WO2021223727A1 - 一种用于农作物杂交育种制种的遗传智能化育制种系统及其应用 - Google Patents

一种用于农作物杂交育种制种的遗传智能化育制种系统及其应用 Download PDF

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WO2021223727A1
WO2021223727A1 PCT/CN2021/092017 CN2021092017W WO2021223727A1 WO 2021223727 A1 WO2021223727 A1 WO 2021223727A1 CN 2021092017 W CN2021092017 W CN 2021092017W WO 2021223727 A1 WO2021223727 A1 WO 2021223727A1
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gat
gene
expression cassette
maintainer
vector
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French (fr)
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安保光
龙湍
李新鹏
曾翔
吴永忠
黄培劲
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海南波莲水稻基因科技有限公司
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Publication of WO2021223727A1 publication Critical patent/WO2021223727A1/zh
Priority to US18/052,800 priority Critical patent/US20230332173A1/en
Priority to ZA2022/12707A priority patent/ZA202212707B/en

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Definitions

  • the invention belongs to the field of agricultural biotechnology, and specifically relates to a genetic intelligent breeding system for crop hybrid breeding and seed production and its application.
  • Heterosis is a phenomenon in which the offspring of hybrids surpass their parents in one or more traits, and it is widespread in the biological world.
  • the utilization of crop heterosis is an important means to increase agricultural production.
  • the core of the industrialization of heterosis utilization lies in the male sterility or male sterility of the female parent.
  • the male sterility of the "three-line method” sterile line is caused by the gene interaction between the nucleus and the cytoplasm, this method has defects: only the restorer line with a specific restorer gene in the nucleus can be restored to the hybrid after the sterile line is crossed. Fertility to produce hybrid rice seeds. Therefore, the "three-line method” has a low utilization rate of germplasm resources, and the homogenization of cytoplasm also has potential risks of diseases and insect pests.
  • the principle of the "two-line method” is to prepare hybrids using light-temperature-sensitive genic male sterile lines and restorer lines.
  • the male sterile line of the sterile line remains sterile at a specific stage of rice development when the environmental temperature is higher than 23.5 degrees, and can be successfully hybridized with the restorer line; if the environmental temperature is lower than 23.5 degrees, the male fertility of the sterile line is restored Normal, will self-bred. Therefore, the "two-line method" only requires sterile lines and restorer lines, saving seed production costs. However, because the fertility conversion of the "two-line method" of the photo-thermo-sensitive genic male sterile line is affected by the light and temperature environment, this technology has great environmental risks in the process of sterile line reproduction and hybrid seed production. The special requirements for temperature, the production season and area are also greatly restricted.
  • Recessive male sterility is different from “three-line” and "two-line”. Its male fertility is controlled by only a pair of recessive nuclear genes and is not affected by light and temperature environment; and because its genes are very rare recessive mutations, Since the above-mentioned sites in the genomes of most varieties are wild-type dominant genes, theoretically almost any variety can be used as a recessive male sterile restorer line with its cross to produce fertile offspring. The discovery of recessive nuclear sterility has a very long history.
  • the F1 generation When it is crossed with wild-type materials, the F1 generation is heterozygous and shows fertility, and the F2 generation appears fertile segregation (in the case of single gene control, the segregation ratio is 3:1), but when the F1 generation self-fertilizes and harvests seeds (ie F2), because the appearance of the seeds is exactly the same, it is impossible to distinguish which part of the seed will be fertile after developing into a plant in the future, and which part will be sterile. Therefore, it has long been limited by the inability to maintain the male sterility of its offspring on a large scale and cannot be used industrially. Therefore, it is particularly necessary to develop a technology that can maintain and propagate recessive nuclear male sterile materials and support commercial production, which is also an urgent need of the majority of breeders.
  • the purpose of the present invention is to provide a method for the maintenance and reproduction of recessive male sterile lines.
  • the present invention provides a GAT vector that mediates and regulates the male fertility of a recessive nuclear male sterile mutant (sterile line) of plants and its application.
  • the invention also provides a crop hybrid breeding technology system (GAT technology system) and application.
  • GAT Genetic Automation Technology, GAT
  • GAT Genetic Automation Technology
  • This technology can successfully use recessive male sterile lines.
  • the core idea is to use modern biotechnology to restore the fertility of crop pollen.
  • Pollen abortion genes, herbicide-sensitive genes, selection marker genes, etc. are closely linked to the GAT vector in a specific order and direction, and are introduced into recessive male sterile lines through high-throughput gene transformation technology to obtain a large number of transformations event.
  • multi-gene transformation often has transformation events that cannot be screened to obtain the desired traits.
  • the present invention obtains 14 initial maintainers with normal functions of each element from 563 transformation events, and creates maintainers of recessive nuclear male sterile lines for use in GAT sterile lines and The production of hybrids has successfully realized the maintenance and reproduction of recessive male sterile lines, and then realized the commercial utilization of recessive male sterile lines.
  • the plant when the gene is in a recessive homozygous state, the plant is in a male sterile state; when the gene is in a heterozygous or dominant homozygous state, the plant is in a male fertile state .
  • the control genes of the recessive nuclear male sterility mutant can be MS1, MS2, MS3, MS5, MS7, MS8, MS9, MS10, MS11, MS12, MS13, MS14, MS17, MS20, MS22, MS23, MS24, MS25, OsCYP704B2, MS27, MS28, MS29, MS30, MS31, MS32, MS33, MS34, MS36, MS37, MS38, MS43, MS45, MS48, MS50 nucleotide sequences and other mutant genes.
  • it is a mutant of OsCYP704B2 (oscyp704b2), and correspondingly, its restorer gene is OsCYP704B2.
  • the present invention first provides a genetically intelligent breeding seed production system for crop hybrid breeding and seed production, called GAT system, containing plant recessive male sterile lines, namely GAT sterile lines, recessive male sterile maintainers , Namely the GAT maintenance system, and the three general restoration systems;
  • the GAT maintainer line contains a GAT vector, which includes five functional element expression cassettes: (1) The plant male fertility restoration gene element expression cassette is used to restore the male fertility of recessive nuclear sterile mutants; (2) ) Plant pollen abortion gene element expression cassette, used to eliminate pollen containing GAT and maintain the heterozygous or hemizygous state of the GAT maintainer line; (3) Gene transformation and maintainer selection element expression cassette, used for gene transformation and GAT Maintainer line purification and purification; (4) Herbicide-sensitive element expression cassette, used to eliminate herbicide-sensitive GAT maintainer pollen and seed escape and GAT sterile line purification; (5) Seed selection element expression cassette, used For seed mechanical sorting; the five functional element expression cassettes are constructed in a plant binary expression vector to obtain a GAT system vector.
  • the GAT vector is introduced into the GAT sterile line to create a GAT maintainer, and the GAT vector exists in the genome of the GAT maintainer as a single copy.
  • the GAT sterile line is a sterile line controlled by a single recessive nuclear gene.
  • the gene locus is recessively homozygous, it is male sterile; heterozygous and dominant homozygous. It is male fertile.
  • the GAT maintainer line is self-bred, and the obtained seeds are 1:1 separated to obtain the GAT maintainer line and the GAT sterile line; the two kinds of seeds are separated by seed selection elements to achieve the GAT maintainer line
  • the GAT maintainer line pollinates the GAT sterile line to make the GAT sterile line sturdy and maintain the male sterility of its offspring, so as to realize the reproduction of the recessive male genic sterile line.
  • GAT sterile line recessive nuclear male sterile seeds
  • GAT retentivity fertile seeds
  • the present invention provides an intelligent seed production vector for crop genetics, called GAT vector, which is obtained by connecting five functional element expression cassettes through linker sequences to construct a plant binary expression vector.
  • the species functional element expression cassettes are respectively for:
  • Plant male fertility restoration gene element expression cassette used to restore the male fertility of recessive genic sterility mutants
  • Plant pollen abortion gene element expression cassette used to eliminate pollen containing GAT and maintain the heterozygous state or hemizygous state of the GAT maintainer line;
  • Herbicide sensitive element expression cassette used to remove pollen and seed exudation of herbicide-sensitive GAT maintainer lines and purification of GAT sterile lines;
  • the seed selection element expression cassette is used for mechanical seed sorting; the five functional element expression cassettes are connected by linker sequences to construct a GAT system vector.
  • the (1) plant male fertility restoring gene element expression cassette is operatively connected in sequence by a promoter, a male fertility restoring gene coding region and a terminator;
  • the male fertility restorer genes are MS1, MS2, MS3, MS5, MS7, MS8, MS9, MS10, MS11, MS12, MS13, MS14, MS17, MS20, MS22, MS23, MS24, MS25, OsCYP704B2, MS27, MS28, MS29, MS30, MS31, MS32, MS33, MS34, MS36, MS37, MS38, MS43, MS45, MS48, MS50, OsCYP704B2 wild-type genes, preferably OsCYP704B2 wild-type genes.
  • the promoter is the 1112bp sequence upstream of the start codon ATG of the rice OsCYP704B2 gene
  • the coding region is the coding region of the OsCYP704B2 gene
  • the terminator is the OsCYP704B2 gene The sequence of 274bp downstream of the stop codon TGA.
  • the function of the expression cassette is to restore the male fertility of the recessive homozygous mutant oscyp704b2 of the OsCYP704B2 gene.
  • the sequence of the (1) plant male fertility restoration gene element expression cassette is shown in SEQ ID NO.6.
  • the (2) plant pollen abortion gene element expression cassette is connected by a plant pollen specific promoter, a abortion gene coding region and a terminator in order to work normally;
  • the preferred promoter is a maize PG47 promoter Promoters, rice PCHF15, OsPC32 promoters, preferably abortion genes are rice ⁇ -amylase gene OsAA, corn ⁇ -amylase gene Zm-AA1, barley ⁇ -amylase gene HvAA1, millet ⁇ -amylase gene SiAA, cell division Oxidase, cysteine protease, gibberellin oxidase, terminator is corn IN2-1, bacterial NosT terminator.
  • the plant pollen abortion gene element expression cassette of the present invention is composed of promoter PG47, Zm-AA1 coding region and terminator IN2-1.
  • the expression cassette Killer sequence is shown in SEQ ID NO. 7; or plant pollen abortion
  • the expression cassette Killer 5400 of the fertility gene element is composed of the promoter PG47, the coding region of OsAA and the Nos terminator (NosT), and the sequence is shown in SEQ ID NO. 8; or the expression cassette Killer Hv of the plant pollen abortion gene element is composed of the promoter It consists of OsPC32, the coding region of barley alpha-amylase gene HvAA1 and NosT, and the sequence is shown in SEQ ID NO.9.
  • This element is to abort the pollen containing the GAT element, to maintain the heterozygous state or hemizygous state of the GAT transformant or the GAT maintainer line and to prevent the drift of the GAT element.
  • the (3) gene transformation and maintainer selection element expression cassette is connected by a promoter, a coding region of a selection marker gene, and a terminator in order to work normally;
  • the preferred promoters are OsUbi promoter, Actin Promoter or any one of the 2180bp sequence upstream of the ATG start codon of the OsALS gene, preferably the coding region of the selection marker gene is OsALSm1, OsALSm2, OsALSm3 sequence, glyphosate resistance gene EPSPSm sequence, glyphosate N-acetyltransferase sequence or Any one of the selection marker genes in the glufosinate resistance gene Bar sequence; the terminator is OsUbiT (SEQ ID NO.24) or NosT terminator.
  • the gene transformation and maintainer selection element expression cassette Marker2 sequence composed of ActinP, OsALSm1 and NosT is shown in SEQ ID NO.10.
  • Another gene transformation and maintainer selection element expression cassette Marker2AAU composed of OsALSP, OsALSm1 and OsUbiT is shown in SEQ ID NO.11.
  • the function of the above-mentioned expression cassette is to screen for resistance markers by gene transformation and screen to distinguish GAT maintainers and sterile lines.
  • the (4) herbicide-sensitive element expression cassette is connected by a promoter, a herbicide-dominant sensitive element and a terminator in order to work normally.
  • the preferred promoter is the ZmUbi promoter
  • the herbicide-sensitive element is The sex-sensitive element is the RNAi structural sequence P450i of the cytochrome P450 gene CYP81A6, and the terminator is the PinII terminator and the NosT terminator.
  • the marker1 sequence of the herbicide sensitive element expression cassette composed of ZmUbiP, P450i and NosT is shown in SEQ ID NO.14, or shown in SEQ ID NO.15 (P450i-2), or shown in SEQ ID NO.16 Shown (P450i-3).
  • the function of this element is to prevent the drift of GAT element, the mixing of GAT maintainer line with other materials, and the use of GAT sterile line for seed production.
  • the (5) seed selection element expression cassette is connected by a promoter, a seed coat coloring gene and a terminator in order to work normally.
  • the promoter is a seed-specific promoter ZZ1.
  • Seed coat coloring genes are deep red fluorescent protein FP635, red fluorescent protein RFP or green fluorescent protein GFP, and the terminator is OS-T28 terminator and NosT terminator.
  • the Marker3ZFN sequence of the seed selection element expression cassette composed of ZZ1P, FP635 and NosT is shown in SEQ ID NO.17.
  • the function of this element is to screen and distinguish GAT maintainer seeds from GAT sterile lines or GAT hybrids.
  • the linker sequence includes: the multiple cloning site MCSI, the sequence is shown in SEQ ID NO.18; the multiple cloning site MCSII, the sequence is shown in SEQ ID NO.19; the multiple cloning site MCSIII , The sequence is shown in SEQ ID NO.20; the multiple cloning site MCSIV, the sequence is shown in SEQ ID NO.21; or the multiple cloning site MCSV, the sequence is shown in SEQ ID NO.22.
  • the final vector of the present invention is pCO307, see Figure 6, and its sequence is shown in SEQ ID NO.25; or the final vector is pC0308, see Figure 7, and its sequence is shown in SEQ ID NO.26, Or the final vector is pCO309, see Figure 8, and its sequence is shown in SEQ ID NO.27.
  • the GAT vector of the present invention is pC1300-MMCK ( Figure 1), pC0308-MMCK ( Figure 2), pC0308-MMMaauCK5400 ( Figure 4A, the nucleotide sequence is shown in SEQ ID NO.1), pC0308- KhvMMaauMCK5400 ( Figure 4B, the nucleotide sequence is shown in SEQ ID NO. 2), pC0308-KhvMaauMCMK5400 ( Figure 4C, the nucleotide sequence is shown in SEQ ID NO. 3), pC0309-KhvMaauMCMK5400 ( Figure 4D, nucleotide sequence The sequence is shown in SEQ ID NO. 4) and pC0307-KhvMaauMCMK5400 ( Figure 3, the nucleotide sequence is shown in SEQ ID NO. 5).
  • the present invention provides a method for constructing the above-mentioned GAT vector, which is obtained by ligating five functional element expression cassettes into a final vector through linker sequences, and the five functional element expression cassettes are respectively:
  • Plant male fertility restoring gene element expression cassette used to restore the male fertility of recessive nuclear sterile mutants;
  • the expression cassette consists of a promoter, a male fertility restoring gene coding region and a terminator in order and can work normally ⁇ ;
  • Plant pollen abortion gene element expression cassette used to eliminate pollen containing GAT and maintain the heterozygous or hemizygous state of the GAT maintainer;
  • the expression cassette is composed of a plant pollen-specific promoter, abortion gene coding region and termination The sub-sequence can be connected normally;
  • Gene transformation and maintenance line selection element expression cassette used for gene transformation and GAT maintenance line removal and purification; the expression cassette is connected by a promoter, a selection marker gene coding region and a terminator in order to work normally;
  • Herbicide-sensitive element expression cassette used to eliminate herbicide-sensitive GAT maintainer pollen and seed exudation and GAT sterile line purification;
  • the expression cassette is composed of a promoter, a herbicide-dominant sensitive element and a terminator Connected in order to work normally;
  • a seed selection element expression cassette for mechanical seed sorting the five functional element expression cassettes are connected to the final vector through a linker sequence to construct a GAT vector; the expression cassette is composed of a promoter, a seed coat coloring gene, and The terminator is connected in order to work normally.
  • the linker sequence includes: the multiple cloning site MCSI, the sequence is shown in SEQ ID NO.18; the multiple cloning site MCSII, the sequence is shown in SEQ ID NO.19; the multiple cloning site MCSIII, The sequence is shown in SEQ ID NO.20; the multiple cloning site MCSIV is shown in SEQ ID NO.21; or the multiple cloning site MCSV is shown in SEQ ID NO.22.
  • the final vector of the present invention is pCO307, see Figure 6, and its sequence is shown in SEQ ID NO.25; or the final vector is pC0308, see Figure 7, and its sequence is shown in SEQ ID NO. 26, or the final vector is pCO309, see Figure 8, and its sequence is shown in SEQ ID NO.27.
  • the invention provides the application of the genetic intelligent breeding seed production system (GAT system) or the GAT vector in plant hybrid breeding and seed production.
  • GAT system genetic intelligent breeding seed production system
  • GAT vector in plant hybrid breeding and seed production.
  • the present invention provides the application of the genetic intelligent breeding system (GAT system) or the GAT vector in the production of non-transgenic hybrids.
  • GAT system genetic intelligent breeding system
  • GAT vector in the production of non-transgenic hybrids.
  • the present invention provides the application of the genetic intelligent breeding system (GAT system) or the GAT vector in the large-scale maintenance and large-scale propagation of plant recessive male sterile lines.
  • GAT system genetic intelligent breeding system
  • GAT vector in the large-scale maintenance and large-scale propagation of plant recessive male sterile lines.
  • the present invention provides the application of the genetic intelligent breeding system (GAT system) or the GAT carrier in the production of new plant varieties with high quality, high yield, wide adaptation and high resistance.
  • GAT system genetic intelligent breeding system
  • GAT carrier the genetic intelligent breeding system
  • the plants include rice, corn, wheat, barley, soybean, cotton, rape, sorghum, millet, oats, rye, barley, pepper, watermelon and the like.
  • the present invention provides a method for maintaining and reproducing the male sterility of recessive nuclear male sterile material (GAT sterile material) in plants, that is, a method for maintaining the male sterility gene in a recessive homozygous state,
  • GAT sterile material recessive nuclear male sterile material
  • the details are as follows: using the genetic intelligent breeding system GAT system, the GAT vector is introduced into the recessive homozygous GAT sterile line to create a GAT transformant containing only a single copy of the GAT vector, and the genotype of the GAT transformant It is recessive homozygous/GAT-.
  • the GAT transformant is pollinated to the GAT sterile line, and the obtained seed genotype is still recessively homozygous, thereby maintaining the male sterility of the offspring of the GAT sterile line.
  • ms represents the recessive male sterility mutant gene
  • MS represents the wild-type gene
  • the GAT element is introduced into the GAT sterile material (the genotype is ms ms) to create a GAT transformant containing only a single copy of the GAT vector (gene The type is ms ms/GAT-). Because the GAT vector contains restoring gene elements, male fertility can be restored.
  • the pollen and female gametes produced by GAT transformants have two types: (ms GAT) and (ms-), (ms GAT) type pollen because it contains GAT The pollen abortion gene elements in the vector will abort, so only (ms-) type pollen survive.
  • the GAT transformant is pollinated to the GAT sterile plant, and the obtained seed genotype is still recessive homozygous (ms ms), which can maintain the male sterility of the offspring of the GAT sterile plant. Since the genetic background of the GAT sterile plant and the GAT transformant is exactly the same except for the GAT elements, all the genotypes and phenotypes of the GAT sterile plant obtained by this method are unchanged.
  • the present invention provides a method for maintaining the GAT element of the maintainer material (GAT transformant or GAT maintainer) of a recessive nuclear male sterile plant in a heterozygous state/hemizygous state, using the genetic intelligence Chemical breeding system, introducing GAT vector into GAT sterile line with recessive homozygous genotype, creating a GAT transformant containing only a single copy of GAT vector.
  • the genotype of this GAT transformant is recessive homozygous/GAT- .
  • Selfing of GAT transformants can obtain two genotype seeds, one is recessive homozygous/--, which is GAT sterile plant; the other is recessive homozygous/GAT-, which is the maintainer material of GAT sterile plant According to the law of inheritance, the two separate 1:1, that is, the GAT locus with the recessive homozygous/GAT- genotype in the selfed progeny of the GAT transformant maintains the heterozygous state/hemizygous state.
  • the present invention provides a method for screening or distinguishing self-bred seeds of GAT transformants.
  • the GAT transformants adopt the genetic intelligent breeding system of the present invention to achieve a recessive homozygous GAT sterile
  • the GAT vector was introduced into the line to create a GAT transformant containing only a single copy of the GAT vector.
  • the genotype of the GAT transformant was recessive homozygous/GAT-.
