WO2020093997A1 - 一种转基因玉米核雄性不育保持系及应用 - Google Patents

一种转基因玉米核雄性不育保持系及应用 Download PDF

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WO2020093997A1
WO2020093997A1 PCT/CN2019/115675 CN2019115675W WO2020093997A1 WO 2020093997 A1 WO2020093997 A1 WO 2020093997A1 CN 2019115675 W CN2019115675 W CN 2019115675W WO 2020093997 A1 WO2020093997 A1 WO 2020093997A1
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expression
gene
transgenic
dna
corn
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沈志成
张先文
王东芳
赵宇
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Zhejiang University
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
    • A01H1/027—Apparatus for pollination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C12N15/8289—Male sterility

Definitions

  • the invention relates to a transgenic corn nuclear male sterility maintenance line and its application.
  • hybrid advantages is one of the most important technologies for increasing grain production.
  • hybrid seeds are usually prepared by crossing two inbred lines.
  • Hybrid seeds have more advantages in yield and resistance than inbred seeds in production.
  • most of the production of hybrid seeds is still done by manual or mechanical detasseling, which is costly and requires a lot of work.
  • the plants may be tasseled again after detasseling, or the detasseling may be incomplete, which may result in self-pollination of the female parent and affect the purity of hybrid seeds.
  • the male sterility system provides a simple and efficient method for preparing hybrid corn seeds.
  • the more mature hybrid technology system is a three-line hybrid seed production system based on nuclear-plasmic interaction male sterility (CMS) inbred lines.
  • CMS nuclear-plasmic interaction male sterility
  • the technology includes 3 main materials: 1) sterile line: male sterile material; 2) maintainer line: can provide pollen for the sterile line, so that the progeny of the sterile line is still the sterile line; 3) restorer line: can Restore the fertility of the sterile line.
  • Sterile lines cross with restorer lines to produce F1, a hybrid used for agricultural production. More specifically, the nuclear-cytoplasmic interaction sterility type is expressed as nuclear-cytoplasmic interaction inheritance.
  • cytoplasmic sterility gene S not only the cytoplasmic sterility gene S, but also the homozygous sterility gene (rf / rf) in the nucleus is required, and both can coexist to make the plant behave as male sterility.
  • the CMS three-line hybrid seed production system has the drawbacks that the recovery line is difficult to find and the technical system is complicated. The area of promotion in corn seed production is still relatively small.
  • nuclear gene recessive sterility genes is another possible technical route for maize hybrid seed production.
  • the nuclear homozygous (ms / ms) plants that control male sterility will only appear as male sterility. Since male sterile plants cannot self-breed, they can only be crossed with hybrid plants (Ms / ms) Male sterile plants (ms / ms) will be obtained. However, male sterile grains (ms / ms) and fertile hybrid grains (Ms / ms) coexist on the same ear and cannot be distinguished simply by physical methods.
  • the transgenic corn maintainer line is a recessive nuclear sterility homozygous line (such as ms45 / ms45) into which the foreign DNA containing the following expression box is introduced: 1) a normal wild-type male fertility Gene (for example, Ms45) whose function is complementary to lead to male nuclear sterility genes (such as ms45) to save fertility; 2) a marker gene for seed color (fluorescent protein gene); 3) an ⁇ expressed in pollen
  • Ms45 normal wild-type male fertility Gene
  • ms45 male nuclear sterility genes
  • homozygous recessive sterility genes such as ms45 / ms45
  • transgenes such as MS45
  • the foreign gene cannot be homozygous. 50% pollen (non-transgenic) without foreign DNA can successfully pollinate female gametes of homozygous nuclear sterility (such as ms45 / ms45), thereby obtaining male sterile corn seeds. These seeds can be used as the female parent for maize hybrid seed production.
  • the fertile seeds that contain the foreign DNA can be distinguished from the male sterile seeds that do not contain the foreign DNA by using fluorescence characteristics. This technique can simplify the process of corn hybrid seed production and reduce costs.
  • Chinese invention patent CN102960234B discloses another method for preparing non-transgenic nuclear-sterile maize by using transgenic sterile maintainer lines.
  • the characteristic of this method is to use the size of the seed instead of the fluorescence of the seed fluorescent protein to distinguish male sterile seeds from male fertile seeds.
