骤三。 实施例 3、 使用实施例 2中的雄性不育保持系对 ms45雄性不育自交系进行 大规模扩繁
以郑 58自交系为例， 将实施例 2中的雄性不育系郑 58 (ms45ms45) 和实 施例 2中的雄性不育保持系郑 58 (Ms45ms45ms45) 播种到田间， 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， 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， 产生的后代中由于含有纯合的转基因成分 （Ms45_MnlRNAi) 的籽粒与杂合的籽 粒无法辨别， 这些籽粒予以丢弃， 而正常大小的籽粒 （大粒） 可以作为不育系。 雄性不育系郑 58 (ms45ms45) 接受了雄性不育保持系郑 58 (Ms45ms45ms45) 的花粉， 其后代中正常大小籽粒的为不含转基因成分的不育系， 而小粒籽粒为 含有转基因成分的保持系。 保持系全部用于下一年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扩繁不育系和保持系， 具体生产流程如附图 5所示。
实施例 4、 利用实施例 3中的雄性不育系大规模生产杂交种
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
(ms45ms45) 自交系，如雄性不育郑 58 (ms45ms45) 配合力高的自交系， 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， 发明 人将雄性不育自交系与野生型自交系隔行播种于田间， 确保繁种周边 300米内 无其他玉米播种， 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。 这样不育系果穗上所产生的种子为杂交种。
实施例 5、 构建含有控制玉米雄性生育能力和控制玉米籽粒胚乳成分的謹 片段 （DNA构建体） 植物转化载体 pMs45_Mc 16-KD y -zein
图 8所示的植物转化载体 pMs45_Mc 16-KD y -zein中含有控制玉米雄性生 育能力和控制玉米籽粒胚乳是否透明的 DNA片段（DNA构建体）和选择标记基因。 其中， 控制玉米雄性生育能力和控制玉米籽粒大小的靈片段名称为 Ms45-Mc 16-KD y -zein,为 pMs45_Mc 16-KD y -zein的 LB和 RB之间的 DNA片段。 Ms45_Mc 16-KD y -zein包含 SEQ ID No : 1的 Ms45表达元件 （第一核苷酸序列） 和 SEQ ID No:6的 MC16-KD Y-醇溶蛋白基因表达元件 （第二核苷酸序列） 。 Ms45表达元件 和 MC16-KD Y-醇溶蛋白基因表达元件紧密相连， 当将 pMs45-Mc 16-KD y -zein 转入植物时， 这两个表达元件在植物中同时存在。 pMs45_Mc 16-KD γ-zein的构 建方法如下：
1、 恢复玉米雄性不育突变体 ms45雄性生育能力的 Ms45野生型等位基因 (Ms45表达元件） 的扩增
同实施例 1的步骤 1。
2、 Mc突变体 16-KD Y-醇溶蛋白基因的人工合成
根据 （CheolSoo Kim et al. 2006) 对该突变体的报道， 及对该序列的说 明 （Gene accession no. DQ826676) 对该基因进行了合成， 合成了 SEQ ID No :6 所示的 MC16-KD Y-醇溶蛋白基因表达元件， 同时在合成该基因时 5' 端添加了 Hindlll酶切位点， 3' 端添加了 BstEII酶切位点。 SEQ ID No:6中， 第 9-1149 位为启动子序列， 第 1244-1780位为 MC16-KD Y-醇溶蛋白基因的编码序列， 编 码 SEQ ID No:7的 Mcl6-KD Y -醇溶蛋白。
3、构建包含雄性生育力基因 Ms45和 Mc突变体 16-KD y -醇溶蛋白基因表达 元件及选择标记基因的植物转化载体 pMs45_Mc 16-KD y -zein
以质粒 pCAMBAI3301为骨架 DNA，构建包含雄性生育力基因 Ms45和 Mcl6_KD Y -醇溶蛋白表达元件及选择标记基因 bar的植物转化载体 pMs45-Mc 16-KD y -zein。 首先利用 BstEII和 Hindlll消化 Mcl6_KD γ -醇溶蛋白表达元件和