  • the self-bred seeds of the GAT transformant showed a 1:1 separation, 50 of which % Is seeds containing GAT vector, the genotype is recessive homozygous/GAT-, which shows fluorescence when observed under excitation light; 50% is seeds that do not contain GAT vector, and the genotype is recessive homozygous, which is observed under excitation light No fluorescence.
  • the present invention provides a method for screening or distinguishing selfed seeds and plants of GAT transformants.
  • the GAT transformants adopt the genetic intelligent breeding system (GAT system) of the present invention, and the genotype is recessive.
  • the GAT vector was introduced into the sexually homozygous GAT sterile line, and a GAT transformant containing only a single copy of the GAT vector was created.
  • the genotype of the GAT transformant was recessive homozygous/GAT-, and the self-bred seeds of the GAT transformant appeared 1:1 separation, 50% of which are seeds and plants containing GAT vector, their genotype is recessive homozygous/GAT-, with high resistance to various herbicides against acetolactate synthase or EPSPS or Bar genes , Including but not limited to bispyrifen, imidazolate, imazapyr, glyphosate, glufosinate or glufosinate; 50% are seeds that do not contain GAT vectors, and the genotype is recessive homozygous and does not have this high resistance .
  • the present invention provides a method for preventing plant pollen drift.
  • the GAT vector of the present invention is transferred into plants, so that when the pollen matures, the plant material containing the GAT vector will contain pollen abortion genetic elements.
  • the specificity leads to the abortion of pollen containing GAT carrier, and ensures the normal development of pollen without GAT carrier, thereby reducing the probability of pollen containing GAT carrier escape.
  • the present invention provides a method for preventing the drift or mixing of GAT seeds or plants, which contains GAT carriers in the seeds or plants, and applies a specific concentration of herbicides at the seed stage or from the seedling stage to the flowering stage, including Bentasone or bensulfuron-methyl or nicosulfuron-methyl can kill materials containing GAT seeds or plants in a specific period, thereby preventing GAT seeds or plants from being mixed into other common materials.
  • the present invention provides a method for producing seeds of sterile lines by using recessive nuclear male sterile lines of plants.
  • the genetic intelligent breeding and production system is adopted to mix and sown GAT maintainers and GAT sterile lines in a certain proportion, and use GAT to maintain seeds.
  • the line is pollinated to the GAT sterile line. After the pollination is completed, herbicides, including bentazone, bensulfuron-methyl or nicosulfuron, are applied to specifically kill the GAT maintainer line, and only the GAT sterile line is retained to harvest seeds.
  • the present invention provides a method for purifying a recessive male sterile line of plants, using the described genetic intelligent breeding and seed production system, the GAT sterile line is coated with seeds or a specific concentration of herbicide is applied from the seedling stage to the flowering stage. Including bentazone or bensulfuron-methyl or nicosulfuron-methyl to ensure the purity of GAT sterile lines.
  • the present invention provides a method for hybrid seed production using plant recessive nuclear male sterile lines, adopting the described genetic intelligent breeding seed production system, using GAT maintainer lines to self-produce GAT maintainer line seeds and GAT sterile line seeds; GAT maintainer line is pollinated to GAT sterile line to produce GAT sterile line seeds; GAT sterile line and conventional materials are used to hybridize to produce common commercial hybrids.
  • the present invention provides a method for cross-breeding using plant recessive male sterile lines, adopting genetic intelligent breeding system, using GAT maintainer line and common material for cross breeding, which can be selected through conventional backcross or through
  • GAT maintainer line and common material for cross breeding which can be selected through conventional backcross or through
  • the pedigree method is used to select GAT maintainers and sterile lines, and the selection process is supplemented by various GAT molecular markers, herbicide screening, seed color selection, etc. to speed up the breeding.
  • the present invention also provides a primer for detecting the GAT vector or the transgenic positive plant containing the GAT vector, and the primer is any one of the following:
  • the primer sequence for detecting the expression cassette of the plant male fertility restoration gene element is shown in SEQ ID NO. 28-29; or
  • the primer sequence for detecting plant pollen abortion gene element expression cassette is shown in SEQ ID NO. 30-31; or
  • the molecular primers for detecting gene transformation and maintainer screening element expression cassettes are shown in SEQ ID NO. 32-33; or
  • the primer sequence for detecting the expression cassette of the herbicide sensitive element is shown in SEQ ID NO. 34-35.
  • the molecular marker primer sequence for detecting the seed selection element expression cassette is shown in the sequence SEQ ID NO. 39-40.
  • the present invention provides a method for detecting or transgenic positive plants containing GAT vectors.
  • SEQ ID NO.28-29 are used as primers for amplification, and the amplified product is digested with HaeIII and electrophoresed, there may be 3 band patterns in the final product: 86bp is the wild-type CYP704B2 band pattern, 84bp is the cyp704b2-3 mutant
  • the band type of 66bp is the band type of the expression cassette of the plant male fertility restoration gene element on the GAT vector. If there are 84bp and 66bp band patterns, but no 88bp band patterns, it indicates that the plant is a male sterile mutant background and the plant male fertility restoring gene element expression cassette exists;
  • SEQ ID NO.30-31 primer is used for amplification, if a 914bp band can be amplified, it indicates that the plant pollen abortion gene element expression cassette exists;
  • SEQ ID NO. 32-33 is used as primers for amplification, if an 831 bp band can be amplified, it indicates that the gene transformation and maintainer selection element expression cassettes exist;
  • SEQ ID NO. 34-35 are used as primers for amplification, if a 923 bp band can be amplified, it indicates the presence of the herbicide sensitive element expression cassette.
  • SEQ ID NO. 36-37 are used as primers for amplification, if a 1412bp band can be amplified, it indicates that the seed selection element expression cassette exists.
  • the present invention provides a method for sorting plants and progeny with different functions.
  • the GAT vector is transformed into plants, and (3) gene transformation and maintainer selection element expression cassettes in the GAT vector are used, and (4) herbicide-sensitive Element expression cassette, (5) Seed screening element expression cassette, based on the combination of chemical herbicide positive and negative bidirectional selection and mechanical color selection to sort plants and progeny with different functions.
  • the positive and negative selection of the chemical herbicide is that the same plant is resistant to one herbicide phenotype and sensitive to another herbicide phenotype.
  • the chemical herbicide positive selection herbicide is bispyribac-methyl, imidazole, imazapyr, glyphosate resistance, glufosinate or glufosinate-ammonium
  • the chemical herbicide negative selection is p-bendasone, Sensitive to bensulfuron or nicosulfuron.
  • the chemical herbicide positive selection herbicides are bispyribac-resistant, imidazole niacin, and imazapyr, and the chemical herbicide negative selection is sensitive to bentazone and bensulfuron-methyl.
  • the present invention constructs a GAT vector, including five functional element expression cassettes: a plant male fertility restoration gene element expression cassette, used to restore the male fertility of recessive genic sterility mutants; a plant pollen abortion gene element expression cassette, used For removing pollen containing GAT and maintaining the heterozygous/hemizygous state of the GAT maintainer; the chemical herbicide positive selection expression cassette is used for gene transformation and the purification of the GAT maintainer; the chemical herbicide negative selection expression cassette, It is used for removing pollen and seed exudation of herbicide-sensitive GAT maintainer lines and purification of GAT sterile lines; seed selection element expression cassettes are used for seed mechanical sorting.
  • the subsequent generations can be used for the subsequent organic combination of seed color selection and maintenance line selection elements and herbicide sensitive elements for the subsequent removal of impurities and purity of GAT maintenance lines and sterile lines, during the seed stage and the vegetative growth period.
  • Separation of recessive male sterile seeds/plants (GAT sterile line) and fertile seeds/plants (GAT retention) can be achieved during the reproductive growth period, successfully solving the large-scale reproduction of recessive male sterility
  • the creative realization of the use of recessive male sterility can realize commercial production.
  • the five key elements in the GAT carrier provided by the present invention are organically combined to play a role, and combined with mechanization and automatic processing, the commercial utilization of the recessive nuclear male sterile line of plants can be successfully realized.
  • the invention can be applied to hybrid breeding and hybrid seed production of plant recessive nuclear sterile materials, thereby obtaining high-quality, high-yield, wide-adapted and high-resistant new plant varieties and their seeds, and has great economic value.
  • Figure 1 is a schematic diagram of the pC1300-MMCK vector.
  • Figure 2 Schematic diagram of pC0308-MMCK vector.
  • Figure 3 is a schematic diagram of pC0307-KhvMaauMCMK5400 vector.
  • Figure 4A is a schematic diagram of pC0308-MMMaauCK5400 vector.
  • Figure 4B Schematic diagram of pC0308-KhvMMaauMCK5400 vector.
  • Figure 4C Schematic diagram of pC0308-KhvMaauMCMK5400 vector.
  • Figure 5A shows the verification result of pC0308-KhvMMaauMCK5400 restriction digestion.
  • Lane 1 is Kpn I single digestion;
  • Lane 2 is Pst I single digestion;
  • Lane 3 is Sma I single digestion;
  • M D2000 DNA Marker.
  • Figure 5B pC0308-KhvMaauMCMK5400 digestion verification results, lane 1 is not digested pC0308-KhvMaauMCMK5400 plasmid; lane 2 is Kpn I single digestion; lane 3 is BamH I single digestion; lane 4 is Sac I single enzyme Cut; Lane 5 is Sma I single digestion; Lane 6 is Bgl II single digestion; M, D15000 DNA Marker.
  • Figure 5C pC0309-KhvMaauMCMK5400 digestion verification results, lane 1 is not digested pC0309-KhvMaauMCMK5400 plasmid; lane 2 is Sac I single digestion; lane 3 is Sph I single digestion; lane 4 is Kpn I single enzyme digestion Cut; Lane 5 is BamH I single digestion; Lane 6 is Xho I single digestion; M, D15000 DNA Marker.
  • Figure 5D pC0307-KhvMaauMCMK5400 digestion verification results, lane 1 is not digested pC0307-KhvMaauMCMK5400 plasmid; lane 2 is Sac I single digestion; lane 3 is BamH I single digestion; lane 4 is Kpn I single enzyme Cut; M, D15000 DNA Marker.
  • Figure 5E shows the results of PCR detection of pC0308-MMMaauCK5400 Agrobacterium colonies.
  • Lanes 1-22 are the detection results of the specific primer SEQ ID NO.34-35, where 1 is the negative water control, 2 is the pC0308-MMMaauCK5400 plasmid control, and 3-22 is For different single colonies, M is D2000 DNA Marker.
  • Figure 6 Schematic diagram of pC0307 vector.
  • Figure 7 Schematic diagram of pC0308 vector.
  • Figure 8 Schematic diagram of pC0309 vector.
  • Figure 9 Schematic diagram of pC1300 vector.
  • Figure 10 Schematic diagram of pUC57-Simple vector.
  • GAT vector colony PCR detection 1-12 is the specific primer SEQ ID NO.30-31 colony PCR detection result, where 1 is the negative water control, 2 is the pC0308-MMMaauCK5400 plasmid control, 3-4 is the pC1300-MMCK, 5- 8 is pC0308-MMCK, 9-12 is pC0308-MMMaauCK5400, 13-24 is the colony PCR detection result of specific primer SEQ ID NO.34-35, the template sequence is the same as 1-12; M: D2000DNA Marker.
  • Figure 12 The result of pC0308-MMMaauCK5400 restriction digestion verification results, M1, D15000 plus DNA Ladder; M2, DNA Marker VI; CK, plasmid; A, Hind III and Sma I; B, Kpn I; C, Hind III and Pst I; D , Hind III and Kpn I.
  • Figure 13 shows the results of PCR detection of pC0308-MMMaauCK5400 Agrobacterium colonies
  • lanes 1-22 are the detection results of specific primer SEQ ID NO.30-31, of which 1 is the negative water control, 2 is the pC0308-MMMaauCK5400 plasmid control, and 3-22 is For different single colonies, M is D2000 DNA Marker.
  • Figure 14 The PCR positive detection result of pC0308-MMMaauCK5400 transgenic plants, the detection primer sequence is as SEQ ID NO. 30-31.
  • M is D2000 DNA Marker
  • the first "-” is the negative control water
  • the second "-” is the negative control wild-type Zhonghua 11
  • "+” is the positive control pC0308-MMMaauCK5400 plasmid
  • 1-18 is the transgenic T0 plant .
  • Figure 15 The PCR positive detection result of pC0308-MMMaauCK5400 transgenic plants, the detection primer sequence is as SEQ ID NO. 32-33.
  • M is D2000 DNA Marker
  • the first "-” is the negative control water
  • the second "-” is the negative control wild-type Zhonghua 11
  • "+” is the positive control pC0308-MMMaauCK5400 plasmid
  • 1-19 is the transgenic T0 plant .
  • Figure 16 shows the detection results of complementary elements of pC1300-MMCK and pC0308-MMMaauCK5400 transgenic T0 plants.
  • the detection primer sequence is shown in SEQ ID NO.28-29. 1 is wild-type Zhonghua 11, and 2 is a homozygous mutant of cyp704b2-3. 3-8 are pC1300-MMCK transgenic plants, and 9-14 are pC0308-MMMaauCK5400 transgenic plants.
  • Figure 17 shows the results of spraying bispyrifen on some of the GAT T0 generation transformants of Example 4.
  • Fig. 18 shows the results of partial spraying of Bentazon on leaves of some GAT T0 generation transformants of Example 4.
  • Figure 19 shows the results of iodine staining of mature pollen of GAT T0 generation transformants, black is fertile pollen; light color is aborted pollen.
  • Figure 21 is the identification diagram of pollen fertility and self-fruiting after the fluorescent seeds and non-fluorescent seeds in the GAT representative line 88-4 develop into plants, respectively.
  • a and C are derived from the rice ears and anthers of the fluorescent seeds developed into plants. Half of the pollen is fertile, and the iodine dye is blue; the other half of the pollen is sterile, and the iodine dye does not color, and the ears of rice can be self-bred and bear fruit.
  • B and D are derived from the panicles and anthers of non-fluorescent seeds that develop into plants. They are pollen-free types, which are typical features of the recessive male sterile mutant of cyp704b2. The panicles are self-fertilizing.
  • Figure 22 shows the results of spraying Bentazon, a key strain of GAT T1 generation.
  • Figure 23 is a diagram showing the results of the mature pollen fertility test of the T1 generation of GAT transformants.
  • A cyp704b2, sterile mutant material, no pollen in microscopic examination;
  • B ZH11, common wild-type material Zhonghua 11, microscopic examination of iodine-stained pollen fertility is normal;
  • C GAT, infertile mutant transferred GAT carrier material, half of the fertility of pollen stained with iodine under microscope was normal, and the other half was not stained.
  • Figure 24 is a graph showing the results of fluorescence identification of seeds of GAT transformants (T1 generation).
  • Figure 25 shows the escape rate detection of the T1 generation GAT maintainer system.
  • Fig. 26 represents the results of identification of pollen fertility and self-fruiting of fluorescent seeds and non-fluorescent seeds in strain 88-4-16 after they have developed into plants.
  • a and C are derived from the panicles and anthers of the fluorescent seeds developed into plants. Half of the pollen is fertile, and the iodine dye is blue; the other half is sterile, and the iodine dye is not colored, and the panicle can be self-bred.
  • B and D are derived from rice ears and anthers developed from non-fluorescent seeds. They are pollen-free types and are typical features of the recessive male sterile mutant of cyp704b2.
  • the GAT vector adopts the strategy of segmented construction with the expression cassette as the unit, and unit splicing. First construct the expression cassettes on the transitional vectors pC1300 ( Figure 9) and pUC57-Simple ( Figure 10), digest them and verify them by sequencing, and then splice the expression cassettes to the final vector.
  • the specific steps of vector construction are as follows:
  • NSPT-Construct V1.81-Marker 1 The sequence of NSPT-Construct V1.81-Marker 1 is cut by the Kpn I restriction site ggtacc, MCSI (sequence shown in SEQ ID NO.18), herbicide sensitive element expression cassette Marker 1 (SEQ ID NO.14), MCSII (SEQ ID NO.19), the spacer sequence tgcagggacccttgccaac, and Hind III restriction site aagctt are connected in sequence.
  • the sequence of MCSI is composed of Pst I, Srf I, Afe I, and Xmn I restriction sites connected in sequence.
  • the sequence of the herbicide sensitive element expression cassette Marker 1 consists of the NosT terminator, an RNAi stem-loop structure sequence of the cytochrome P450 gene CYP81A6, and the ZmUbiP promoter connected in sequence.
  • An RNAi stem-loop structure sequence of the cytochrome P450 gene CYP81A6 consists of a reverse stem sequence composed of a CYP81A6 coding region, a loop sequence composed of a rice intron, and a forward stem sequence complementary to the CYP81A6 coding region, which are sequentially connected Clearly.
  • the sequence of MCSII is composed of Hpa I, PshA I, BspE I, and Pac I restriction sites connected in sequence.
  • NSPT-Construct V1.9-Marker 2 The sequence of NSPT-Construct V1.9-Marker 2 consists of EcoR I restriction site gaattc, Pst I restriction site ctgcag, spacer sequence ggacccttgccaaca, multiple cloning site MCSII (sequence shown in SEQ ID NO.19), The transformation and maintenance line screening element expression cassette Marker2 (SEQ ID NO.10), MCSIII (SEQ ID NO.20), the spacer sequence tgcagtcccaaggcttccg, and the Hind III restriction site aagctt are sequentially connected.
  • the sequence of MCSII is composed of Hpa I, PshA I, BspE I, and Pac I.
  • the sequence of gene transformation and maintainer selection element expression cassette Marker2 is composed of NosT terminator, ALS gene coding region sequence OsALSm1, ActinP
  • the promoters are connected in sequence
  • the sequence of MCSIII is composed of BsrG I, Bae I, AsiS I, and FspAI restriction sites connected in sequence.
  • NSPT-Construct V1.81-Complementation uses Sac I+Hind III double enzyme digestion to connect to pC1300 to produce pC1300-Complementation.
  • the sequence of NSPT-Construct V1.81-Complementation consists of Sac I restriction site gagctc, Pst I restriction site ctgcag, spacer sequence tcccaaggcttccga, multiple cloning site MCSIII (SEQ ID NO.20), plant male fertility restorer gene Element expression cassette Complementation (SEQ ID NO.6), MCSIV (SEQ ID NO.21), spacer sequence tgcagcctgttgccaggga, and Hind III restriction site aagctt are connected in sequence.
  • the sequence of MCSIII is composed of BsrG I, Bae I, AsiS I, and FspA I restriction sites connected in sequence.
  • Plant male fertility restoring gene element expression cassette Complementation consists of a 1112bp sequence upstream of the ATG start codon of the rice OsCYP704B2 gene, a codon-optimized coding region of the OsCYP704B2 gene, and a 274bp sequence downstream of the stop codon TGA of the OsCYP704B2 gene.
  • the sequence of MCSIV is composed of Swa I, BstB I, Mlu I, and Rsr II restriction sites connected in sequence.
  • NSPT-Construct V1.81-Killer uses Nde I+EcoR V double enzyme digestion and connect it into pUC57-Simple to produce pUC57-Simple-Killer.
  • the sequence of NSPT-Construct V1.81-Killer consists of Nde I restriction site catatg, spacer sequence cagggacccttgccaaca, Nru I restriction site tcgcga, Pac I restriction site ttaattaa, Pst I restriction site ctgcag, spacer sequence cctgttgccagggaa , Multiple cloning site MCSIV (SEQ ID NO.21), plant pollen abortion gene element expression cassette Killer (SEQ ID NO.7), spacer sequence tcgacgcggccgatccccgg, Stu I restriction site aggcct, Sac I restriction site gagctc , Multiple cloning site MCSV (SEQ ID NO.22), spacer sequence
  • the sequence of MCSIV is composed of Swa I, BstB I, Mlu I, and Rsr II restriction sites connected in sequence.
  • the expression cassette Killer of plant pollen abortion gene elements consists of promoter PG47, Zm-AA1 coding region and terminator IN2-1 sequence.
  • the sequence of MCSV is composed of Avr II, Pml I, SnaB I, and Alo I restriction sites connected in sequence.
  • Killer 5400 uses BstB I+Avr II double enzyme digestion to connect to pUC57-Simple-Killer, and replace Killer with the expression cassette Killer 5400 to produce pUC57-Simple-Killer 5400.
  • Killer 5400 fragment consists of multiple cloning site MCSIV (SEQ ID NO.21), plant pollen abortion gene element expression cassette Killer 5400 (SEQ ID NO.8), and multiple cloning site MCSV (SEQ ID NO.22) in sequence Connected.
  • Plant pollen abortion gene element expression cassette Killer 5400 includes PG47 promoter, rice alpha-amylase gene OsAA (ie 5400) coding region, NosT terminator and other parts.