PCAMBIA3301 (国际农业分子生物学应用中心 CAMBIA, Australia) ， Mc 16-KD Y -醇溶蛋白表达元件和 PCAMBAI3301大片段进行连接， 检测阳性克隆， 连个片 段进行连接， 检测阳性克隆， 然后再利用 EcoRI和 Smal双酶切阳性克隆和步骤 1的 Ms45野生型等位基因， 回收目标条带， 将两片段进行连接， 检测阳性克隆， 获得包含雄性生育力基因 Ms45和 Mc 16-KD y -醇溶蛋白表达元件及选择标记基 因 bar的植物转化载体 pMs45_Mc 16-KD y -zein,构建完成的载体如（图 8所示）。 pMs45-Mc 16-KD y -zein是将 pCAMBAI3301的 EcoRI和 Smal识别位点间的片段 替换为 SEQ ID No: 1所示的 Ms45基因表达元件且将 pCAMBAI3301的 BstEII和 Hindlll识别位点间的片段替换为 SEQ ID No： 6所示的 Mcl6_KD γ -醇溶蛋白基 因表达元件得到的重组表达载体。
实施例 6、 制备 Ms45_Mc 16-KD γ -zein杂合且 ms45纯合的第二植株 一、 用实施例 5的植物转化载体 pMs45_Mc 16-KD y -zein转化玉米 本发明通过农杆菌侵染玉米幼胚的方法获得转基因植株。 将实施例 5的植 物转化载体 pMs45_Mc 16-KD γ -zein转化根癌农杆菌 EHA105, 再用含有目的基 因的农杆菌侵染自交系 ΗΠΙΑ和 ΗΠΙΒ的杂交 F1代玉米幼胚， 将被农杆菌侵染 的幼胚放在选择培养基上进行多次筛选， 获得抗性愈伤， 将抗性愈伤再生成苗， 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一 些制种母本及 Ms45雄性不育材料进行杂交， 并观察表型。 具体的实验方法同实 施例 2步骤一。
二、 将 Ms45Ms45野生型自交系转变为 ms45ms45纯合隐性自交系
以 ms45纯合隐性突变体 (Maize Genetics Cooperation Stock Center, 9051) 为母本， 与玉米自交系郑 58 (zheng58) (河南秋乐种业科技股份有限 公司） 杂交， 获得的 F1继续与玉米自交系郑 58回交， 对获得的 BC1群体进行 基因型分析， 鉴定 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， 其他位点均为郑 58的单株进行自交， 从而获得 ms45纯合隐性自交系郑 58 (郑 58 (ms45ms45) ) ， 该自交系即可作 为不育系， 将其称为第一植株。 具体的实验方法同实施例 2步骤二。 三、 Ms45_Mc 16-KD y -zein杂合且 ms45纯合的第二植株 将 ms45纯合隐性自交系 （母本） 与步骤一获得的 TO代 pMs45_Mc 16-KD y -zein转化玉米植株 （父本） 杂交然后再以 ms45纯合隐性自交系为轮回亲本进 行多代回交， 将步骤一获得的 TO代 pMs45-Mc 16-KD y -zein转化玉米植株转变 为含有 Ms45_Mc 16-KD γ -zein且 ms45位点为纯合隐性且 Ms45_Mc 16-KD y -zein 杂合的自交系， 该自交系即为 Ms45_Mc 16-KD γ -zein杂合且 ms45纯合的第二 植株。
为达到以上目的， 将步骤一获得的 50株 TO代 pMs45_Mc 16-KD γ -zein转 化玉米植株 （父本） 分别与步骤二获得的 ms45纯合隐性自交系郑 58 (郑 58 ( ms45ms45 ) ) (母本） 杂交， 从杂交后代中挑选胚乳不透明的籽粒播种到田 间后喷施 200mM的双丙胺磷， 对存活的植株继续与步骤二获得的 ms45纯合隐性 自交系郑 58 (郑 58 ( ms45ms45 ) ) 回交， 如此回交 5_6代后， 利用分子标记筛 选转基因位点 （Ms45_Mc 16-KD y -zein ) 为杂合， Ms45位点为隐性纯合， 其他 位点均为郑 58背景的单株， 该单株即为 Ms45_Mc 16-KD γ -zein杂合且 ms45纯 合的第二植株， 将该第二植株命名为郑 58 ( Ms45ms45ms45 ) 第二植株。