  • the sequence of the Marker 3 ZFN fragment is formed by connecting the Pst I restriction site ctgcag, the seed selection element expression cassette Marker 3 ZFN (SEQ ID NO.17), the spacer sequence g, and the Xma I restriction site cccggg in sequence.
  • the seed selection element expression cassette Marker 3 ZFN sequentially includes the NosT terminator, the coding region of the deep red fluorescent protein FP635 gene, and the ZZ1P promoter.
  • Auto-link pC0308-M_MaauCK5400 cut it with AsiSA I after auto-linking, fill in the end with high-fidelity Taq enzyme, and then ligate it with the blunt end of Marker 1 after filling in the end to obtain the transition vector pC0308-Marker 3-Marker 2aau-Marker 1-Complementation-Killer5400 (pC0308-MMaauMCK5400). Marker 1 The transcription direction remains unchanged.
  • Synthesize Killer Hv fragments use Pst I single enzyme digestion to join the transition vector pC0308-MMaauMCK5400 produced in step 19 to obtain the GAT vector pC0308-Killer Hv-Marker 3-Marker 2aau-Marker 1-Complementation-Killer5400(pC0308-KhvMMaauMCK5400 , Figure 4B, SEQ ID NO. 2).
  • the transcription direction of Killer Hv is opposite to that of Marker3.
  • Killer Hv The sequence of Killer Hv is cut by Pst I restriction site ctgcag, plant pollen abortion gene element expression cassette Killer Hv (SEQ ID NO.9), AsiS I restriction site gcgatcgc, SrfI restriction site gcccgggc, Pac I restriction The site ttaattaa and the Pst I restriction site ctgcag are connected in sequence.
  • Plant pollen abortion gene element expression cassette Killer Hv consists of promoter OsPC32, HvAA1 coding region and terminator IN2-1 sequence.
  • KhvMaauMCMK5400 Connect KhvMaauMCMK5400 to pC0309 and pC0307 respectively to obtain the GAT vector pC0309-Killer Hv-Marker 2aau-Marker 1-Complementation-Marker 3-Killer5400 (pC0309-KhvMaauMCMK5400, Figure 4D, SEQ ID NO.4) and pC0307-Killer 2Hauv307-Marker -Marker 1-Complementation-Marker 3-Killer5400 (pC0307-KhvMaauMCMK5400, Figure 3, SEQ ID NO.5).
  • the constructed GAT vectors pC0308-MMMaauCK5400, pC0308-KhvMMaauMCK5400, pC0308-KhvMaauMCMK5400, pC0309-KhvMaauMCMK5400 and pC0307-KhvMaauMCMK5400 vectors were subjected to restriction digestion and sequencing verification.
  • Kpn I, Hind III and Sma I, Hind III and Pst I, Hind III and Kpn I for pC0308-MMMaauCK5400 choose Kpn I, Pst I and Sma I for pC0308-KhvMMaauMCK5400, choose Kpn I, BamH I, Sac I, Sma I and Bgl II were used for pC0308-KhvMaauMCMK5400, Sac I, Sph I, Kpn I, BamH I, and Xho I were selected for pC0309-KhvMaauMCMK5400, and Sac I, BamH I, and Kpn I were selected for pC0307-KhvMaauMCMK5400.
  • the digestion system is 1 ⁇ l of 10 ⁇ Buffer, 3 ⁇ l of plasmid DNA, 0.2 ⁇ l of DNA restriction endonuclease, and 10 ⁇ l of ddH 2 O. Enzyme digestion conditions are incubation at 37°C for 10-15 minutes, and then inactivation at 70°C for 5 minutes. The digested product was detected by electrophoresis in 1% agarose gel.
  • the plasmid size of pC0308-MMMaauCK5400 is 24548bp; after Hind III and Sma I double digestion, the band size of spot A is 8287bp+16261bp; the band size of spot B after Kpn I single digestion is 9114bp +15434bp; Hind III and Pst I double digestion, the band size of C spotting hole is 6295bp+18253bp; Hind III and Kpn I double digestion, the size of D spotting hole is 3010bp+9114bp+12424bp.
  • the result of restriction digestion was exactly in line with expectations. Select the plasmid with the correct restriction enzyme digestion for sequencing, and the sequence is exactly the same as expected. The sequence is shown in SEQ ID NO.1.
  • the plasmid size of pC0308-KhvMMaauMCK5400 is 28365bp; the size of lane 1 after Kpn I single restriction digestion is 4870bp+10564bp+12931bp, of which the 10564bp and 12931bp bands are too large to separate, and the overlap is a bright one.
  • the size of lane 2 after Pst I single digestion is 3807bp+24558bp; the size of lane 3 after single digestion with Sma I is 28365bp.
  • the result of restriction digestion was exactly in line with expectations. Select the plasmid with the correct restriction enzyme digestion for sequencing, and the sequence is exactly the same as expected. The sequence is shown in SEQ ID NO.2.
  • the plasmid size of pC0308-KhvMaauMCMK5400 is 28361bp; Lane 1 is the undigested plasmid control; the band size of Lane 2 after Kpn I single digestion is 2858bp+10564bp+14939bp; after BamH I single digestion 3 The size of lane No.
  • the plasmid size of pC0309-KhvMaauMCMK5400 is 28723bp; lane 1 is the undigested plasmid control; the band size of lane 2 after Sac I single digestion is 270bp+1376bp+5928bp+21149bp; SphI single digestion The size of the last lane 3 is 4317bp+4837bp+5770bp+13799bp; the size of lane 4 after Kpn I single digestion is 2858bp+10926bp+14939bp; the size of lane 5 after BamH I single digestion is 2053bp+ 6895bp+19775bp; after Xho I single restriction digestion, the size of lane 6 is 76bp+1709bp+1935bp+5430bp+19573bp.
  • the result of restriction digestion was exactly in line with expectations. Select the plasmid with the correct restriction enzyme digestion for sequencing, and the sequence is exactly the same as
  • the plasmid size of pC0307-KhvMaauMCMK5400 is 28469bp; Lane 1 is the undigested plasmid control; Lane 2 is 270bp+1376bp+5928bp+20895bp after Sac I single digestion; BamH I single digestion The size of the last lane 3 is 2053bp+6895bp+19521bp; the size of the lane 4 after Kpn I single restriction digestion is 2858bp+10672bp+14939bp. The result of restriction digestion was exactly in line with expectations. Select the plasmid with the correct restriction enzyme digestion for sequencing, and the sequence is exactly the same as expected. The sequence is shown in SEQ ID NO.5.
  • Agrobacterium EHA105 stored at -80°C was streaked on a YEP plate containing rifampicin (25 ⁇ g/ml) + streptomycin (50 ⁇ g/ml) and cultured at 28°C.
  • the specific primers SEQ ID NO.30-31 and SEQ ID NO.34-35 were used to verify the colony PCR of the Agrobacterium clone transformed by pC0308-MMMaauCK5400 ( Figure 13 and Figure 5E), and the 914bp and 923bp target fragments can be expanded . Select positive clones, shake the bacteria for 36-48h, and save the bacterial solution for infection.
  • the engineered Agrobacterium obtained in this example was transformed into the above-mentioned callus by Agrobacterium-mediated genetic transformation, after co-cultivation for 3 days, washed 5-6 times, and transferred to the selection medium containing Bispyri-methyl ( N 6 +2.4-D2mg/L+CH 0.6g/L+Pro0.5g/L+ Sucrose 30g/L+ Phytagel 3g/L+Cn 500mg/L+ Bispyribacter 0.3-0.6 ⁇ m/L or Hygromycin 50mg/L ), cultivate in the dark at 30°C for 30-50 days, and screen for resistant callus;
  • the screened resistant callus is transferred to the differentiation medium containing bispyri-methyl (MS+KT 2mg/L+NAA 0.5-2mg/L+sorbitol 20-30g/L+sucrose 30g/L+Phytagel 3g/L+ Bispyrifen 0.1-0.3 ⁇ m/L), differentiate for 25-30d to obtain positive seedlings;
  • Rooting The positive seedlings obtained after differentiation are transferred to the rooting medium containing Bispyrifen resistance (1/2 MS+sucrose 20g/L+ Paclobutrazol 0.5-1mg/L+ Phytagel 3g/L+ Bispyrifen 0.15-0.5 ⁇ m/L) , And finally obtain positive transgenic plants after rooting for 7-15 days;
  • Seedling refinement and transplanting Open the bottle top sealing film of the transformed strain with vigorous root growth, add sterile water to cover the medium 1-2cm thick, and place it in contact with air at room temperature for 2-3 days, then transplant it to Greenhouse cultivation. A total of 574 GAT transformed strains were screened and 563 strains survived 7 to 14 days after the transformation material was transplanted. While transplanting the GAT transformed lines, a certain number of ZH11 with 2 leaves and 1 heart stage was transplanted as a control.
  • the leaves of the transgenic plant obtained in Example 2 were used to extract total genomic DNA using the CTAB method.
  • the extraction method is as follows:
  • ddH 2 O sterilized water
  • the specific primers SEQ ID NO.30-31 ( Figure 14) and SEQ ID NO.32-33 ( Figure 15) were used to perform PCR positive detection on the total DNA of the T0 transgenic plant of pC0308-MMMaauCK5400. Select plants that can simultaneously amplify 914bp and 831bp bands for use.
  • the amplified regions of the specific primers SEQ ID NO. 28 and SEQ ID NO. 29 contain the cyp704b2-3 mutant mutation site, and it can be observed in polyacrylamide gel electrophoresis that there is a 2-base deletion in the cyp704b2-3 mutant background plant Mutation; the primer amplification region also contains an A ⁇ C SNP introduced in the CYP704B2 coding region (CDS position 660) in the expression cassette of the plant male fertility restoration gene element when constructing the vector.
  • the SNP will introduce a HaeIII restriction site, so that the plant male fertility restoring gene element expression cassette can be cleaved by HaeIII, while the wild-type CYP704B2 cannot be cleaved by HaeIII.
  • 86bp is the wild-type genotype of the rice genome
  • 84bp is the cyp704b2-3 mutant genotype
  • 66bp is the transformed fragment. genotype.
  • the above method was used to identify plants with a genetic background of cyp704b2-3 homozygous mutant and containing GAT vector.
  • a 600mg/L bispyripyr solution was prepared with 10% bispyribac-methyl emulsion (Nongmei Li), and the 563 GATT0 generation and wild-type control strains obtained in Example 2 were sprayed.
  • ZH11 3-5 leaf stage seedlings, continuous observation after spraying. Three days after spraying, the leaves of the control ZH11 appeared withered and yellow. Seven days after spraying, the control ZH11 withered yellow was on the verge of death, most of the GAT transformed plants grew normally, and some of them showed yellowing or growth was inhibited.
  • the control ZH11 14 days after spraying, the control ZH11 was completely dead, but the GAT transformed strain showed three types: normal growth, growth inhibition, and near death or irreversible death.
  • the normal-growing line is the high-resistance line of bispyribac-methyl, with a total of 184, indicating that the selection of marker elements in these lines is more efficient;
  • the growth-inhibited line is the medium-resistant line of bispyri-methyl, with a total of 163.
  • the efficiency of screening marker elements in these strains is average; irreversibly dead or dying strains are bispyribac-methyl non-resistant or low-resistant strains, and there are 216 strains in total. In these lines, the efficiency of screening marker elements is poor ( Figure 17).
  • the inventors screened each phenotype and found that the GAT transformed strains behaved as the probability of normal growth of the bispyribac-methyl strain.
  • the success rate is 32.68%, which is much higher than the current success rate of conversion events after a three-function element assembly or a four-function element assembly in the field.
  • the success rate of a single trait phenotype is 30%-50%.
  • the probability that all elements meet the expectations should be 30% to the Nth- Between 50% to the power of N, N is the number of functional elements.
  • Bentazon mother liquor (Changzhou Precision Biological Technology Co., Ltd.) was used to prepare 3g/L Bentazon solution and sprayed.
  • the surviving GAT T0 generation (347 strains) and wild-type control ZH11 plants were applied to the streaked area of the leaves of the GAT T0 generation (347 strains) and the wild-type control ZH11 plant in the spraying experiment, and the observation was continued after spraying.
  • the pollen was stained with iodine during the flowering of the GAT transformants to detect the pollen fertility of the GAT transformants. Because the GAT vector contains restoring gene elements, if the restoring gene elements work normally, male fertility can be restored. There are two types of pollen (ms/GAT) and (ms/-), (ms/GAT) type pollen because it contains The pollen abortion gene element in the GAT vector, if it works normally, the pollen will abort, so only (ms/-) type pollen survive.
  • the specific methods of iodine staining microscopy are as follows:
  • abortion pollen 1:1 separation, which is the gene separation and separation in the laws of genetics. The law of free combination. Therefore, if there is a 1:1 separation phenotype, it indicates that the above-mentioned components work normally and are in line with expectations. Fertile pollen of the other lines: Aborted pollen does not meet the 1:1 segregation ratio, and skewed segregation appears, indicating that the work efficiency of restoration gene elements and pollen abortion gene elements in these lines is poor or the genome contains multiple copies.
  • the two seeds (fluorescent seeds and non-fluorescent seeds) of the superior strain obtained in step 4 were respectively germinated and transplanted to the seedling stage to observe their fertility.
  • the results are shown in Figure 21.
  • Plants derived from fluorescent seeds can be self-bred, and seeds are harvested ( Figure 21, A and C); plants derived from non-fluorescent seeds are sterile and cannot self-bred ( Figure 21, B and D). It shows that the GAT system successfully achieved the reproduction of recessive nuclear male sterile material (the recessive nuclear male sterile mutant containing cyp704b2 in this example) and the maintenance of the sterility of the recessive nuclear male sterile material.
  • tissue culture was used to screen the key strains and candidate T1 generations. If the GAT vector exists in the form of a single copy in the genome and the pollen abortion gene elements are working normally, the T1 generation of this strain will show a 1:1 separation, that is, 50% of the GAT vector contains the GAT vector, and it is resistant to bispyribac-methyl.
  • Bispyrifen can germinate normally under screening pressure; 50% does not contain GAT carrier, does not have the resistance to Bispyrifen, and cannot germinate under the screening pressure of Bispyrifen. Therefore, 1/2MS medium + 3 ⁇ m Bispyrifen was prepared to screen key GAT strains and candidate strains, and the germination results are shown in Table 2.
  • 10 strains meet the germination ratio of 1:1; one strain has a germination ratio of close to 1:1, indicating that the selection marker elements in these 11 strains work normally and are genetically stable across generations, which also indicates that the selection has been passed.
  • the marker element can effectively distinguish two different types of seeds or seedlings (namely GAT sterile line and GAT maintainer line) separated from the selfed progeny of GAT transformants; the germination ratio of other lines does not match, indicating that there may be abnormal work efficiency or generation of elements. Instability genetic phenomenon.
  • ZH11 resistance control
  • CK+(P450i2-30) is sensitive positive control
  • Example 4-3 In order to identify the working efficiency of pollen abortion gene elements in GAT transformants of the T1 generation, the pollen of the other half of the plants with good detection efficiency in Examples 5-1 and 5-2 was stained with iodine to detect GAT transformation during the flowering of rice. Plant pollen fertility. Similar to the pollen fertility of the T0 generation in Example 4, if the GAT vector in the T1 generation is in the form of a single copy in the genome, and the pollen abortion gene element works normally, the fertile pollen and the aborted pollen are separated 1:1. The specific method of iodine staining microscopic examination refers to Example 4-3.
  • the fluorescence of self-harvested seeds of the above-mentioned strains was further tested to detect the working efficiency of the seed selection elements in the T1 generation GAT transformants.
  • the results showed that the seed coats of some seeds of the T1 generation seeds of all the lines showed strong deep red fluorescence (GAT in Fig. 24).
  • the results of chi-square analysis showed that the seeds of T1 generation of 6 lines met the expected separation ratio of 1:1 (shown in Table 5); the fluorescence separation ratio of seeds of 2 lines was close to 1:1. This indicates that the pollen abortion gene elements and seed fluorescent elements in these lines work normally and can be inherited stably.
  • seed selection elements can effectively distinguish two types of seeds (ie GAT sterile line and GAT maintainer line); another 5 lines have a seed fluorescence separation ratio that does not meet 1:1, indicating that there may be abnormal working efficiency of seed elements or pollen abortion gene elements or intergenerational unstable genetic phenomena.
  • the above-mentioned strains can meet the requirements of GAT maintainers, and can be used as excellent initial GAT maintainers for variety selection and sterile line and hybrid seed production.
  • the pollen and seeds in the excellent initial maintainer line of GAT meet the 1:1 separation ratio, which preliminarily indicates that the pollen abortion element in GAT works normally.
  • this example uses the excellent initial maintainer line of GAT to pollinate Ordinary sterile rice materials, test whether the hybrid seeds are resistant to bispyribac-methyl (the method is the same as in Example 5), if yes, pollen containing GAT has escaped; if not, it indicates that the pollen abortion element in GAT works efficiently Good, can effectively prevent the escape of GAT-containing transformed pollen.
  • the GAT strains (23-2, 88-4) were selected as the male parent to pollinate the male sterile line 1907, and 221 and 373 hybrid seeds were obtained, respectively.
  • the hybrid seeds were screened with bispyrifen. After 21 days of observation, it was found that under the medium without the selection pressure (1/2MS), the hybrid seeds germinated normally, but under the medium with the selection pressure (1/2MS+3uM BS) , The hybrid seeds are consistent with non-transgenic ZH11, 9311, MH63, can not germinate (see Figure 25), the germination rate is 0% (see Table 6), indicating that the hybrid seeds do not contain GAT elements, pollen containing GAT failed to pollinate Ordinary materials once again prove that the pollen abortion element in GAT works normally and can be inherited stably.
  • the two kinds of seeds (fluorescent seeds and non-fluorescent seeds) of the superior strain obtained in step 4 were respectively germinated and transplanted to the seedling stage to observe their fertility, as shown in Figure 26.
  • Plants from fluorescent seeds can be self-bred, and seeds are harvested ( Figure 26, A and C); plants from non-fluorescent seeds are sterile and cannot self-bred ( Figure 26, B and D).
  • Figure 26, A and C plants from non-fluorescent seeds are sterile and cannot self-bred
  • Examples 1-5 prove that this application has successfully realized the reproduction of recessive genic male sterile materials and the maintenance of the sterility of recessive genic male sterile materials by using the GAT system.
  • This example is the transformation of recessive nuclear sterility, that is, the transformation of GAT sterile line.
  • the dominant homozygous CYP704B2 in H28B is replaced with the mutant recessive homozygous cyp704B2-3, but the rest is maintained through backcrossing.
  • cyp704b2-3 is a rice CYP704B2 gene mutant. It is obtained by replacing the GGG after the 794th base of the rice CYP704B2 gene with a T. The mutation site is located in the third exon (disclosed in Chinese Patent CN 105002191 B).
  • H28B is an approved traditional three-line sterile line variety and belongs to a three-line maintainer line, it is a dominant homozygous CYP704B2 locus, and does not contain GAT vector elements.
  • the specific transfer implementation steps are as follows :
  • F 1 as the parent and recipient parent, such as H28B, backcross to obtain BC 1 F 1 .
  • Plant BC1F1 uses the primers of SEQ ID NO.28-29 to detect the cyp704b2-3 genotype.
  • Select cyp704b2-3 heterozygous genotype that is, plants with 86bp and 84bp bands can be amplified at the same time.
  • genotypes such as 100, or 200, etc.
  • a set of genotypes (such as 100, or 200, etc.) between the cyp704b2-3 mutant and the reincarnation parent genome to have polymorphisms, and evenly distributed molecular markers (can be but not limited to SSR, SNP, INDEL, EST, RFLP, AFLP, RAPD, SCAR and other type markers), the genetic background of the single plant selected in step 3 is identified, and the genotype of the recurrent parent is highly similar (such as greater than 88% similarity, or 2%). The selection rate, etc.) of the plants.
  • step 4 Use the plant selected in step 4 and the recipient parent, such as H28B, to backcross to obtain BC 2 F 1 .
  • Plant BC 2 F 1 repeat steps 3 and 4, and select plants that are heterozygous for cyp704b2-3 genotype and have a high genetic background recovery rate (such as greater than 98%, or 2% selection rate, etc.), and harvest them from crosses BC 2 F 2 .
  • Plant BC 2 F 2 repeat steps 3 and 4, select the plants with the cyp704b2-3 genotype heterozygous and the highest genetic background homozygous rate, and harvest them from the cross BC 2 F 3 .
  • the cyp704b2-3 homozygous genotype plants isolated from the progeny of BC 2 F 3 are the cyp704b2-3 recessive genic male sterile line, and BC 2 F 3 is used to preserve the germplasm resources of the cyp704b2-3 recessive genic male sterile line.
  • the letter G is used to name the recessive nuclear sterile line.
  • the cyp704b2-3 homozygous recessive nuclear sterile line of H28B in this example is named H28G.
  • H28B is used as a breeding example, but it is not limited to H28B, and it can be any rice material.
  • the GAT maintenance line is obtained, that is, cyp704B2-3 is obtained and contains GAT vector elements, but other traits maintain the original donor traits, such as using H28B (H28B itself is a dominant homozygous genotype of CYP704B2 and does not contain GAT vector elements ), through continuous backcrossing with H28B and molecular marker-assisted selection, the other traits are the same as H28B, but cyp704B2-3 is recessively homozygous and contains GAT vector elements.
  • GAT retention is named H28T. This process is called Transform the GAT maintainer line.
  • the homozygous mutation at the CYP704B2 locus was selected from the transgenic plants of the GAT large vector obtained in Example 4.
  • the GAT transgene PCR positive test was positive, and the plants with the phenotype controlled by each GAT element were used as the donor for GAT maintainer transfer.
  • Body parent The cyp704b2-3 heterozygous plant obtained in Example 6, for example, the heterozygous plant of H28G, was selected as the recipient parent.
  • the donor plant and the recipient plant were crossed, backcrossed and selfed.
  • Plant BC1F1 use primers with sequences such as SEQ ID NO.28-29 to PCR amplify the genomic DNA of the BC1F1 plant, and then digest the amplified product with HaeIII, and determine the genotype of the plant based on the band of the digested product. Select plants with 84bp and 66bp bands, that is, plants with cyp704b2-3 gene and GAT transgene.
  • genotypes such as 100, or 200, etc.
  • a set of genotypes (such as 100, or 200, etc.) between the cyp704b2-3 mutant and the reincarnation parent genome to have polymorphisms, and evenly distributed molecular markers (can be but not limited to SSR, SNP, INDEL, EST, RFLP, AFLP, RAPD, SCAR and other type markers), the genetic background of the single plant selected in step 3 is identified, and the genotype of the recurrent parent is highly similar (such as greater than 88% similarity, or 2%). The selection rate, etc.) of the plants.
  • step 4 Use the plant selected in step 4 and the cyp704b2-3 heterozygous recipient parent, such as cyp704b2-3 heterozygous H28G, to backcross to obtain BC 2 F 1 .
  • Plant BC 2 F 1 repeat steps 3 and 4, select plants with a high genetic background recovery rate (such as greater than 98%, or 2% selection rate, etc.), and harvest them from the cross BC 2 F 2 .
  • a high genetic background recovery rate such as greater than 98%, or 2% selection rate, etc.
  • Plant BC 2 F 2 repeat steps 3 and 4, select the plant with the highest genetic background homozygous rate, and collect it from the cross BC 2 F 3 , which is the GAT maintainer line.
  • the GAT maintenance system is named with the letter T.
  • the GAT maintenance system of H28G in this embodiment is named H28T.
  • H28B is used as a breeding example, but it is not limited to H28B, and it can be any rice material.
  • the GAT maintainer seeds are planted in the legally transgenic area, and the seeds are harvested by selfing and fruiting.
  • the dark red fluorescent seeds are screened by the fluorescent seed sorter, which are the GAT maintainer seeds, which can be used for self-reproduction or pollination of the GAT maintainer.
  • GAT sterile line is used for sterile line seed production.
  • Example 9 GAT maintenance system removes impurities and maintains purity
  • GAT maintainer line is sowed, spray 30-90mg/m2 of bispyribac-methyl or 50-100mg/L of diazepam or 185-750mg/L imidazolium nicotinic acid in the seedling stage for impurity removal and purity; spraying from the tillering stage to the booting stage 60-120mg/m2 of bispyribac-methyl or 100-200mg/L of imazapyr or 500-1500mg/L imidazole nicotinic acid for impurity removal; spray 120-300mg/m2 of bispyribac-methyl or 200-750mg during the flowering period /L of imazapyr or 1000-3000mg/L imidazole nicotinic acid for impurity removal and purity;
  • Spraying the above-mentioned herbicides can not only kill non-GAT maintainer materials, but also help weeding in the field. It plays an important role in maintaining line production and removing impurities and maintaining purity.
  • GAT maintainer seeds are planted in legally transgenic areas, self-bred and harvested, and non-fluorescent seeds are screened by a fluorescent seed sorting machine. They are GAT sterile seeds, which can be used as the female parent to be used with other varieties (fathers). This) seed production can also be used to reproduce GAT sterile lines.
  • the GAT sterile line seeds obtained in Example 10 were mixed or inter-sown with the GAT maintainer line seeds to the legal transgenic area.
  • the GAT maintainer line will loose powder to give the GAT sterile line to solidify, and then spray 1 -3g/m 2 of bentazone or 500-3000mg/L bensulfuron-methyl kills the GAT maintainer line, harvests all seeds, and uses a fluorescent seed sorter to screen and obtain non-fluorescent seeds, which is the GAT sterile line
  • the seeds can be used as the male parent for seed production with other varieties, and can also be used to propagate sterile lines.
  • GAT sterile lines are sown, and 0.1-1g/m 2 of Bentazone or 100-800mg/L bensulfuron-methyl is sprayed at the seedling stage for impurity removal and purity; 0.5-1.5g/ is sprayed from the tillering stage to the booting stage m 2 of Bentasone or 500-2000mg/L Bensulfuron-methyl for impurity removal; spray 1-3g/m 2 of Bentasone or 500-3000mg/L Bensulfuron-methyl for impurity removal during the flowering period Keep pure
  • Spraying the above herbicides can not only kill the GAT maintainer material, maintain the purity of the GAT sterile line, and maintain the purity of the GAT sterile line, but also help weeding in the field, killing two birds with one stone.
  • the GAT sterile lines obtained in Examples 10 and 11 were crossed with the male parent to produce seeds, and the seeds on the sterile lines were harvested after pollination, all of which were non-transgenic hybrid seeds.
  • the invention provides a genetic and intelligent breeding seed production system for crop hybrid breeding and seed production and its application.
  • the system of the present invention contains a GAT system vector, which includes five functional element expression cassettes: a plant male fertility restoration gene element expression cassette for restoring the male fertility of recessive genic sterile mutants; plant pollen abortion gene elements
  • the expression cassette is used to eliminate pollen containing GAT and maintain the heterozygous or hemizygous state of the GAT maintainer; the chemical herbicide positive selection expression cassette is used for gene transformation and the purification of the GAT maintainer; the negative chemical herbicide
  • the selection expression cassette is used to eliminate the pollen and seed escape of herbicide-sensitive GAT maintainer lines and the purification of GAT sterile lines; the seed selection element expression cassette is used for mechanical seed sorting.
  • the invention can be used for hybrid breeding and hybrid seed production of plant recessive nuclear male sterile materials, thereby obtaining high-quality, high-yield, wide-adaptation and high-resistance new plant varieties and their seeds, and has good economic value and application prospects.

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Abstract

一种用于农作物杂交育种制种的遗传智能化育制种系统及其应用。该系统含有GAT系统载体，该载体包括五种功能元件表达盒：植物雄性育性恢复基因元件表达盒，用于恢复隐性核不育突变体的雄性育性；植物花粉败育基因元件表达盒，用于清除含GAT的花粉及保持GAT保持系的杂合状态或半合子状态；化学除草剂正向选择表达盒，用于基因转化及GAT保持系除杂提纯；化学除草剂负向选择表达盒，用于清除除草剂敏感的GAT保持系花粉及种子外逸及GAT不育系除杂提纯；种子筛选元件表达盒，用于种子机械分选。该系统可用于植物隐性核雄性不育材料的杂交育种和杂交制种，从而获得优质高产广适高抗的植物新品种及其种子。

Description

一种用于农作物杂交育种制种的遗传智能化育制种系统及其应用
交叉引用
本申请要求2020年5月7日提交的专利名称为“一种用于农作物杂交育种制种的遗传智能化育制种系统及其应用”的第202010379287.9号中国专利申请的优先权，其全部公开内容通过引用整体并入本文。
技术领域
本发明属于农业生物技术领域，具体涉及一种用于农作物杂交育种制种的遗传智能化育制种系统及其应用。
背景技术
杂种优势是杂交后代在一种或多种性状上超越双亲的现象，普遍存在于生物界中。农作物杂种优势利用是农业增产重要手段，杂种优势利用产业化的核心在于母本的雄性不育性或雄性不育系。
以水稻为例，中国是世界上杂交水稻产业化最成功的国家，在杂交水稻育制种研究领域处于世界领先地位。目前我国杂交水稻育制种方法主要分为“三系法”和“两系法”，各有其优缺点。“三系法”自70年代开始应用于水稻，其主要原理是，利用不育系、保持系和恢复系来配制杂交种。其基本流程是利用保持系自交繁殖保持系，不育系与保持系杂交繁殖不育系，恢复系与不育系杂交产生杂交种子。由于“三系法”不育系的雄性不育是由细胞核与细胞质的基因互作造成的，导致该方法存在缺陷：只有细胞核含有特定恢复基因的恢复系与不育系杂交后才能恢复杂交种育性，从而生产杂交水稻种子。因而“三系法”种质资源利用率较低，且细胞质的同质化也具有潜在病虫害风险。“两系法”的原理是，利用光温敏核不育系与恢复系配制杂交种。该不育系在水稻发育特定时期，环境温度高于23.5度时不育系的雄性保持不育，可与恢复系成功配制杂交种；若环境温度低于23.5度，不育系雄性育性恢复正常，将自交繁殖。因此，“两系法”只需要不育系与恢复系，节省制种成本。但是，由于“两系法”的光温敏核不育系育性转换受到光温环境影响，导致该技术在不育系繁殖以及杂交种制种过程中都存在极大的环境风险，同时由于对温度的特殊要求，生产季节和区域也大大受到限制。
隐性核雄性不育不同于“三系”、“两系”，其雄性育性仅受一对隐性核基因控制，不受光温环境影响；且由于其基因为非常稀有的隐性突变，由于绝大多数品种的基因组上述位点为野生型的显性基因，故理论上几乎任何品种都可以作为隐性核雄性不育的恢复系与其杂交产生可育后代。隐性核不育的发现已经有非常长久的历史，当其与 野生型材料杂交时，其F1代为杂合状态表现为可育，F2代出现育性分离(单基因控制情况下，分离比为3:1)，但在F1代自交结实收获种子(即F2)时，因种子外观完全一样，无法区分哪一部分种子将来发育成株后是可育，哪一部分是不育的。因此，长期以来受限于无法规模化保持其后代雄性不育性而不能产业化利用。因此，研发一种能够保持和繁殖隐性核雄性不育材料并配套商业化生产的技术，就显得尤为必要，这也是广大育种人员的迫切需求。
发明内容
本发明的目的是提供一种隐性核雄性不育系的保持和繁殖产业化的方法。
为实现该目的，本发明提供了一种介导调控植物隐性核雄性不育突变体(不育系)雄性育性的GAT载体及其应用。本发明还提供一种农作物杂交育制种技术体系(GAT技术体系)和应用。GAT(Genetic Automation Technology，GAT)技术是一种新型的杂交种子育制种技术，该技术可成功利用隐性核雄性不育系，其核心思路是利用现代生物技术，将农作物花粉育性恢复基因、花粉败育基因、除草剂敏感基因、筛选标记基因等按特定顺序和方向紧密连锁地构建在GAT载体上，通过高通量基因转化技术导入到隐性核雄性不育系中，获得大量转化事件。由于多基因转化过程中经常出现部分基因转入、各转基因元件无法同时发挥功能及转基因沉默等问题，故多基因转化经常出现无法筛选获得均达目的性状的转化事件。本发明经过对各功能元件的筛选，从563个转化事件中筛选获得14个各元件均发挥正常功能的初始保持系，创造隐性核雄性不育系的保持系，用于GAT不育系和杂交种的生产，从而成功实现隐性核雄性不育系的保持和繁殖，进而实现隐性核雄性不育系的商业化利用。