以上述郑 58 ( Ms45ms45ms45 )第二植株为父本， 与步骤二获得的 ms45纯合 隐性自交系郑 58 (郑 58 ( ms45ms45 ) ) (母本） 杂交， 产生的后代不但有雄性 不育系郑 58 ( ms45ms45 ) ， 而且还有雄性不育郑 58的保持系郑 58
(Ms45ms45ms45 ) ， 并且郑 58 ( ms45ms45 ) 的籽粒正常， 胚乳透明 （图 9中 B 所示） ， 而其保持系郑 58 ( Ms45ms45ms45 ) 籽粒为胚乳不透明 （图 9中 A所示 籽粒） 。 具体选育过程见附图 10。
四、 分析步骤一获得的 TO代 pMs45-Mc 16-KD y -zein转化玉米植株 针对植株整体形态对来自步骤一的 T0代 pMs45_Mc 16-KD y -zein转化玉米 植株及其后代加以评估， 针对花粉和籽粒表型着重进行分析。 除了籽粒之外， 在 T0代 pMs45-Mc 16-KD γ -zein转化玉米植株与非转基因对照植株之间没有观 察到其他形态的不同。 当将 T0代 pMs45_Mc 16-KD γ -zein转化玉米植株与 ms45 雄性不育材料进行杂交时， 杂交后代中含有转基因成分的植株表现为可育， 图 9 中 C所示， 而非转基因植株表现为完全不育。 这表明 Ms45基因的表达互补了隐 性纯合 ms45雄性不育表现， 同时， 含有 Mc突变体 16-KD Y -醇溶蛋白基因的转 基因籽粒表现为胚乳不透明， 表型同 Mc突变体， 图 9中 A所示。 这表明 Mc显 性突变体等位基因在转基因植株中均能正常行使功能。 同时对通过籽粒表型鉴 定的胚乳不透明籽粒及正常籽粒播种到田间， 这些籽粒均能够正常发芽， 出芽 率与正常籽粒没有显著差异。 当植株生长至 4-5片叶时， 喷施 200mM的双丙胺 磷， 胚乳不透明籽粒均能正常成活， 并且生长不受到抑制。 而正常籽粒出的苗 全部死亡。 这表明选择标记基因 bar、 Ms45雄性生育力基因及 Mc 16-KD y -醇溶 蛋白基因均能正常行使功能， 并且这三个基因连锁遗传。 当转基因植株与雄性 不育突变体 ms45杂交产生的后代中， 正常籽粒： 胚乳不透明籽粒为 1 : 1。 图 9的具体实验方法同步骤三。
实施例 7、 使用实施例 6中的雄性不育保持系对 ms45雄性不育自交系进行 大规模扩繁
以郑 58自交系为例， 将实施例 6中的雄性不育系郑 58 ( ms45ms45 ) 和实 施例 6中的雄性不育保持系郑 58 ( Ms45ms45ms45 )播种到田间， 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， 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， 产生的后代中由于含有纯合的转基因成分的籽粒与杂合的籽粒无法辨别， 这些 籽粒予以丢弃， 而正常的籽粒可以作为不育系。 不育系材料接受了保持系的花 粉， 其后代中正常籽粒的为不含转基因成分的不育系， 而胚乳不透明籽粒为含 有转基因成分的保持系。 保持系全部用于下一年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扩繁不育系和保持系， 具体生产流程如附图 10所示。
实施例 8、 利用实施例 7中的雄性不育系大规模生产杂交种
在实施例 7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 ( ms45ms45 ) 自交系，如雄性不育郑 58 ( ms45ms45 ) 配合力高的自交系， 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， 将雄 性不育自交系与野生型自交系隔行播种于田间， 确保繁种周边 300米内无其他 玉米播种， 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。 这样不育系果穗上所产生的种子为杂交种。
工业应用
本发明的扩繁植物雄性不育系方法采用了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。 本发明的扩繁植物雄性不育系方法利用了可以分辨籽粒形状 （如大小、 长短、 宽窄、 薄厚等） 或胚乳主要营养物质成分 （如淀粉含量、 油份含量、 是 否粉质胚乳等） 的核苷酸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， 可通过籽粒形状 （如大小、 长短、 宽窄、 薄厚等） 或胚乳营养物质成 分 （如淀粉含量、 油份含量、 是否粉质胚乳等） 高效分辨转基因籽粒中的可育 籽粒及不育籽粒。 利用本发明方法产生的纯合隐性雄性不育植株可用于生产杂 交种。