本发明所述的隐性核雄性不育突变体，其基因呈隐性纯合状态时，植株呈现雄性不育状态；该基因为杂合或显性纯合状态时，植株呈现雄性可育状态。该隐性核雄性不育突变体的控制基因可为MS1、MS2、MS3、MS5、MS7、MS8、MS9、MS10、MS11、MS12、MS13、MS14、MS17、MS20、MS22、MS23、MS24、MS25、OsCYP704B2、MS27、MS28、MS29、MS30、MS31、MS32、MS33、MS34、MS36、MS37、MS38、MS43、MS45、MS48、MS50核苷酸序列等的突变基因。优选的，为OsCYP704B2的突变体(oscyp704b2)，对应的，其恢复基因为OsCYP704B2。
本发明首先提供一种用于农作物杂交育种制种的遗传智能化育制种系统，称为GAT系统，含有植物隐性核雄性不育系，即GAT不育系，隐性核雄性不育保持系，即GAT保持系，和普通恢复系三个系别；
其中的GAT保持系中含有GAT载体，该载体包括五种功能元件表达盒：(1)植 物雄性育性恢复基因元件表达盒，用于恢复隐性核不育突变体的雄性育性；(2)植物花粉败育基因元件表达盒，用于清除含GAT的花粉及保持GAT保持系的杂合状态或半合子状态；(3)基因转化及保持系筛选元件表达盒，用于基因转化及GAT保持系除杂提纯；(4)除草剂敏感元件表达盒，用于清除除草剂敏感的GAT保持系花粉及种子外逸及GAT不育系除杂提纯；(5)种子筛选元件表达盒，用于种子机械分选；所述五种功能元件表达盒构建于植物双元表达载体中，得到GAT系统载体。
上述GAT系统中，将GAT载体导入GAT不育系创制GAT保持系，且GAT载体以单拷贝形式存在于GAT保持系基因组中。
上述GAT系统中，所述GAT不育系为单隐性细胞核基因控制的不育系，当基因位点为隐性纯合状态时表现为雄性不育；杂合状态及显性纯合状态表现为雄性可育。
进一步地，在所述的GAT系统中，GAT保持系自交结实，所获种子呈1:1分离得到GAT保持系和GAT不育系；两种种子通过种子筛选元件实现分离，实现GAT保持系的自我繁殖；GAT保持系向GAT不育系授粉使得GAT不育系结实并保持其后代的雄性不育性，从而实现隐性雄性核不育系的繁殖。通过种子筛选可规模化将隐性核雄性不育种子(GAT不育系)与可育种子(GAT保持性)分离，辅以保持系筛选元件及除草剂敏感元件的功能进行后续的GAT保持系及不育系除杂保纯，创造性的解决了现有技术存在的隐性雄性核不育系的规模化繁殖与保持问题，从而使得隐性核雄性不育的产业化成为可能。
本发明提供了一种农作物遗传智能化育制种载体，称为GAT载体，该载体由五种功能元件表达盒通过接头序列连接构建于植物双元表达载体中得到，所述物种功能元件表达盒分别为：
(1)植物雄性育性恢复基因元件表达盒，用于恢复隐性核不育突变体的雄性育性；
(2)植物花粉败育基因元件表达盒，用于清除含GAT的花粉及保持GAT保持系的杂合状态或半合子状态；
(3)基因转化及保持系筛选元件表达盒，用于基因转化及GAT保持系除杂提纯；
(4)除草剂敏感元件表达盒，用于清除除草剂敏感的GAT保持系花粉及种子外逸及GAT不育系除杂提纯；
(5)种子筛选元件表达盒，用于种子机械分选；所述五种功能元件表达盒通过接头序列连接构建得到GAT系统载体。
本发明的GAT载体中，所述(1)植物雄性育性恢复基因元件表达盒由启动子、 雄性育性恢复基因编码区及终止子依序可正常工作的相连；
所述雄性育性恢复基因为MS1、MS2、MS3、MS5、MS7、MS8、MS9、MS10、MS11、MS12、MS13、MS14、MS17、MS20、MS22、MS23、MS24、MS25、OsCYP704B2、MS27、MS28、MS29、MS30、MS31、MS32、MS33、MS34、MS36、MS37、MS38、MS43、MS45、MS48、MS50、OsCYP704B2基因的野生型基因，优选OsCYP704B2基因的野生型基因。
优选地，本发明所述(1)植物雄性育性恢复基因元件表达盒中，启动子为水稻OsCYP704B2基因起始密码子ATG上游1112bp序列，编码区为OsCYP704B2基因的编码区，终止子为OsCYP704B2基因终止密码子TGA下游274bp序列。该表达盒的作用是恢复OsCYP704B2基因的隐性纯合突变体oscyp704b2的雄性育性。
所述(1)植物雄性育性恢复基因元件表达盒的序列如SEQ ID NO.6所示。
本发明的GAT载体中，所述(2)植物花粉败育基因元件表达盒由植物花粉特异启动子、败育基因编码区及终止子依序可正常工作的相连；优选启动子为玉米PG47启动子、水稻PCHF15、OsPC32启动子，优选败育基因为水稻α-淀粉酶基因OsAA、玉米α-淀粉酶基因Zm-AA1、大麦α-淀粉酶基因HvAA1、小米α-淀粉酶基因SiAA、细胞分裂素氧化酶，半胱氨酸蛋白酶，赤霉素氧化酶，终止子为玉米IN2-1、细菌NosT终止子。
优选地，本发明所述植物花粉败育基因元件表达盒由启动子PG47、Zm-AA1编码区和终止子IN2-1组成的表达盒Killer序列如SEQ ID NO.7所示；或植物花粉败育基因元件表达盒Killer 5400，由启动子PG47、OsAA编码区和Nos终止子(NosT)组成，序列如SEQ ID NO.8所示；或植物花粉败育基因元件表达盒Killer Hv，由启动子OsPC32、大麦α-淀粉酶基因HvAA1编码区和NosT组成，序列如SEQ ID NO.9所示。
该元件的作用是使得含GAT元件的花粉败育，用于维持GAT转化株或者GAT保持系的杂合状态或半合子状态及防止GAT元件漂移。
本发明的GAT载体中，所述(3)基因转化及保持系筛选元件表达盒由启动子、筛选标记基因编码区及终止子依序可正常工作的相连；优选启动子为OsUbi启动子、Actin启动子或OsALS基因起始密码子ATG上游2180bp序列的任一个，优选筛选标记基因编码区为OsALSm1、OsALSm2、OsALSm3序列、草甘膦抗性基因EPSPSm序列、草甘膦N-乙酰转移酶序列或草丁膦抗性基因Bar序列中的任一个筛选标记基因；终止子为OsUbiT(SEQ ID NO.24)或NosT终止子。
优选地，由ActinP、OsALSm1和NosT组成的基因转化及保持系筛选元件表达盒Marker2序列如SEQ ID NO.10所示。另一个由OsALSP、OsALSm1和OsUbiT组成的基因转化及保持系筛选元件表达盒Marker2AAU序列如序列SEQ ID NO.11所示。或EPSPS表达盒，其序列如序列SEQ ID NO.12所示，或Bar表达盒，其序列如序列SEQ ID NO.13所示。上述表达盒的作用是基因转化筛选抗性标记及筛选区分GAT保持系和不育系。
本发明的GAT载体中，所述(4)除草剂敏感元件表达盒由启动子、除草剂显性敏感元件及终止子依序可正常工作的相连，优选启动子为ZmUbi启动子，除草剂显性敏感元件为细胞色素P450基因CYP81A6的RNAi结构序列P450i，终止子为PinII终止子、NosT终止子。
优选地，由ZmUbiP、P450i和NosT组成的除草剂敏感元件表达盒Marker1序列如SEQ ID NO.14所示，或如SEQ ID NO.15所示(P450i-2)，或如SEQ ID NO.16所示(P450i-3)。该元件的作用是防止GAT元件漂移、GAT保持系混杂入其他材料及用于GAT不育系制种。
本发明的GAT载体中，所述(5)种子筛选元件表达盒由启动子、种皮显色基因及终止子依序可正常工作的相连，优选地，启动子为种子特异性启动子ZZ1启动子，种皮显色基因为深红色荧光蛋白FP635，红色荧光蛋白RFP或绿色荧光蛋白GFP，终止子为OS-T28终止子、NosT终止子。
优选地，由ZZ1P、FP635和NosT组成的种子筛选元件表达盒Marker3ZFN序列如SEQ ID NO.17所示。该元件的作用是筛选区分GAT保持系种子与GAT不育系或GAT杂交种。
本发明所述GAT载体，所述接头序列包括：多克隆位点MCSI，序列如SEQ ID NO.18所示；多克隆位点MCSII，序列如SEQ ID NO.19所示；多克隆位点MCSIII，序列如SEQ ID NO.20所示；多克隆位点MCSIV，序列如SEQ ID NO.21所示；或多克隆位点MCSV，序列如SEQ ID NO.22所示。
优选地，本发明所述终载体为pC0307，见图6，其序列如SEQ ID NO.25所示；或所述终载体为pC0308，见图7，其序列如SEQ ID NO.26所示，或所述终载体为pC0309，见图8，其序列如SEQ ID NO.27所示。
优选地，本发明所述GAT载体为pC1300-MMCK(图1)、pC0308-MMCK(图2)、、pC0308-MMMaauCK5400(图4A，核苷酸序列如SEQ ID NO.1所示)、pC0308-KhvMMaauMCK5400(图4B，核苷酸序列如SEQ ID NO.2所示)、 pC0308-KhvMaauMCMK5400(图4C，核苷酸序列如SEQ ID NO.3所示)、pC0309-KhvMaauMCMK5400(图4D，核苷酸序列如SEQ ID NO.4所示)和pC0307-KhvMaauMCMK5400(图3，核苷酸序列如SEQ ID NO.5所示)。
本发明提供了上述GAT载体的构建方法，将五种功能元件表达盒通过接头序列连接构建于终载体中得到，所述五种功能元件表达盒分别为：
(1)植物雄性育性恢复基因元件表达盒，用于恢复隐性核不育突变体的雄性育性；该表达盒由启动子、雄性育性恢复基因编码区及终止子依序可正常工作的相连；
(2)植物花粉败育基因元件表达盒，用于清除含GAT的花粉及保持GAT保持系的杂合状态或半合子状态；该表达盒由植物花粉特异启动子、败育基因编码区及终止子依序可正常工作的相连；
(3)基因转化及保持系筛选元件表达盒，用于基因转化及GAT保持系除杂提纯；该表达盒由启动子、筛选标记基因编码区及终止子依序可正常工作的相连；
(4)除草剂敏感元件表达盒，用于清除除草剂敏感的GAT保持系花粉及种子外逸及GAT不育系除杂提纯；该表达盒由启动子、除草剂显性敏感元件及终止子依序可正常工作的相连；
(5)种子筛选元件表达盒，用于种子机械分选；所述五种功能元件表达盒通过接头序列连接于终载体上构建得到GAT载体；该表达盒由启动子、种皮显色基因及终止子依序可正常工作的相连。
上述构建方法中，优选地，接头序列包括：多克隆位点MCSI，序列如SEQ ID NO.18所示；多克隆位点MCSII，序列如SEQ ID NO.19所示；多克隆位点MCSIII，序列如SEQ ID NO.20所示；多克隆位点MCSIV，序列如SEQ ID NO.21所示；或多克隆位点MCSV，序列如SEQ ID NO.22所示。
上述构建方法中，优选地，本发明所述终载体为pC0307，见图6，其序列如SEQ ID NO.25所示；或所述终载体为pC0308，见图7，其序列如SEQ ID NO.26所示，或所述终载体为pC0309，见图8，其序列如SEQ ID NO.27所示。
本发明提供了所述的遗传智能化育制种系统(GAT系统)或所述的GAT载体在植物杂交育种、制种中的应用。
本发明提供了所述的遗传智能化育制种系统(GAT系统)或所述的GAT载体在生产非转基因杂交种中的应用。
本发明提供了所述的遗传智能化育制种系统(GAT系统)或所述的GAT载体在植物隐性核雄性不育系的规模化保持及规模化繁殖中的应用。
本发明提供了所述的遗传智能化育制种系统(GAT系统)或所述的GAT载体在生产优质高产广适高抗植物新品种中的应用。
本发明中，所述植物包括水稻、玉米、小麦、大麦、大豆、棉花、油菜、高粱、小米、燕麦、黑麦、青稞、辣椒、西瓜等。
进一步地，本发明提供了一种保持及繁殖植物隐性核雄性不育材料(GAT不育材料)的雄性不育性的方法，即保持其雄性不育基因处于隐性纯合状态的方法，具体如下：采用遗传智能化育制种系统GAT系统，向基因型为隐性纯合的GAT不育系中导入GAT载体，创制只含单拷贝GAT载体的GAT转化株，该GAT转化株的基因型为隐性纯合/GAT-。使GAT转化株授粉给GAT不育系，获得的种子基因型仍为隐性纯合状态，从而保持GAT不育系后代的雄性不育性。
具体地，以ms代表隐性核雄性不育突变基因，MS代表野生型基因，将GAT元件导入GAT不育材料(基因型为ms ms)，创制只含单拷贝GAT载体的GAT转化株(基因型为ms ms/GAT-)。因GAT载体中含恢复基因元件，故可恢复雄性育性，GAT转化株产生的花粉和雌配子均有两种类型：(ms GAT)和(ms-)，(ms GAT)类型花粉由于含有GAT载体中的花粉败育基因元件，会败育，因此只有(ms-)类型花粉存活。故GAT转化株授粉给GAT不育株，获得的种子基因型仍为隐性纯合状态(ms ms)，可保持GAT不育株后代的雄性不育性。由于GAT不育株与GAT转化株除GAT元件外遗传背景完全相同，故由此方法获得的GAT不育株所有基因型不变，表型也相同。
本发明提供了一种用于维持植物隐性核雄性不育株的保持系材料(GAT转化株或GAT保持系)的GAT元件呈杂合状态/半合子状态的方法，采用所述的遗传智能化育制种系统，向基因型为隐性纯合的GAT不育系中导入GAT载体，创制只含单拷贝GAT载体的GAT转化株，该GAT转化株的基因型为隐性纯合/GAT-。GAT转化株自交可获得两种基因型种子，一种为隐性纯合/--，是GAT不育株；另一种为隐性纯合/GAT-，是GAT不育株保持系材料，根据遗传定律，二者呈1：1分离，即GAT转化株自交后代中基因型为隐性纯合/GAT-的GAT座位保持杂合状态/半合子状态。
本发明提供一种用于筛选或区分GAT转化株自交种子的方法，所述GAT转化株是采用本发明所述的遗传智能化育制种系统，向基因型为隐性纯合的GAT不育系中导入GAT载体，创制只含单拷贝GAT载体的GAT转化株，该GAT转化株的基因型为隐性纯合/GAT-，GAT转化株自交结实的种子呈现1:1分离，其中50％为包含GAT载体的种子，基因型为隐性纯合/GAT-，在激发光下观察呈现荧光；50％为不包含GAT载体的种子，基因型为隐性纯合，在激发光下观察无荧光。
本发明提供了一种用于筛选或区分GAT转化株自交种子及植株的方法，所述GAT转化株是采用本发明所述的遗传智能化育制种系统(GAT系统)，向基因型为隐性纯合的GAT不育系中导入GAT载体，创制只含单拷贝GAT载体的GAT转化株，该GAT转化株的基因型为隐性纯合/GAT-，GAT转化株自交结实的种子呈现1:1分离，其中50％为包含GAT载体的种子及其植株，其基因型为隐性纯合/GAT-，具有对针对乙酰乳酸合酶或EPSPS或Bar基因的各类除草剂高抗性，包括但不限于双草醚、咪唑乙烟酸、灭草烟、草甘膦、草丁膦或草铵膦；50％为不包含GAT载体的种子，基因型为隐性纯合不具有此高抗性。
本发明提供了一种用于防止植物花粉漂移的方法，将本发明所述的GAT载体转入植物中，使得含GAT载体的植物材料在花粉成熟时，因其含有花粉败育基因元件，会特异性导致含GAT载体的花粉败育，而保证不含GAT载体的花粉正常发育散粉，从而降低含GAT载体的花粉外逸的概率。
本发明提供一种用于防止GAT种子或植株漂移或混杂的方法，使所述种子或植株中含有GAT载体，在种子时期通过包衣或在苗期至开花期施用特定浓度的除草剂，包括苯达松或苄嘧磺隆或烟嘧磺隆，可在特定时期杀死含GAT种子或植株的材料，从而防止GAT种子或植株混杂入其他普通材料中。
本发明提供一种利用植物隐性核雄性不育系生产不育系种子方法，采用所述的遗传智能化育制种系统，将GAT保持系与GAT不育系按一定比例混合播种，利用GAT保持系授粉给GAT不育系，授粉完成后，施用除草剂，包括苯达松或苄嘧磺隆或烟嘧磺隆，特异性的杀死GAT保持系，仅保留GAT不育系收获种子。
本发明提供一种提纯植物隐性核雄性不育系的方法，采用所述的遗传智能化育制种系统，GAT不育系通过种子包衣或在苗期至开花期施用特定浓度的除草剂，包括苯达松或苄嘧磺隆或烟嘧磺隆，可保证GAT不育系纯度。
本发明提供一种利用植物隐性核雄性不育系进行杂交制种方法，采用所述的遗传智能化育制种系统，利用GAT保持系自交生产GAT保持系种子和GAT不育系种子；利用GAT保持系授粉给GAT不育系生产GAT不育系种子；利用GAT不育系和常规材料杂交组配生产普通商业化杂交种。
本发明提供了一种利用植物隐性核雄性不育系进行杂交育种方法，采用遗传智能化育制种系统，利用GAT保持系与普通材料杂交选育，可通过常规回交选育，也可通过系谱法选育GAT保持系及不育系，选育过程辅以GAT各项分子标记、除草剂筛选、种子色选等加速选育。
本发明还提供了用于检测所述的GAT载体或含有GAT载体的转基因阳性植株的引物，所述引物为以下任一：
检测植物雄性育性恢复基因元件表达盒的引物序列如SEQ ID NO.28-29所示；或
检测植物花粉败育基因元件表达盒的引物序列如SEQ ID NO.30-31所示；或
检测基因转化及保持系筛选元件表达盒的分子引物如SEQ ID NO.32-33所示；或
检测除草剂敏感元件表达盒的引物序列如SEQ ID NO.34-35所示。
检测种子筛选元件表达盒的分子标记引物序列如序列SEQ ID NO.39-40所示。
本发明提供了一种检测或含有GAT载体的转基因阳性植株的方法。
若用SEQ ID NO.28-29作引物扩增，扩增产物用HaeIII酶切后电泳，最终产物中可能出现3种带型：86bp为野生型CYP704B2的带型，84bp为cyp704b2-3突变体的带型，66bp为GAT载体上植物雄性育性恢复基因元件表达盒的带型。如果出现84bp和66bp带型，而没有88bp带型，表明该植株为雄性不育突变体背景，且植物雄性育性恢复基因元件表达盒存在；
若用SEQ ID NO.30-31引物进行扩增，如果能扩增出914bp条带，表明植物花粉败育基因元件表达盒存在；
若用SEQ ID NO.32-33作为引物进行扩增，如果能扩增出831bp条带表明基因转化及保持系筛选元件表达盒存在；
若用SEQ ID NO.34-35作为引物进行扩增，如果能扩增出923bp条带，表明除草剂敏感元件表达盒存在。
若用SEQ ID NO.36-37作为引物进行扩增，如果能扩增出1412bp条带，表明种子筛选元件表达盒存在。
本发明提供了一种分选不同功能的植株和后代的方法，所述的GAT载体转入植株，利用GAT载体中的(3)基因转化及保持系筛选元件表达盒，(4)除草剂敏感元件表达盒，(5)种子筛选元件表达盒，基于化学除草剂正负双向选择与机械色选相结合，分选不同功能的植株和后代。
优选地，所述化学除草剂正负双向选择是同一种植株对一种除草剂表型抗性，对另一种除草剂表型敏感。
更优选地，化学除草剂正向选择除草剂是抗双草醚、咪唑乙烟酸、灭草烟、抗草甘膦、抗草丁膦或草铵膦，化学除草剂负向选择是对苯达松、苄嘧磺隆或烟嘧磺隆敏感。
更优选地，化学除草剂正向选择除草剂是抗双草醚、咪唑乙烟酸、灭草烟，化学 除草剂负向选择是对苯达松、苄嘧磺隆敏感。
本发明构建了GAT载体，包括五种功能元件表达盒：植物雄性育性恢复基因元件表达盒，用于恢复隐性核不育突变体的雄性育性；植物花粉败育基因元件表达盒，用于清除含GAT的花粉及保持GAT保持系的杂合状态/半合子状态；化学除草剂正向选择表达盒，用于基因转化及GAT保持系除杂提纯；化学除草剂负向选择表达盒，用于清除除草剂敏感的GAT保持系花粉及种子外逸及GAT不育系除杂提纯；种子筛选元件表达盒，用于种子机械分选。在育种实践中，由于隐性核雄性不育材料是不育的，无法产生种子，长期保存很困难，若杂交保留的话，后代会出现育性性状分离，而在种子时期无法判定究竟哪些为可育种子，哪些为不育种子，导致保种、选种不准确，因此操作非常复杂，更不用说用于规模化生产，故至今仍未实现利用隐性核雄性不育的产业化利用。本申请将GAT载体导入GAT不育系(隐性核雄性不育系)创制GAT保持系，建立了可保持和繁育隐性核雄性不育系的遗传智能化育制种系统。利用该系统，在其后代中可通过种子色选及保持系筛选元件及除草剂敏感元件等的功能有机组合进行后续的GAT保持系及不育系除杂保纯，在种子时期、营养生长期及生殖生长期均可实现隐性核雄性不育种子/植株(GAT不育系)和可育种子/植株(GAT保持性)的分离，成功地解决了隐性核雄性不育的规模化繁殖与保持问题，创造性的实现了隐性核雄性不育的利用从而可实现商业化生产。本发明提供的GAT载体中的5个关键元件有机结合发挥作用并结合机械化、自动化加工可成功实现植物隐性核雄性不育系的商业化利用。本发明可应用于植物隐性核不育材料的杂交育种和杂交制种，从而获得优质高产广适高抗植物新品种及其种子，具有巨大的经济价值。
附图说明
图1为pC1300-MMCK载体示意图。
图2 pC0308-MMCK载体示意图。
图3为pC0307-KhvMaauMCMK5400载体示意图。
图4A为pC0308-MMMaauCK5400载体示意图。
图4B pC0308-KhvMMaauMCK5400载体示意图。
图4C pC0308-KhvMaauMCMK5400载体示意图。
图4DpC0309-KhvMaauMCMK5400载体示意图。
图5A pC0308-KhvMMaauMCK5400酶切验证结果图，1号泳道为Kpn I单酶切；2号泳道为Pst I单酶切；3号泳道为Sma I单酶切；M，D2000 DNA Marker。
图5B pC0308-KhvMaauMCMK5400酶切验证结果图，1号泳道未酶切 pC0308-KhvMaauMCMK5400质粒；2号泳道为Kpn I单酶切；3号泳道为BamH I单酶切；4号泳道为Sac I单酶切；5号泳道为Sma I单酶切；6号泳道为Bgl II单酶切；M，D15000 DNA Marker。
图5C pC0309-KhvMaauMCMK5400酶切验证结果图，1号泳道未酶切pC0309-KhvMaauMCMK5400质粒；2号泳道为Sac I单酶切；3号泳道为Sph I单酶切；4号泳道为Kpn I单酶切；5号泳道为BamH I单酶切；6号泳道为Xho I单酶切；M，D15000 DNA Marker。
图5D pC0307-KhvMaauMCMK5400酶切验证结果图，1号泳道未酶切pC0307-KhvMaauMCMK5400质粒；2号泳道为Sac I单酶切；3号泳道为BamH I单酶切；4号泳道为Kpn I单酶切；M，D15000 DNA Marker。
图5E转pC0308-MMMaauCK5400农杆菌菌落PCR检测结果图，1-22泳道为特异引物SEQ ID NO.34-35检测结果，其中1为阴性水对照，2为pC0308-MMMaauCK5400质粒对照，3-22为不同单菌落，M是D2000 DNA Marker。
图6 pC0307载体示意图。
图7 pC0308载体示意图。
图8 pC0309载体示意图。
图9 pC1300载体示意图。
图10 pUC57-Simple载体示意图。
图11 GAT载体菌落PCR检测1-12为特异引物SEQ ID NO.30-31菌落PCR检测结果，其中1为阴性水对照，2为pC0308-MMMaauCK5400质粒对照，3-4为pC1300-MMCK、5-8为pC0308-MMCK、9-12为pC0308-MMMaauCK5400、13-24为特异引物SEQ ID NO.34-35的菌落PCR检测结果，其模板顺序与1-12相同；M：D2000DNA Marker。
图12 pC0308-MMMaauCK5400酶切验证结果图，M1，D15000 plus DNA Ladder；M2，DNA Marker VI；CK，质粒；A，Hind III和Sma I；B，Kpn I；C，Hind III和Pst I；D，Hind III和Kpn I。
图13转pC0308-MMMaauCK5400农杆菌菌落PCR检测结果图，1-22泳道为特异引物SEQ ID NO.30-31检测结果，其中 1为阴性水对照，2为pC0308-MMMaauCK5400质粒对照，3-22为不同单菌落，M是D2000 DNA Marker。
图14 pC0308-MMMaauCK5400转基因植株PCR阳性检测结果图，检测引物序列如SEQ ID NO.30-31。M为D2000 DNA Marker，第一个“-”为阴性对照水，第二个“-” 为阴性对照野生型中花11，“+”为阳性对照pC0308-MMMaauCK5400质粒，1-18为转基因T0植株。
图15 pC0308-MMMaauCK5400转基因植株PCR阳性检测结果图，检测引物序列如SEQ ID NO.32-33。M为D2000 DNA Marker，第一个“-”为阴性对照水，第二个“-”为阴性对照野生型中花11，“+”为阳性对照pC0308-MMMaauCK5400质粒，1-19为转基因T0植株。
图16为pC1300-MMCK、pC0308-MMMaauCK5400转基因T0植株的互补元件检测结果图，检测引物序列如SEQ ID NO.28-29。1为野生型中花11，2为cyp704b2-3纯合突变体，3-8为pC1300-MMCK转基因植株，9-14为pC0308-MMMaauCK5400转基因植株。
图17为实施例4的部分GAT T0代转化株喷施双草醚结果。
图18为实施例4的部分GAT T0代转化株叶片分段喷施苯达松结果。
图19为GAT T0代转化株成熟花粉碘染结果，黑色为可育花粉；浅色为败育花粉。
图20 GAT转化株(T0代)种子荧光鉴定结果图。
图21为GAT代表株系88-4中的荧光种子和非荧光种子分别发育成株后花粉育性及自交结实鉴定图，A和C来源于荧光种子发育成株的稻穗和花药，其中有一半花粉可育，碘染呈蓝色；另一半花粉不育，碘染不着色，稻穗可自交结实。B和D来源于非荧光种子发育成株的稻穗和花药，为无花粉类型，是cyp704b2隐性核雄性不育突变体的典型特征，稻穗自交不结实。
图22为GAT T1代重点株系苯达松喷施结果。
图23为GAT转化株T1代成熟花粉育性检测结果图。A：cyp704b2，为不育突变体材料，镜检无花粉；B：ZH11，为普通野生型材料中花11，镜检碘染花粉育性正常；C：GAT，为不育突变体中转入GAT载体材料，镜检碘染花粉育性有一半育性正常，另一半花粉不染色。
图24为GAT转化株(T1代)种子荧光鉴定结果图。
图25为T1代GAT保持系逃逸率检测。
图26代表株系88-4-16中的荧光种子和非荧光种子分别发育成株后花粉育性及自交结实鉴定结果图。A和C来源于荧光种子发育成株的稻穗和花药，其中有一半花粉可育，碘染呈蓝色；另一半花粉不育，碘染不着色，稻穗可自交结实。B和D来源于非荧光种子发育成株的稻穗和花药，为无花粉类型，是cyp704b2隐性核雄性不育 突变体的典型特征，稻穗自交不结实。
具体实施方式
下面结合具体实施方式对本发明进行详细说明。
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例中所用技术手段为本领域技术人员所熟知的常规手段，所用原料均为市售商品。
实施例1 GAT载体构建与验证
一、GAT载体的构建
GAT载体采取以表达盒为单元的分段构建，单元拼合的策略。先在过渡载体pC1300(图9)和pUC57-Simple(图10)上构建表达盒并酶切和测序验证，然后将表达盒拼接到终载体上。载体构建的具体步骤如下：
1、合成MCS pC0307片段(SEQ ID NO.38)，用Sac II和Pme I连入pC1300后获得pC0307(图6)。
2、合成MCS pC0308片段(SEQ ID NO.39)，用Sac II和Sph I连入pC1300后获得pC0308(图7)。
3、合成MCS pC0309片段(SEQ ID NO.40)，用Sac II和Pme I连入pC1300后获得pC0309(图8)。
4、合成DNA片段NSPT-Construct V1.81-Marker 1，用Kpn I+Hind III双酶切连入pC1300，产生pC1300-Marker 1。NSPT-Construct V1.81-Marker 1的序列由Kpn I酶切位点ggtacc，MCSI(序列如SEQ ID NO.18所示)，除草剂敏感元件表达盒Marker 1(SEQ ID NO.14)，MCSII(SEQ ID NO.19)，间隔序列tgcagggacccttgccaac，和Hind III酶切位点aagctt顺次连接而成。其中MCSI的序列由Pst I、Srf I、Afe I、Xmn I酶切位点顺次连接组成。除草剂敏感元件表达盒Marker 1的序列由NosT终止子，细胞色素P450基因CYP81A6的一种RNAi茎环结构序列，ZmUbiP启动子顺次连接组成。细胞色素P450基因CYP81A6的一种RNAi茎环结构序列由一段CYP81A6编码区构成的反向茎序列、一个水稻内含子构成的环序列和与所述CYP81A6编码区互补的正向茎序列顺次连接而成。MCSII的序列由Hpa I、PshA I、BspE I、Pac I酶切位点顺次连接组成。
5、合成DNA片段NSPT-Construct V1.9-Marker 2，用EcoR I+Hind III双酶切连入pC1300，产生pC1300-Marker 2。NSPT-Construct V1.9-Marker 2的序列由EcoR I酶切位点gaattc，Pst I酶切位点ctgcag，间隔序列ggacccttgccaaca，多克隆位点MCSII(序列如SEQ ID NO.19所示)，基因转化及保持系筛选元件表达盒Marker2(SEQ ID  NO.10)，MCSIII(SEQ ID NO.20)，间隔序列tgcagtcccaaggcttccg，和Hind III酶切位点aagctt顺次连接而成。其中MCSII的序列由Hpa I、PshA I、BspE I、Pac I酶切位点顺次连接组成，基因转化及保持系筛选元件表达盒Marker2的序列由NosT终止子，ALS基因编码区序列OsALSm1，ActinP启动子顺次连接组成，MCSIII的序列由BsrG I、Bae I、AsiS I、FspAI酶切位点顺次连接组成。
6、合成DNA片段NSPT-Construct V1.81-Complementation，用Sac I+Hind III双酶切连入pC1300，产生pC1300-Complementation。NSPT-Construct V1.81-Complementation的序列由Sac I酶切位点gagctc，Pst I酶切位点ctgcag，间隔序列tcccaaggcttccga，多克隆位点MCSIII(SEQ ID NO.20)，植物雄性育性恢复基因元件表达盒Complementation(SEQ ID NO.6)，MCSIV(SEQ ID NO.21)，间隔序列tgcagcctgttgccaggga，和Hind III酶切位点aagctt顺次连接而成。其中MCSIII的序列由BsrG I、Bae I、AsiS I、FspA I酶切位点顺次连接组成。植物雄性育性恢复基因元件表达盒Complementation由水稻OsCYP704B2基因起始密码子ATG上游1112bp序列，经过密码子优化的OsCYP704B2基因的编码区，OsCYP704B2基因终止密码子TGA下游274bp序列组成。MCSIV的序列由Swa I、BstB I、Mlu I、Rsr II酶切位点顺次连接组成。
7、合成DNA片段NSPT-Construct V1.81-Killer，用Nde I+EcoR V双酶切连入pUC57-Simple，产生pUC57-Simple-Killer。NSPT-Construct V1.81-Killer的序列由Nde I酶切位点catatg，间隔序列cagggacccttgccaaca，Nru I酶切位点tcgcga，Pac I酶切位点ttaattaa，Pst I酶切位点ctgcag，间隔序列cctgttgccagggaa，多克隆位点MCSIV(SEQ ID NO.21)，植物花粉败育基因元件表达盒Killer(SEQ ID NO.7)，间隔序列tcgacgcggccgatcccccgg，Stu I酶切位点aggcct，Sac I酶切位点gagctc，多克隆位点MCSV(SEQ ID NO.22)，间隔序列tggcactggccgtcgtttt，Hind III酶切位点aagctt，EcoR I酶切位点gaattc以及间隔序列ggcgcgccgggccca顺次连接而成。其中MCSIV的序列由Swa I、BstB I、Mlu I、Rsr II酶切位点顺次连接组成。植物花粉败育基因元件表达盒Killer由启动子PG47、Zm-AA1编码区和终止子IN2-1序列组成。MCSV的序列由Avr II、Pml I、SnaB I、Alo I酶切位点顺次连接组成。
8、用Pac I+Hind III酶切pC1300-Marker 1和pUC57-Simple-Killer，将表达盒Killer连入pC1300-Marker 1产生pC1300-Marker 1-Killer。
9、用BsrG I+Hind III酶切pC1300-Marker 2和pC1300-Complementation，将表达盒Complementation连入pC1300-Marker 2产生pC1300-Marker 2-Complementation。
10、用Pac I+Swa I酶切pC1300-Marker 1-Killer和pC1300-Marker 2-Complementation，将相连的两个表达盒Marker 2-Complementation连入pC1300-Marker 1-Killer产生GAT载体pC1300-Marker 1-Marker 2-Complementation-Killer(pC1300-MMCK，图1)。
11、用Pst I+Hind III酶切pC1300-MMCK、pC0308、pC0309，将四个相连的表达盒Marker 1-Marker 2-Complementation-Killer分别连入pC0308、pC0309产生GAT载体pC0308-MMCK(图2)。
12、合成DNA片段Killer 5400，用BstB I+Avr II双酶切连入pUC57-Simple-Killer，用表达盒Killer 5400替代Killer，产生pUC57-Simple-Killer 5400。Killer 5400片段由多克隆位点MCSIV(SEQ ID NO.21)，植物花粉败育基因元件表达盒Killer 5400(SEQ ID NO.8)，以及多克隆位点MCSV(SEQ ID NO.22)顺次连接而成。植物花粉败育基因元件表达盒Killer 5400包括PG47启动子、水稻α-淀粉酶基因OsAA(即5400)编码区以及NosT终止子等部分。
13、用Swa I+SnaB I双酶切pUC57-Simple-Killer 5400和pC0308-MMCK，回收表达盒Killer 5400和酶切产物pC0308-MMC，将Killer 5400连入pC0308-MMC产生pC0308-Marker 1-Marker 2-Complementation-Killer5400(pC0308-MMCK5400)。
14、合成DNA片段Marker 3 ZFN，用Pst I+Xma I双酶切连入pC0308-MMCK5400，产生pC0308-Marker 3-Marker 1-Marker 2-Complementation-Killer5400(pC0308-MMMCK)。Marker 3 ZFN片段的序列由Pst I酶切位点ctgcag，种子筛选元件表达盒Marker 3 ZFN(SEQ ID NO.17)，间隔序列g，以及Xma I酶切位点cccggg顺次连接而成。种子筛选元件表达盒Marker 3 ZFN顺次包括NosT终止子、深红色荧光蛋白FP635基因编码区，ZZ1P启动子。
15、合成OsALSP片段(SEQ ID NO.23)，用Nco I+BsrG I双酶切连入pC1300-NSPT-Construct V1.9-Marker 2，产生pC1300-Marker 2 AAN。合成DNA片段OsUbiT(SEQ ID NO.24)，用Pac I+Kpn I双酶切连入pC1300-Marker 2 AAN，产生另一个基因转化及保持系筛选元件表达盒Marker 2 AAU(SEQ ID NO.11)(即本申请的基因转化及保持系筛选元件表达盒)，相应载体为pC1300-Marker 2 AAU。Marker 2 AAU表达盒包括OsUbiT终止子，ALS基因编码区序列OsALSm1，OsALSP启动子。
16、用Pac I+BsrG I双酶切pC1300-Marker 2 AAU，回收Marker 2 AAU表达盒。用Pac I+BsrG I双酶切pC0308-MMMCK5400，回收较大的片段与Marker 2 AAU连接产生GAT载体pC0308-Marker 3-Marker 1-Marker 2aau-Complementation-Killer5400 (pC0308-MMMaauCK5400，图4A，SEQ ID NO.1)。
17、用Xma I+BspE I双酶切步骤16产生的载体pC0308-MMMaauCK5400，可获得一个较小片段Marker 1和一个较大片段pC0308-M_MaauCK5400。用高保真Taq酶补平上述片段末端，并分别回收。将pC0308-M_MaauCK5400自连，自连后用AsiSA I酶切，再用高保真Taq酶补平末端，然后与补平末端后的Marker 1平端连接，得到过渡载体pC0308-Marker 3-Marker 2aau-Marker 1-Complementation-Killer5400(pC0308-MMaauMCK5400)。Marker 1转录方向保持不变。
18、合成Killer Hv片段，用Pst I单酶切连入步骤19中产生的过渡载体pC0308-MMaauMCK5400，得到GAT载体pC0308-Killer Hv-Marker 3-Marker 2aau-Marker 1-Complementation-Killer5400(pC0308-KhvMMaauMCK5400，图4B，SEQ ID NO.2)。Killer Hv的转录方向与Marker3的转录方向相反。Killer Hv的序列由Pst I酶切位点ctgcag，植物花粉败育基因元件表达盒Killer Hv(SEQ ID NO.9)，AsiS I酶切位点gcgatcgc，SrfI酶切位点gcccgggc，Pac I酶切位点ttaattaa，Pst I酶切位点ctgcag顺次连接而成。植物花粉败育基因元件表达盒Killer Hv由启动子OsPC32、HvAA1编码区和终止子IN2-1序列组成。
19、用Pst I+Pac I双酶切步骤20中合成的Killer Hv片段并回收。同时也用Pst I+Pac I双酶切步骤19中产生的过渡载体pC0308-MMaauMCK5400，并回收酶切产物中最大的片段pC0308-_MaauMCK5400。将酶切后的Killer Hv连入pC0308-_MaauMCK5400，获得过渡载体pC0308-Killer Hv-Marker 2aau-Marker 1-Complementation-Killer5400(pC0308-KhvMaauMCK5400)。
20、用Pst I+Pac I双酶切步骤19中产生的过渡载体pC0308-MMaauMCK5400，回收酶切产物中的较小片段Marker 3 ZFN，用高保真Taq酶补平。用Swa I酶切步骤21中获得的过渡载体pC0308-KhvMaauMCK5400，将补平后的Marker 3 ZFN连入，获得GAT载体pC0308-Killer Hv-Marker 2aau-Marker 1-Complementation-Marker 3-Killer5400(pC0308-KhvMaauMCMK5400，图4C，SEQ ID NO.3)。Marker 3 ZFN的转录方向保持不变。
21、用Pst I+SnaB I双酶切步骤22中获得的GAT载体pC0308-KhvMaauMCMK5400，回收酶切产物中最大的片段KhvMaauMCMK5400。同时用Pst I+SnaB I分别双酶切pC0309和pC0307，回收线性化的pC0309和pC0307。将KhvMaauMCMK5400分别连入pC0309和pC0307获得GAT载体pC0309-Killer Hv-Marker 2aau-Marker 1-Complementation-Marker 3-Killer5400 (pC0309-KhvMaauMCMK5400，图4D，SEQ ID NO.4)和pC0307-Killer Hv-Marker 2aau-Marker 1-Complementation-Marker 3-Killer5400(pC0307-KhvMaauMCMK5400，图3，SEQ ID NO.5)。
二、GAT载体的验证
对上述构建好的GAT载体pC0308-MMMaauCK5400、pC0308-KhvMMaauMCK5400、pC0308-KhvMaauMCMK5400、pC0309-KhvMaauMCMK5400和pC0307-KhvMaauMCMK5400载体进行酶切和测序验证。
分别取1μl上述质粒与50μl大肠杆菌感受态细胞混合，1.8KV电击转化，转化产物涂布于含有卡那霉素的LA培养板上，37℃培养16h左右，挑取单菌落，使用特异引物进行菌液PCR检测(图11)。特异引物序列如SEQ ID NO.30和SEQ ID NO.31及序列SEQ ID NO.34和SEQ ID NO.35所示。选择阳性单菌落接种含有卡那霉素的LB培养基进行扩大培养，37℃培养16h左右提取质粒。方法如下：
1：将混浊的菌液倒入2ml离心管中，瞬时离心30s，沉淀菌体。
2：倒掉上清液，加200μl Solution I，震荡悬浮菌液，室温1min。
3：加入300μl Solution II，快速颠倒数次，冰浴1min。
4：加入400μl Solution III，温和振荡，冰浴2min冷凝，瞬时离心30s。
5：吸上清，12000rpm离心2min，将上清转移至1.5ml EP管中，加入0.6倍体积异丙醇，冰浴2min。
6：瞬时离心1min，弃上清。
7：加入70％乙醇500μl漂洗沉淀2次，晾干。
8：加入50μl ddH 2O(已加RNase A，浓度为1％)回融。
选择Kpn I、Hind III和Sma I、Hind III和Pst I、Hind III和Kpn I对pC0308-MMMaauCK5400，选择Kpn I、Pst I和Sma I对pC0308-KhvMMaauMCK5400，选择Kpn I、BamH I、Sac I、Sma I、Bgl II对pC0308-KhvMaauMCMK5400，选择Sac I、Sph I、Kpn I、BamH I、Xho I对pC0309-KhvMaauMCMK5400，选择Sac I、BamH I、Kpn I对pC0307-KhvMaauMCMK5400进行酶切验证。酶切体系为10×Buffer 1μl，质粒DNA 3μl，DNA限制性内切酶0.2μl，用ddH 2O补足10μl。酶切条件为37℃孵育10-15min，然后70℃灭活5min。酶切产物在1％琼脂糖胶中电泳检测。
如图12所示，pC0308-MMMaauCK5400质粒大小为24548bp；Hind III和Sma I双酶切后A点样孔条带大小为8287bp+16261bp；Kpn I单酶切后B点样孔条带大小 为9114bp+15434bp；Hind III和Pst I双酶切后C点样孔条带大小为6295bp+18253bp；Hind III和Kpn I双酶切后D点样孔条带大小为3010bp+9114bp+12424bp。酶切结果与预期完全相符。选择酶切正确的质粒测序，序列与预期完全相符。序列如SEQ ID NO.1所示。
如图5A所示，pC0308-KhvMMaauMCK5400质粒大小为28365bp；Kpn I单酶切后1号泳道条带大小为4870bp+10564bp+12931bp，其中10564bp和12931bp条带由于太大而没有分开，重叠为一条明亮的带；Pst I单酶切后2号泳道条带大小为3807bp+24558bp；Sma I单酶切后3号泳道条带大小为28365bp。酶切结果与预期完全相符。选择酶切正确的质粒测序，序列与预期完全相符。序列如SEQ ID NO.2所示。