Claims

权利要求
1、 一种方法， 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；
(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， 所述构建体在所述第二植株中以杂合状 态存在， 所述构建体包含：
1、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籽粒形状或胚乳营养 物质成分， 通过肉眼或仪器可以分辨含有该构建体的籽粒和不含有该构建体的 籽粒；
所述第一核苷酸序列与所述第二核苷酸序列紧密相连， 这两个核苷酸序列 在植株中同时存在；
(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。
2、 根据权利要求 1所述的方法， 其特征在于： 所述方法为扩繁植物雄性不 育系的方法； 和 /或，
所述籽粒形状为大小、 长短、 宽窄或 /和薄厚； 所述胚乳营养物质成分为是 否粉质胚乳、 淀粉含量或 /和油份含量。
3、 根据权利要求 2所述的方法， 其特征在于： 所述植物、 所述第一植株和 所述第二植株均为玉米、 水稻、 高粱、 小麦、 大豆、 棉花或向日葵。
4、 根据权利要求 3所述的方法， 其特征在于： 所述第一植株为玉米雄性不 育突变体 ms45; 和 /或，
所述第一核苷酸序列为 Ms45表达元件， 所述 Ms45表达元件在所述第一植 株中表达 SEQ ID No : 4所示的蛋白质 Ms45。
5、根据权利要求 4所述的方法，其特征在于：所述 Ms45表达元件中的 Ms45 编码序列是 SEQ ID No : 8。
6、 根据权利要求 5所述的方法， 其特征在于： 所述 Ms45表达元件的核苷 酸序列是 SEQ ID No : l o
7、 根据权利要求 1所述的方法， 其特征在于： 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籽粒的大小。
8、 根据权利要求 7所述的方法， 其特征在于： 所述第二核苷酸序列为干扰 SEQ ID No : 5所示的蛋白质表达的 DNA片段。
9、 根据权利要求 8所述的方法， 其特征在于： 所述干扰 SEQ ID No : 5所示 的蛋白质表达的 DNA片段为 SEQ - X - SEQ fi向；
所述 850 ^的核苷酸序列为 SEQ ID No : 2的第 14-276位； 所述 SEQ ^的序 列与所述 SEQ 的序列反向互补； 所述 X是所述 SEQ 与所述 SEQ ^之间的间 隔序列， 在序列上， 所述 X与所述 SEQ 及所述 SEQ^均不互补。
10、 根据权利要求 9所述的方法， 其特征在于： 所述 SEQ ^- X - SEQ^ 的核苷酸序列为 SEQ ID No: 2的 SEQ ID No :2的第 14-663位。
11、 根据权利要求 1所述的方法， 其特征在于： 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是否粉质胚乳。
12、 根据权利要求 11所述的方法， 其特征在于： 所述第二核苷酸序列编码 SEQ ID No: 7所示的 Mcl6_KD γ-醇溶蛋白。
13、 根据权利要求 12所述的方法， 其特征在于： 所述第二核苷酸序列中的 MC16-KDY-醇溶蛋白基因的编码序列为 SEQ ID No:6的第 1244-1780位。
14、根据权利要求 13所述的方法，其特征在于：所述第二核苷酸序列如 SEQ ID No :6所示。
15、 一种靈构建物， 该构建物如权利要求 1所述的构建体。
16、 一种植物， 该植物如权利要求 1所述的第二植株。
17、 由权利要求 16所述的植物产生的再生细胞的组织培养物。
18、 由权利要求 17所述的组织培养物产生的原生质体。
19、 一种植物， 所述植物是利用权利要求 1所述方法产生的纯合隐性的雄 性不育植株。
20、 利用权利要求 1所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。
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