如图5B所示，pC0308-KhvMaauMCMK5400质粒大小为28361bp；1号泳道为未酶切质粒对照；Kpn I单酶切后2号泳道条带大小为2858bp+10564bp+14939bp；BamH I单酶切后3号泳道条带大小为2053bp+6895bp+19413bp；Sac I单酶切后4号泳道条带大小为270bp+1376bp+5928bp+20787bp；Sma I单酶切后5号泳道条带大小为28361bp；Bgl II单酶切后6号泳道条带大小为2194bp+7867bp+8375bp+9925bp。酶切结果与预期完全相符。选择酶切正确的质粒测序，序列与预期完全相符。序列如SEQ ID NO.3所示。
如图5C所示，pC0309-KhvMaauMCMK5400质粒大小为28723bp；1号泳道为未酶切质粒对照；Sac I单酶切后2号泳道条带大小为270bp+1376bp+5928bp+21149bp；Sph I单酶切后3号泳道条带大小为4317bp+4837bp+5770bp+13799bp；Kpn I单酶切后4号泳道条带大小为2858bp+10926bp+14939bp；BamH I单酶切后5号泳道条带大小为2053bp+6895bp+19775bp；Xho I单酶切后6号泳道条带大小为76bp+1709bp+1935bp+5430bp+19573bp。酶切结果与预期完全相符。选择酶切正确的质粒测序，序列与预期完全相符。序列如SEQ ID NO.4所示。
如图5D所示，pC0307-KhvMaauMCMK5400质粒大小为28469bp；1号泳道为未酶切质粒对照；Sac I单酶切后2号泳道条带大小为270bp+1376bp+5928bp+20895bp；BamH I单酶切后3号泳道条带大小为2053bp+6895bp+19521bp；Kpn I单酶切后4号泳道条带大小为2858bp+10672bp+14939bp。酶切结果与预期完全相符。选择酶切正确的质粒测序，序列与预期完全相符。序列如SEQ ID NO.5所示。
实施例2农杆菌介导的GAT载体遗传转化水稻
1.实施例1构建的GAT载体转化农杆菌及验证
取-80℃保存的农杆菌EHA105于含利福平(25μg/ml)+链霉素(50μg/ml)YEP 平板划线，28℃培养。挑取单菌落接种于5ml含上述抗生素的YEP液体培养基中，220rpm，28℃振荡培养12-16hr。取2ml菌液转接于100ml含上述抗生素YEP液体培养基中，28℃，220rpm振荡培养至OD 600＝0.5。冰上预冷10min，4℃，5000rpm离心10min(冷冻离心机预冷到4℃)。无菌去离子水洗2次(每次10ml)，10％无菌甘油洗1次，4℃，5000rpm离心10min，菌体重悬于3ml 10％无菌甘油中。取1μl实施例1中得到的测序正确的GAT质粒pC0308-MMMaauCK5400加入50μl农杆菌感受态细胞中，1.8KV电击转化。涂布于含有卡那霉素、利福平和链霉素的YEP培养板上，28℃培养48h左右，挑取单菌落摇菌过夜。
使用特异性引物SEQ ID NO.30-31及SEQ ID NO.34-35对pC0308-MMMaauCK5400转化的农杆菌单克隆进行菌落PCR验证(如图13和图5E)，可扩出914bp和923bp目的片段。挑选阳性克隆，摇菌36-48h，保存菌液用于侵染。
2.农杆菌介导遗传转化
诱导：将中花11(ZH11)背景且携带纯合隐性雄性不育基因Oscyp704b2-3的种子经次氯酸钠消毒后置于诱导培养基(N 6+2.4-D 3mg/L+CH 0.6g/L+Pro0.5g/L+蔗糖30g/L+Phytagel 3g/L)上，28℃常温暗培养30-40d，得到诱导的愈伤后继代培养30-40d；
筛选：将本实施例得到的工程农杆菌，通过农杆菌介导的遗传转化法转化上述愈伤组织，共培养3d后，清洗5-6遍，转移至含双草醚抗性筛选培养基(N 6+2.4-D2mg/L+CH 0.6g/L+Pro0.5g/L+蔗糖30g/L+Phytagel 3g/L+Cn 500mg/L+双草醚0.3-0.6μm/L或潮霉素50mg/L)上，30℃暗培养30-50d，筛选获得抗性愈伤；
分化：筛选获得的抗性愈伤转移至含双草醚抗性分化培养基(MS+KT 2mg/L+NAA 0.5-2mg/L+山梨醇20-30g/L+蔗糖30g/L+Phytagel 3g/L+双草醚0.1-0.3μm/L)上，分化25-30d获得阳性苗；
生根：经分化获得的阳性苗转移至含双草醚抗性生根培养基(1/2 MS+蔗糖20g/L+多效唑0.5-1mg/L+Phytagel 3g/L+双草醚0.15-0.5μm/L)上，生根7-15d最后获得阳性转基因植株；
炼苗及移栽：将根系生长旺盛的转化株系开启瓶口封口膜，加无菌水覆盖培养基1-2cm厚，置于室温下与空气接触进行炼苗2-3d后，移栽至温室栽培。筛选共获得GAT转化株系共574株，转化材料移栽7-14天后，存活563株。移栽GAT转化株系的同时，移栽一定数量的2叶1心期ZH11作为对照。
实施例3 GAT T0代转化材料分子鉴定
在五叶期取实施例2中获得的转基因植株叶片用CTAB法提取基因组总DNA， 抽提方法如下：
取2-4cm的叶片放入研钵，加入1.5％CTAB溶液800-900ul，进行研磨，然后将研磨后的液体转入1.5ml的离心管中，放在冰上或低温冰箱中待用；样品水浴65℃，30min，期间颠倒混匀数次；在通风橱中，用玻璃移液管加入氯仿与异戊醇溶液(氯仿：异戊醇＝24：1，即500ml氯仿加入异戊醇22ml，轻轻混匀)650ul，混匀后放在摇床上震荡30min或手摇10min左右，此时可看到明显分层；将摇好的样品进行8000-10000rpm离心8min；吸上清400ul左右，转入一新的离心管中，加入-20℃预冷的95％乙醇800ul，轻轻颠倒混匀，放入-20℃冰箱冷冻30min；取出-20℃冷冻的样品，12000rpm离心10min，去掉上清；加入75％的乙醇，静置1min左右，去掉上清，然后风干；加入200-300ul的灭菌水(ddH 2O)，溶解风干的样品DNA，待用。
使用特异引物SEQ ID NO.30-31(图14)和SEQ ID NO.32-33(图15)对pC0308-MMMaauCK5400的T0转基因植株的总DNA进行PCR阳性检测。选取能同时扩增出914bp和831bp条带的植株备用。
取上述通过PCR阳性检测筛选到的pC0308-MMMaauCK540的T0转基因阳性植株的DNA，先用特异引物SEQ ID NO.28和SEQ ID NO.29进行PCR扩增，然后用HaeIII酶切扩增产物，最后用6％的SDS-PAGE凝胶检测。如图16所示，选取只能显示出84bp和66bp条带的植株，用于后续表型鉴定。
特异引物SEQ ID NO.28和SEQ ID NO.29的扩增区域包含cyp704b2-3突变体变异位点，可在聚丙烯酰胺凝胶电泳中观察出cyp704b2-3突变体背景植株存在2碱基缺失突变；引物扩增区域内还包含构建载体时在植物雄性育性恢复基因元件表达盒中CYP704B2的编码区(CDS第660位)引入的一个A→C的SNP。该SNP会引入一个HaeIII的酶切位点，使植物雄性育性恢复基因元件表达盒能被HaeIII酶切，而野生型CYP704B2是不能被HaeIII酶切的。用上述引物对扩增转GAT载体植株的DNA，并用HaeIII酶切后,可能有三种片段：86bp为水稻自身基因组的野生型基因型，84bp为cyp704b2-3突变体基因型，66bp为转化片段的基因型。用上述方法以对鉴定出遗传背景为cyp704b2-3纯合突变体，且含有GAT载体的植株。
实施例4 GAT T0代转化材料表型鉴定
1.除草剂筛选——双草醚抗性表型鉴定
为了检测GAT系统中筛选标记元件的工作效率，用10％双草醚乳液(日本农美利)配制600mg/L双草醚溶液，喷施实施例2中获得的563株GATT0代和野生型对照ZH11 3-5叶期幼苗，喷施后连续观察。喷施后3天，对照ZH11出现叶片枯黄。喷 施后7天，对照ZH11枯黄濒临死亡，GAT转化株系多数植株生长正常，部分出现黄化或生长受到抑制。喷施后14天，对照ZH11已完全死亡，但GAT转化株系呈现正常生长、生长受抑制及濒临死亡或不可逆死亡三种类型。其中正常生长株系为双草醚高抗株系，共有184株，表明在这些株系中筛选标记元件工作效率较高；生长受抑制株系为双草醚中抗株系，共有163株，在这些株系中筛选标记元件工作效率一般；不可逆死亡株系或濒临死亡株系为双草醚无抗或低抗株系，共有216株，在这些株系中筛选标记元件工作效率较差(图17)。
本实验筛选获得双草醚抗性株系，用于后面保持系与不育系的筛选区分，以及保持系的除杂保纯。在这个过程中，可以看到，功能元件整合在一个载体上，功能元件越多，越不容易出现能达到所有功能元件对应表型都得到表现的转化事件，这也印证了本领域目前达成的共识，即：同一载体整合的元件越多，越难达到同时符合的性状，即功能元件越多，转化事件出现预期表型的几率就越低。然而在本发明构建的GAT系统中筛选标记元件工作效率过程中，发明人对每一个表型都进行了筛选，发现GAT转化株系表现为双草醚高抗株系的正常生长株系的几率为32.68％，这个成功率远远高于本领域目前三功能元件集合或四功能元件集合后的转化事件成功率。因为实际转基因事件中，单一性状表型的成功比例是30％-50％，理论而言，对于多功能元件集合的转化事件，所有元件都符合预期的概率应当是在30％的N次方-50％的N次方之间，N为功能元件的个数。
2.除草剂筛选——苯达松敏感表型鉴定
为了检测GAT系统中除草剂敏感元件的工作效率，在双草醚抗性表型鉴定完成后，用48％苯达松母液(常州精度生物科技有限公司)配制3g/L苯达松溶液，喷施上一双草醚喷施实验中存活的GAT T0代(347个株系)和野生型对照ZH11植株叶片划线区域，喷后连续观察。喷施后3d，部分GAT T0转化株系叶尖卷曲枯黄；喷施后7d-14d，，有144个株系叶片出现不可逆枯萎，属于高度敏感株系，表明在这些株系中除草剂敏感元件工作效率较高；有81个株系前期叶片枯萎严重但逐渐恢复生长，属于中度敏感株系，在这些株系中除草剂敏感元件工作效率一般；还有122个株系喷施苯达松前后无明显变化，属于低敏或无表型株系，在这些株系中除草剂敏感元件工作效率较差(图18)。同时具有双草醚高抗和苯达松高敏的株系有86株。基于对上述双草醚抗性株系转化事件的类似分析，可知本实验证实了GAT系统中除草剂敏感元件的工作效率高，创制的GAT育制种系统能够被快速、准确地鉴定其对应的表型。
双草醚和苯达松除草剂喷施实验后观察这些GAT转化株和对照ZH11无明显的形态上的不同，继续生长至开花进行下一步实验。
3.花粉育性鉴定
为了鉴定恢复基因元件和花粉败育基因元件的工作效率，在GAT转化株开花时对花粉进行碘染以检测GAT转化株花粉育性。因GAT载体中含恢复基因元件，若恢复基因元件正常工作，可恢复雄性育性，产生的花粉有两种类型(ms/GAT)和(ms/-)，(ms/GAT)类型花粉由于含有GAT载体中的花粉败育基因元件，若其正常工作则该花粉会败育，因此只有(ms/-)类型花粉存活。因此，若GAT转化株仅含一个拷贝GAT载体，且恢复基因元件和花粉败育基因元件正常工作，则其花粉呈现可育花粉：败育花粉＝1:1分离。因此在碘染时约50％为蓝黑色可育花粉，50％为不染色的不育花粉。碘染镜检具体方法如下：
(1)配制碘化钾染色液(取2g KI溶于5-10mL蒸馏水中，然后加入1g I 2(用适量的无水乙醇溶解)，待全部溶解后，再加蒸馏水定容至300mL。贮于棕色瓶中备用，使用时按碘化钾：去离子水＝1:1的比例稀释成碘染工作液)。
(2)花粉采集：取充分成熟将要散粉的花药，剥除颖壳，取出花药，置于载玻片上。
(3)镜检：取约70μl的碘染工作液滴于花药上，用镊子将花药充分捣碎，使花粉粒释放，盖上盖玻片，于低倍显微镜下观察。凡被染成蓝黑色的花粉粒为可育花粉粒，呈浅黄色的为败育的花粉粒。
碘染镜检结果表明：中花11背景的cyp704b2-3突变体没有花粉(如图19的A)；对照ZH11，绝大部分花粉可染成蓝黑色，是雄性可育花粉(图19的B)，共有102个株系的GAT转化株约50％花粉可染成蓝黑色，表现为正常育性；约50％花粉不能染成蓝黑色，表现为败育花粉(如图19的C中GAT所示)，即可育花粉：败育花粉符合1:1的分离比例，表明在这些株系中恢复基因元件和花粉败育基因元件呈单拷贝存在且其工作效率较高，这是因为若GAT转化株仅含一个拷贝GAT载体，且恢复基因元件和花粉败育基因元件正常工作，则其花粉呈现可育花粉：败育花粉＝1:1分离，这是遗传学定律中的基因分离和自由组合规律。因此如果出现1:1分离的表型，则表明上述元件工作效率正常，符合预期。其余株系可育花粉：败育花粉不符合1:1的分离比，出现偏分离，表明在这些株系中恢复基因元件和花粉败育基因元件的工作效率较差或基因组中含多拷贝。
4.种子荧光鉴定
除草剂表型和花粉染色表型均优良的株系共有26株，见表1，上述株系自交结实收获T0代的种子(T1代)。按花粉育性鉴定结果，若GAT转化株仅含一个拷贝GAT载体，且恢复基因元件和花粉败育基因元件正常工作，其自交结实的种子也将呈现1:1分离，即其中50％为包含GAT载体的种子(基因型为ms ms/GAT-)在560-595nm激发光下观察呈现深红色荧光；50％为不包含GAT载体的种子(基因型为ms ms)在560-595nm激发光下观察无荧光。结果表明：对照ZH11种子无荧光(图20中WT)。而GAT转化株的部分种子呈现深红色荧光(图20中的GAT)。卡方分析表明，其中有18株GAT转化株的T0代种子符合荧光种子：无荧光种子＝1:1分离(表1所示)，表明在这些株系中种子筛选元件呈单拷贝存在且其工作效率较高。1株分离比例接近1:1，可能由于T0代结实率略低原因导致，故也作为优良转化株系。另有7株不符合1:1分离，呈现偏分离，且荧光较弱，表明在这些株系中种子筛选元件工作效率较差或基因组中含多拷贝。
表1 GAT转化株的T0代种子荧光汇总
Figure PCTCN2021092017-appb-000001
Figure PCTCN2021092017-appb-000002
5.育性鉴定及隐性核雄性不育系与保持系的筛选分离
将步骤4中获得的优良株系的两种种子(荧光种子和非荧光种子)，分别发芽、移栽至成苗时期观测其育性，结果见图21。来自荧光种子的植株可自交结实，收获种子(图21的A和C)；来自非荧光种子的植株表现为不育，无法自交结实(图21的B和D)。表明通过GAT系统成功实现了隐性核雄性不育材料(本实施例中含cyp704b2的隐性核雄性不育突变体)的繁殖以及隐性核雄性不育材料不育性的保持。
实施例5 GAT T1代转化材料表型鉴定
为鉴定GAT系统在不同代际间稳定性及工作效率，分别对T0代获得的18个优良株系中种子量较多的14个株系进行下列实验：
1.除草剂筛选——双草醚组培筛选验证
为鉴定筛选标记元件在GAT T1代的工作效率，利用组培发芽筛选重点株及候选株T1代。若GAT载体在基因组中呈单拷贝形式存在，且花粉败育基因元件正常工作时，该株系T1代将呈现1:1分离，即50％含GAT载体，具备对双草醚抗性，在双草醚筛选压力下可正常发芽；50％不含GAT载体，不具备对双草醚抗性，在双草醚筛选压力下无法发芽。因此，配制1/2MS培养基+3μm双草醚筛选GAT重点株和候选株，发芽结果见表2。其中有10个株系符合发芽比例1:1；1个株系发芽比例接近1:1，表明这11个株系中筛选标记元件工作效率正常且在代际间遗传较稳定，也说明通过筛选标记元件可有效区分GAT转化株自交后代分离出的两种不同类型种子或幼苗(即GAT不育系和GAT保持系)；其余株系发芽比例不符合，表明可能存在元件工作效率异常或代际间不稳定遗传现象。
表2 GAT重点株T0代种子BS培养基筛选结果汇总
Figure PCTCN2021092017-appb-000003
2.除草剂筛选——苯达松表型鉴定
为鉴定除草剂敏感元件在T1代GAT转化株的工作效率，用48％苯达松母液(常州精度生物科技有限公司)配制3g/L苯达松溶液，喷施实施例5-1中双草醚筛选出的阳性株系中的部分植株，喷后连续观察。其中呈双草醚抗性株系中有6个株系整株喷施14d后均枯萎死亡，呈高度敏感；有5个株系有个别单株敏感度略差，但经过较长时间后最终植株也死亡；有2个株系中半数以上单株敏感度较差，出现分离，表明可能存在元件工作效率异常或代际间不稳定遗传现象，具体结果见表3和图22。
表3 GAT重点株T1代幼苗苯达松筛选结果汇总
Figure PCTCN2021092017-appb-000004
Figure PCTCN2021092017-appb-000005
ZH11为抗性对照，CK+(P450i2-30)为敏感阳性对照
3.花粉育性鉴定
为了鉴定花粉败育基因元件在T1代GAT转化株的工作效率，在水稻开花时对实施例5-1和5-2中检测效率良好的株系另外半数植株的花粉进行碘染以检测GAT转化株花粉育性。和实施例4中T0代花粉育性相同，若T1代中GAT载体在基因组中呈单拷贝形式存在，且花粉败育基因元件正常工作，可育花粉与败育花粉呈1：1分离。碘染镜检具体方法参照实例4-3，结果显示，对照ZH11绝大多数花粉可染成蓝黑色，是完全可育的(如图23的B中所示)；突变体cyp704b2无花粉(如图23的A中所示)，而T1代大部分GAT转化株系花粉出现半数染色，即可育花粉：败育花粉符合1:1的分离比例(如图23中C图所示)。其中10个T1代GAT转化株育性保持和T0完全一致(表4)，全部未发生分离，表明花粉败育基因元件工作效率正常且在代际间稳定遗传；另外4个株系中有单株出现花粉不符合1:1分离，表明可能存在元件工作效率异常或代际间不稳定遗传现象。
表4 GAT转化株的T1代植株花粉育性鉴定
Figure PCTCN2021092017-appb-000006
Figure PCTCN2021092017-appb-000007
4.种子荧光鉴定
进一步检测上述株系自交收获种子的荧光情况，以检测种子筛选元件在T1代GAT转化株中的工作效率。结果表明，全部株系T1代种子中部分种子的种皮呈现较强的深红色荧光(图24中的GAT)。卡方分析结果显示其中有6个株系的T1代种子符合预期分离比1:1(表5所示)；有2个株系种子荧光分离比接近1:1。这表明在这些株系中花粉败育基因元件和种子荧光元件工作效率正常且能稳定遗传，也说明通过种子筛选元件可有效区分GAT转化株系自交种子后代中分离的两种类型种子(即GAT不育系和GAT保持系)；另有5个株系种子荧光分离比不符合1:1，表明可能存在种子元件或花粉败育基因元件工作效率异常或代际间不稳定遗传现象。
表5 GAT转化株的T1代种子荧光鉴定
Figure PCTCN2021092017-appb-000008
Figure PCTCN2021092017-appb-000009
综合上述结果，在GAT转化株T1代中各元件均正常工作且GAT载体呈单拷贝存在的株系有11-1、23-2、53-3、77-2、88-4、93-2、23等，95-2、4、175-4等作为候选，上述株系可满足GAT保持系各项要求，可作为优良初始GAT保持系进行品种选育与不育系、杂交种种子生产。
5.花粉漂移鉴定
GAT优良初始保持系中花粉和种子符合1:1分离比，初步表明GAT中花粉败育元件工作效率正常，为进一步检测其花粉是否是否外逸，本实例通过GAT优良初始保持系株系授粉给普通不育系水稻材料，检测所得杂交种子是否具备双草醚抗性(方法同实施例5)，若是，则含GAT的花粉有逃逸；若无，则表明GAT中的花粉败育元件工作效率良好，可有效防止含GAT的转化花粉逃逸。挑选GAT株系(23-2、88-4)作为父本授粉给母本不育系1907，分别获得221、373粒杂交种子。用双草醚筛选杂交种子，21d后观察发现，在不添加筛选压的培养基下(1/2MS)，杂交种子发芽正常，而在添加筛选压的培养基下(1/2MS+3uM BS)，杂交种子均与非转基因ZH11、9311、MH63一致，不能发芽(见图25)，发芽率为0％(如表6)，说明杂交种子中不含GAT元件，含GAT的花粉未能授粉给普通材料，再次证明GAT中的花粉败育元件工作正常，且能稳定遗传。
表6 GAT花粉逃逸率检测
Figure PCTCN2021092017-appb-000010
Figure PCTCN2021092017-appb-000011
6.育性鉴定及隐性核雄性不育系与保持系的筛选分离
将步骤4中获得的优良株系的两种种子(荧光种子和非荧光种子)，分别发芽、移栽至成苗时期观测其育性，如图26所示。来自荧光种子的植株可自交结实，收获种子(图26的A和C)；来自非荧光种子的植株表现为不育，无法自交结实(图26的B和D)。表明通过GAT系统成功实现了隐性核雄性不育材料(本实施例中含cyp704b2的隐性核雄性不育突变体)的繁殖以及隐性核雄性不育材料不育性的保持以及GAT系统的代际稳定遗传。
综上，通过实施例1-5的结果证明，本申请利用GAT系统成功实现了隐性核雄性不育材料的繁殖以及隐性核雄性不育材料不育性的保持。结合种子荧光色选系统及保持系筛选元件及除草剂敏感元件等的功能有机组合进行后续的GAT保持系及不育系除杂保纯，在种子时期、营养生长期及生殖生长期均可实现隐性核雄性不育种子/植株(GAT不育系)和可育种子/植株(GAT保持性)的分离，本申请成功地解决了隐性核雄性不育的规模化繁殖与保持问题，从而可成功实现隐性核雄性不育材料产业化。
实施例6 GAT不育系转育
本实施例是隐性核不育转育，即GAT不育系转育，将H28B中的显性纯合CYP704B2替换为突变型隐性纯合cyp704B2-3，但通过回交转育仍保持其余H28B性状，从而最终将三系保持系H28B转育为GAT不育系，这一过程称为GAT不育系转育。
cyp704b2-3是一种水稻CYP704B2基因突变体，其为水稻CYP704B2基因第794个碱基之后的GGG被1个T替换得到，该突变位点位于第3个外显子(公开于中国专利CN 105002191 B中)。用cyp704B2-3突变体与育性正常的受体，进行杂交、回交和自交，并在此过程中用分子标记进行cyp704b2-3基因和遗传背景选择，最终获得目标受体背景下带有纯合cyp704b2-3基因的隐性核不育系。下面以H28B(H28B是审定的传统三系不育系的品种，属三系保持系，其为CYP704B2位点为显性纯合，且不包含GAT载体元件)为例，具体转育实施步骤如下：
1、以受体亲本，如H28B，为父本与含cyp704b2-3的纯合突变体杂交获得F 1。
2、以F 1为母本与受体亲本，如H28B，回交获得BC 1F 1。
3、种植BC1F1，使用序列如SEQ ID NO.28-29的引物检测cyp704b2-3基因型。选择cyp704b2-3杂合基因型，即同时能扩增出86bp和84bp条带的植株。
4、使用一组基因型(例如100个，或200个等)在cyp704b2-3突变体和轮回亲本基因组之间存在多态性，且分布均匀的分子标记(可以是但不限于SSR、SNP、INDEL、EST、RFLP、AFLP、RAPD、SCAR等类型标记)，对步骤3中选出的单株进行遗传背景鉴定，选取与轮回亲本基因型相似度高(如大于88％相似度，或2％中选率等)的植株。
5、用步骤4中选出的植株与受体亲本，如H28B，回交获得BC 2F 1。
6、种植BC 2F 1，重复步骤3和步骤4，选出cyp704b2-3基因型杂合，遗传背景回复率高(如大于98％，或2％中选率等)的植株，收自交种BC 2F 2。
7、种植BC 2F 2，重复步骤3和步骤4，选出cyp704b2-3基因型杂合，遗传背景纯合率最高的植株，收自交种BC 2F 3。BC 2F 3后代中分离的cyp704b2-3纯合基因型植株即cyp704b2-3隐性核不育系，BC 2F 3用于保存cyp704b2-3隐性核不育系种质资源。用字母G来命名隐性核不育系，例如本实施例中H28B的cyp704b2-3纯合隐性核不育系被命名为H28G。
以上仅以H28B作为转育实施例，但并不限于H28B，可以为任何水稻材料。
实施例7 GAT保持系转育
本实施例体现获得GAT保持系，即获得cyp704B2-3且包含GAT载体元件，但其他性状保持原有供体性状，比如利用H28B(H28B本身为CYP704B2显性纯合基因型且不包含GAT载体元件)，通过与H28B的不断回交转育及分子标记辅助选择，获得其他性状与H28B相同，但cyp704B2-3隐性纯合且包含GAT载体元件的GAT保持性，命名为H28T，这一过程称为GAT保持系转育。
在实施例4中获得的GAT大载体的转基因植株中选择CYP704B2位点纯合突变，GAT转基因PCR阳性检测为阳性，每个GAT元件控制的表型都出现的植株作为GAT保持系转育的供体亲本。选择实施例6中获得的cyp704b2-3杂合型植株，例如H28G的杂合植株为受体亲本。将供体植株和受体植株进行杂交、回交和自交，并在此过程中用分子标记对cyp704b2-3基因，GAT元件和遗传背景进行选择，最终获得H28B背景下带有纯合cyp704b2-3基因和GAT元件的GAT保持系。具体实施步骤如下：
1、用供体亲本与cyp704b2-3杂合基因型受体亲本，如cyp704b2-3杂合型的H28G杂交获得F 1。
2、用F 1与cyp704b2-3杂合型受体亲本，如cyp704b2-3杂合型H28G回交，回交获得BC 1F 1。
3、种植BC1F1，使用序列如SEQ ID NO.28-29的引物对BC1F1植株的基因组DNA进行PCR扩增，然后用HaeIII酶切扩增产物，根据酶切产物的条带判断植株的基因型。选择带有84bp和66bp条带的植株，即带有cyp704b2-3基因和GAT转基因的植株。
4、使用一组基因型(例如100个，或200个等)在cyp704b2-3突变体和轮回亲本基因组之间存在多态性，且分布均匀的分子标记(可以是但不限于SSR、SNP、INDEL、EST、RFLP、AFLP、RAPD、SCAR等类型标记)，对步骤3中选出的单株进行遗传背景鉴定，选取与轮回亲本基因型相似度高(如大于88％相似度，或2％中选率等)的植株。
5、用步骤4中选出的植株与cyp704b2-3杂合型受体亲本，如cyp704b2-3杂合型H28G，回交获得BC 2F 1。
6、种植BC 2F 1，重复步骤3和步骤4，选出遗传背景回复率高(如大于98％，或2％中选率等)的植株，收自交种BC 2F 2。
7、种植BC 2F 2，重复步骤3和步骤4，选出遗传背景纯合率最高的植株，收自交种BC 2F 3，即GAT保持系。用字母T来命名GAT保持系，例如本实施例中H28G的GAT保持系被命名为H28T。
以上仅以H28B作为转育实施例，但并不限于H28B，可以为任何水稻材料。
实施例8 GAT保持系生产
GAT保持系种子种植于法定转基因区域，自交结实收获种子，通过荧光种子分选机，筛选获得带深红色荧光的种子，即为GAT保持系种子，后续可用于GAT保持系自我繁殖或授粉给GAT不育系进行不育系制种。
实施例9 GAT保持系除杂保纯
GAT保持系播种，在苗期喷施30-90mg/m2的双草醚或50-100mg/L的灭草烟或185-750mg/L咪唑乙烟酸进行除杂保纯；在分蘖期至孕穗期喷施60-120mg/m2的双草醚或100-200mg/L的灭草烟或500-1500mg/L咪唑乙烟酸进行除杂保纯；在开花期喷施120-300mg/m2的双草醚或200-750mg/L的灭草烟或1000-3000mg/L咪唑乙烟酸进行除杂保纯；
喷施上述除草剂，不但可以杀死非GAT保持系材料，还有助于田间除草，对保持系生产及除杂保纯有重要作用，一举两得。
实施例10 GAT不育系生产(1)
GAT保持系种子种植于法定转基因区域，自交结实收获种子，通过荧光种子分选机，筛选获得无荧光的种子，即为GAT不育系种子，后续可作为母本用于与其他品种(父本)制种，也可用于再繁殖GAT不育系。
实施例11 GAT不育系生产(2)
从实施例10中获得的GAT不育系种子，与GAT保持系种子按混播或间播至法定转基因区域，待至开花期，GAT保持系会散粉给GAT不育系结实，散粉后喷施1-3g/m 2的苯达松或500-3000mg/L苄嘧磺隆杀死GAT保持系，收获所有种子，并利用荧光种子分选机，筛选获得无荧光的种子，即为GAT不育系种子，后续可用于与其他品种作为父本制种，也可用于繁殖不育系。
实施例12 GAT不育系除杂保纯
GAT不育系播种，在苗期喷施0.1-1g/m 2的苯达松或100-800mg/L苄嘧磺隆进行除杂保纯；在分蘖期至孕穗期喷施0.5-1.5g/m 2的苯达松或500-2000mg/L苄嘧磺隆进行除杂保纯；在开花期喷施1-3g/m 2的苯达松或500-3000mg/L苄嘧磺隆进行除杂保纯；
喷施上述除草剂，不但可以杀死GAT保持系材料，保持GAT不育系的纯度，保持GAT不育系的纯度，还有助于田间除草，一举两得。
实施例13 GAT杂交种生产
将实施例10、11中获得的GAT不育系，与父本杂交制种，授粉后收获不育系上结的种子，均为非转基因杂交种子。
工业实用性
本发明提供一种用于农作物杂交育种制种的遗传智能化育制种系统及其应用。本发明的系统含有GAT系统载体，该载体包括五种功能元件表达盒：植物雄性育性恢复基因元件表达盒，用于恢复隐性核不育突变体的雄性育性；植物花粉败育基因元件表达盒，用于清除含GAT的花粉及保持GAT保持系的杂合状态或半合子状态；化学除草剂正向选择表达盒，用于基因转化及GAT保持系除杂提纯；化学除草剂负向选择表达盒，用于清除除草剂敏感的GAT保持系花粉及种子外逸及GAT不育系除杂提纯；种子筛选元件表达盒，用于种子机械分选。本发明可用于植物隐性核雄性不育材料的杂交育种和杂交制种，从而获得优质高产广适高抗的植物新品种及其种子，具有较好的经济价值和应用前景。

Claims (25)

  1. 一种用于农作物杂交育种制种的遗传智能化育制种系统，称为GAT系统，其特征在于，含有植物隐性核雄性不育系，即GAT不育系，隐性核雄性不育保持系，即GAT保持系，和普通恢复系三个系别；
    其中的GAT保持系中含有GAT载体，该载体包括五种功能元件表达盒：(1)植物雄性育性恢复基因元件表达盒，用于恢复隐性核不育突变体的雄性育性；(2)植物花粉败育基因元件表达盒，用于清除含GAT的花粉及保持GAT保持系的杂合状态或半合子状态；(3)基因转化及保持系筛选元件表达盒，用于基因转化及GAT保持系除杂提纯；(4)除草剂敏感元件表达盒，用于清除除草剂敏感的GAT保持系花粉及种子外逸以及GAT不育系除杂提纯；(5)种子筛选元件表达盒，用于种子机械分选；所述五种功能元件表达盒构建于终载体上，得到GAT系统载体。
  2. 根据权利要求1所述的遗传智能化育制种系统，其特征在于，将GAT载体导入GAT不育系创制GAT保持系，且GAT载体以单拷贝形式存在于GAT保持系基因组中；
    所述GAT不育系为单隐性细胞核基因控制的不育系，当基因位点为隐性纯合状态时表现为雄性不育；杂合状态及显性纯合状态表现为雄性可育；
    GAT保持系自交结实，所获种子呈1:1分离得到GAT保持系和GAT不育系；两种种子通过种子筛选元件实现分离，实现GAT保持系的自我繁殖；GAT保持系向GAT不育系授粉使得GAT不育系结实并保持其后代的雄性不育性，从而实现隐性雄性核不育系的繁殖。
  3. 一种农作物遗传智能化育制种载体，称为GAT载体，该载体由五种功能元件表达盒通过接头序列连接构建于终载体上得到，所述物种功能元件表达盒分别为：
    (1)植物雄性育性恢复基因元件表达盒，用于恢复隐性核不育突变体的雄性育性；
    (2)植物花粉败育基因元件表达盒，用于清除含GAT的花粉及保持GAT保持系的杂合状态或半合子状态；
    (3)基因转化及保持系筛选元件表达盒，用于基因转化及GAT保持系除杂提纯；
    (4)除草剂敏感元件表达盒，用于清除除草剂敏感的GAT保持系花粉及种子外逸及GAT不育系除杂提纯；
    (5)种子筛选元件表达盒，用于种子机械分选；所述五种功能元件表达盒通过接头序列连接构建得到GAT系统载体。
  4. 根据权利要求3所述的载体，其特征在于，所述(1)植物雄性育性恢复基因元件表达盒由启动子、雄性育性恢复基因编码区及终止子依序可正常工作的相连；
    所述雄性育性恢复基因为MS1、MS2、MS3、MS5、MS7、MS8、MS9、MS10、MS11、MS12、MS13、MS14、MS17、MS20、MS22、MS23、MS24、MS25、OsCYP704B2、MS27、MS28、MS29、MS30、MS31、MS32、MS33、MS34、MS36、MS37、MS38、MS43、MS45、MS48、MS50、或OsCYP704B2基因的野生型基因，启动子和终止子分别为其各自基因的启动子和终止子，优选OsCYP704B2基因；优选所述(1)植物雄性育性恢复基因元件表达盒的序列如SEQ ID NO.6所示。
  5. 根据权利要求3所述的载体，其特征在于，所述(2)植物花粉败育基因元件表达盒由植物花粉特异启动子、信号肽及败育基因编码区及终止子依序可正常工作的相连；优选启动子为玉米PG47启动子、水稻PCHF15、OsPC32启动子，优选败育基因为水稻α-淀粉酶基因OsAA、玉米α-淀粉酶基因Zm-AA1、大麦α-淀粉酶基因HvAA1、小米α-淀粉酶基因SiAA、细胞分裂素氧化酶，半胱氨酸蛋白酶，赤霉素氧化酶，终止子为玉米IN2-1或NosT终止子；优选所述(2)植物花粉败育基因元件表达盒的序列如SEQ ID NO.7所示，或如SEQ ID NO.8所示，或如SEQ ID NO.9所示。
  6. 根据权利要求3所述的载体，其特征在于，所述(3)基因转化及保持系筛选元件表达盒由启动子、筛选标记基因编码区及终止子依序可正常工作的相连；优选启动子为Actin启动子或OsALS基因起始密码子ATG上游2180bp序列的任一个，优选筛选标记基因编码区为OsALSm1、OsALSm2、OsALSm3序列、草甘膦抗性基因EPSPSm序列、草甘膦N-乙酰转移酶序列或草丁膦抗性基因Bar序列中的任一个；终止子为OsUbiT终止子、或NosT终止子；优选所述基因转化及保持系筛选元件表达盒的序列如SEQ ID NO.10所示；或如SEQ ID NO.11所示，或如SEQ ID NO.12所示，或如SEQ ID NO.13所示。
  7. 根据权利要求3-6任一所述的载体，其特征在于，所述(4)除草剂敏感元件表达盒由启动子、除草剂显性敏感元件及终止子依序可正常工作的相连，优选启动子为ZmUbi启动子，除草剂显性敏感元件为细胞色素P450基因CYP81A6的RNAi结构序列P450i，终止子为PinII终止子、NosT终止子；
    优选所述(4)除草剂敏感元件表达盒为P450i-1、P450i-2或P450i-3，其序列分别如SEQ ID NO.14，SEQ ID NO.15，SEQ ID NO.16所示。
  8. 根据权利要求3-6任一所述的载体，其特征在于，所述(5)种子筛选元件表达盒由启动子、种皮显色基因及终止子依序可正常工作的相连，优选地，启动子为种 子特异性启动子ZZ1启动子，种皮显色基因为深红色荧光蛋白FP635，红色荧光蛋白RFP或绿色荧光蛋白GFP，终止子为OS-T28终止子、NosT终止子；优选所述种子筛选元件表达盒序列如SEQ ID NO.17所示。
  9. 根据权利要求3-6任一所述的载体，其特征在于，所述接头序列包括：多克隆位点MCSI，序列如SEQ ID NO.18所示；多克隆位点MCSII，序列如SEQ ID NO.19所示；多克隆位点MCSIII，序列如SEQ ID NO.20所示；多克隆位点MCSIV，序列如SEQ ID NO.21所示；或多克隆位点MCSV，序列如SEQ ID NO.22所示。
  10. 根据权利要求3-9任一所述的载体，其特征在于，所述终载体为pC0307，其序列如SEQ ID NO.25所示，或所述终载体为pC0308，其序列如SEQ ID NO.26所示，或所述终载体为pC0309，其序列如SEQ ID NO.27所示。
  11. 根据权利要求3-10任一所述的载体，其特征在于，所述GAT载体为pC0308-MMMaauCK5400、pC0308-KhvMMaauMCK5400、pC0308-KhvMaauMCMK5400、pC0309-KhvMaauMCMK5400和pC0307-KhvMaauMCMK5400；其核苷酸序列分别如SEQ ID NO.1-5所示。
  12. 权利要求3-11任一所述载体的构建方法，其特征在于，将五种功能元件表达盒通过接头序列连接构建于终载体中得到，所述五种功能元件表达盒分别为：
    (1)植物雄性育性恢复基因元件表达盒，用于恢复隐性核不育突变体的雄性育性；该表达盒由启动子、雄性育性恢复基因编码区及终止子依序可正常工作的相连；
    (2)植物花粉败育基因元件表达盒，用于清除含GAT的花粉及保持GAT保持系的杂合状态或半合子状态；该表达盒由植物花粉特异启动子、败育基因编码区及终止子依序可正常工作的相连；
    (3)基因转化及保持系筛选元件表达盒，用于基因转化及GAT保持系除杂提纯；该表达盒由启动子、筛选标记基因编码区及终止子依序可正常工作的相连；
    (4)除草剂敏感元件表达盒，用于清除除草剂敏感的GAT保持系花粉及种子外逸及GAT不育系除杂提纯；该表达盒由启动子、除草剂显性敏感元件及终止子依序可正常工作的相连；
    (5)种子筛选元件表达盒，用于种子机械分选；所述五种功能元件表达盒通过接头序列连接构建得到GAT系统载体；该表达盒由启动子、种皮显色基因及终止子依序可正常工作的相连。
  13. 权利要求1-2任一所述的遗传智能化育制种系统或权利要求3-11任一所述的载体的以下任一中应用：
    (1)在植物杂交育种及制种中的应用；
    (2)在生产非转基因杂交种中的应用；
    (3)在植物隐性核雄性不育系的规模化保持及规模化繁殖中的应用。
  14. 一种用于保持植物隐性核雄性不育株的基因型状态的方法，其特征在于：采用权利要求1-2任一所述的遗传智能化育制种系统，向基因型为隐性纯合的GAT不育系中导入GAT载体，创制只含单拷贝GAT载体的GAT转化株或GAT保持系，该GAT转化株或GAT保持系的基因型为隐性纯合/GAT-，使GAT转化株或GAT保持系授粉给GAT不育系，获得的种子基因型仍为隐性纯合状态，从而保持GAT不育系后代的雄性不育性，并成功繁殖GAT不育系。
  15. 一种用于保持GAT转化株或保持系中GAT座位保持杂合状态或半合子状态的方法，采用权利要求1-2任一所述的遗传智能化育制种系统，向基因型为隐性纯合的GAT不育系中导入GAT载体，创制只含单拷贝GAT载体的GAT转化株或GAT保持系，该GAT转化株或GAT保持系的基因型为隐性纯合/GAT-；GAT转化株或GAT保持系自交繁殖，产生两种基因型种子，一种为隐性纯合/GAT-，为GAT保持系；一种为隐性纯合/--，为GAT不育株；根据遗传定律，二者呈1：1分离，即GAT转化株或GAT保持系自交后代中基因型为隐性纯合/GAT-的GAT座位仍保持杂合状态或半合子状态。
  16. 一种用于筛选或区分GAT转化株自交种子及植株的方法，所述GAT转化株是采用权利要求1-2任一所述的遗传智能化育制种系统，向基因型为隐性纯合的GAT不育系中导入GAT载体，创制只含单拷贝GAT载体的GAT转化株，该GAT转化株的基因型为隐性纯合/GAT-，其特征在于：GAT转化株自交结实的种子呈现1:1分离，其中50％为包含GAT载体的种子，基因型为隐性纯合/GAT-，在激发光下观察呈现荧光；50％为不包含GAT载体的种子，基因型为隐性纯合，不含GAT元件，在激发光下观察无荧光；在种子或植株水平，基因型为隐性纯合/GAT–时具有对针对乙酰乳酸合酶或EPSPS或Bar基因的各类除草剂高抗性，包括但不限于双草醚、咪唑乙烟酸、灭草烟、草甘膦、草丁膦或草铵膦；50％为不包含GAT载体的种子，基因型为隐性纯合，不含GAT元件，不具有此高抗性。
  17. 一种用于防止GAT植物花粉漂移的方法，其特征在于：将权利要求3-11任一所述的载体转入植物中，使得含GAT载体的植物材料在花粉成熟时，因其含有花粉败育基因元件，会特异性导致含GAT载体的花粉败育，而保证不含GAT载体的花粉正常发育散粉，从而降低含GAT载体的花粉外逸的概率。
  18. 一种用于防止GAT种子或植株漂移或混杂的方法，其特征在于：使所述种子或植株中含有将权利要求3-11任一所述的载体，在种子时期通过包衣或在苗期至开花期施用特定浓度的除草剂，包括苯达松或苄嘧磺隆或烟嘧磺隆，可在特定时期杀死含GAT种子或植株的材料，从而防止GAT种子或植株混杂入其他普通材料中。
  19. 一种利用植物隐性核雄性不育系生产不育系种子方法，其特征在于：采用权利要求1-2任一所述的遗传智能化育制种系统，将GAT保持系与GAT不育系按一定比例混合播种，利用GAT保持系授粉给GAT不育系，授粉完成后，施用除草剂，包括苯达松或苄嘧磺隆或烟嘧磺隆，特异性的杀死GAT保持系，仅保留GAT不育系收获种子。
  20. 一种提纯植物隐性核雄性不育系的方法，其特征在于：采用权利要求1-2任一所述的遗传智能化育制种系统，GAT不育系通过种子包衣或在苗期至开花期施用特定浓度的除草剂，包括苯达松或苄嘧磺隆或烟嘧磺隆，可保证GAT不育系纯度。
  21. 一种利用植物隐性核雄性不育系进行杂交制种方法，其特征在于：采用权利要求1-2任一所述的遗传智能化育制种系统，利用GAT保持系自交生产GAT保持系种子和GAT不育系种子；利用GAT保持系授粉给GAT不育系生产GAT不育系种子；利用GAT不育系和常规材料杂交组配生产普通商业化杂交种。
  22. 一种利用植物隐性核雄性不育系进行杂交育种方法，其特征在于：采用权利要求1-2任一所述的遗传智能化育制种系统，利用GAT保持系与普通材料杂交选育，可通过常规回交选育，也可通过系谱法选育GAT保持系及不育系，选育过程辅以GAT各项分子标记、除草剂筛选、种子色选等加速选育。
  23. 一种利用权利要求1-2任一所述的遗传智能化育制种系统制备不含GAT元件的商业化杂交种的方法，其特征在于：利用GAT保持系与常规材料A杂交，获得F1杂种，后续可通过自交获得F2，在F2代中辅以GAT分子标记、除草剂筛选、种子色选等选育包含纯合隐性核不育位点及GAT元件的材料，通过连续自交获得高世代稳定材料，从中获得新的GAT保持系和GAT不育系，新选育的GAT不育系可以和普通恢复系杂交组配选育普通商业化杂交种，该杂交种不含GAT元件，为常规商业化杂交种。
  24. 一种检测或含有GAT载体的转基因阳性植株的方法，其特征在于，用以下任一对引物对待测样品的基因组进行PCR检测，
    检测植物雄性育性恢复基因元件表达盒的引物序列如SEQ ID NO.28-29所示；或
    检测植物花粉败育基因元件表达盒的引物序列如SEQ ID NO.30-31所示；或
    检测基因转化及保持系筛选元件表达盒的分子引物如SEQ ID NO.32-33所示；或
    检测除草剂敏感元件表达盒的引物序列如SEQ ID NO.34-35所示；
    检测种子筛选元件表达盒的分子标记引物序列如序列SEQ ID NO.36-37所示；
    若用SEQ ID NO.28-29作引物扩增，扩增产物用HaeIII酶切后电泳，最终产物中可能出现3条带型：86bp为原基因组中OsCYP704B2基因野生型带型，84bp为oscyp704b2-3突变体带型，66bp为GAT载体带型；如果出现84bp和66bp带型，而没有88bp带型，表明该植株为雄性不育突变体背景，且植物雄性育性恢复基因元件表达盒存在；
    若用SEQ ID NO.30-31引物进行扩增，如果能扩增出914bp条带，表明植物花粉败育基因元件表达盒存在；
    若用SEQ ID NO.32-33作为引物进行扩增，如果能扩增出831bp条带表明基因转化及保持系筛选元件表达盒存在；
    若用SEQ ID NO.34-35作为引物进行扩增，如果能扩增出923bp条带，表明除草剂敏感元件表达盒存在；
    若用SEQ ID NO.36-37作为引物进行扩增，如果能扩增出1412bp条带，表明种子筛选元件表达盒存在。
  25. 一种分选不同功能的植株和后代的方法，其特征在于，将权利要求3-11任一所述的GAT载体转入植株，利用GAT载体中的(3)基因转化及保持系筛选元件表达盒，(4)除草剂敏感元件表达盒，(5)种子筛选元件表达盒，基于化学除草剂正负双向选择与机械色选相结合，分选不同功能的植株和后代；
    所述化学除草剂正负双向选择是同一种植株对一种除草剂表现抗性，对另一种除草剂表现敏感；
    优选地，化学除草剂正向选择除草剂是抗双草醚、咪唑乙烟酸、灭草烟，抗草甘膦、抗草丁膦或草铵膦，化学除草剂负向选择是对苯达松、苄嘧磺隆或烟嘧磺隆敏感；
    机械色选是通过种子的不同荧光进行筛选分离含GAT载体的种子或不含GAT载体的种子。
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