  • the technical scheme is to construct an interference expression frame of an endosperm-specific cell wall invertase (InCW2) gene (Cheng et al. Plant Cell 8: 971-983) that interferes with the specific expression of corn endosperm and silence the gene. After InCW2 is silenced, the corn kernels become smaller. This technique is very simple to distinguish between male sterile seeds and male fertile seeds.
  • InCW2 endosperm-specific cell wall invertase
  • the present invention provides a technical route that uses two selective traits at the same time to obtain male sterile seeds of higher purity through two screenings.
  • the method of the present invention is more capable of improving the purity of the male sterile line corn and preventing the transgenic restoration line from entering the field production system than the existing method.
  • the present invention creatively applies the selective killing technology of transgenic corn to improve the purity of the male sterile line corn.
  • Inhibiting the expression of the herbicide-tolerant gene CYP81A9 by RNA interference in maize can result in the transgenic corn being sensitive to herbicides such as nicosulfuron.
  • These genetically modified corns can be selectively killed by herbicides such as nicosulfuron (Li, Jing, et al., 2013, Plos One 8 (12): e81645).
  • the purpose of the present invention is to provide the construction and application of a transgenic maize nuclear male sterility maintenance line.
  • the characteristics of the seeds of this transgenic maintainer line are the use of two or more markers distinguished from the male sterile line seeds, which can further reduce and prevent the risk of the transgenic maintainer line seeds being mixed into commercial hybrids.
  • the present invention comprehensively utilizes technology including seed fluorescent color labeling, seed shape size labeling technology and herbicide sensitive labeling (transgenic corn can be selectively killed), while the previous technology only uses one label, such as fluorescent protein labeling (Wu et al. ., Plant Biotechnology, 14: 1046-1054; US Patent 7,612,251), or a marker to control the size of seeds (Chinese Patent CN102960234B).
  • the present invention requires the use of male nuclear sterility corn materials.
  • Table 1 exemplifies some of the male nuclear sterility corn materials that have been reported so far. The genes they control have been cloned or can be cloned.
  • Male sterility materials with known sterility genes can also be used to obtain nuclear male sterility corn materials by gene editing methods. For example, ms45 and ms26 male sterile mutants (Svitashev et al., Nature Communications 7, 13274) can be obtained through gene editing.
  • the present invention takes a male sterile mutant ms45 in Table 1 as an example to provide a method for preparing hybrid seeds using recessive nuclear sterile corn. The following description and explanation provide a more detailed description, but this is not intended to limit the scope of the present invention.
  • the invention provides a transgenic maize nuclear male sterility maintenance line, which is constructed by introducing an exogenous T-DNA vector into the maize genome, and the exogenous T-DNA vector except the male sterility
  • the Ms45 expression box of the fertility restoration gene ie, the fertility restoration expression box of the sterile line
  • it also contains at least two of the following three functional fragments: 1) An expression box that expresses the fluorescent protein in the endosperm of the seed (ie, maintains the line seed Compared with the sterile line seeds, it has a significantly different expression box in the color visible to the naked eye); 2) A functional fragment that can inhibit the expression of the corn herbicide resistance gene CYP81A9 (nsf1) (ie, the endogenous herbicide resistance gene of the plant) Silencing or knocking out the expression box), 3) A functional fragment that can inhibit the expression of endosperm-specific cell wall invertase (InCW2) specific expression of maize endosperm (Expression box for significant differences in shape visible to
  • the foreign T-DNA vector includes the following two expression boxes in addition to the fertility recovery gene of male nuclear sterility: 1) an expression box for expressing a fluorescent protein in the seed endosperm; 2) one A functional fragment that can inhibit the expression of maize herbicide resistance gene CYP81A9.
  • the foreign T-DNA vector contains at least the following two expression boxes in addition to the fertility recovery gene of male nuclear sterility: 1) an expression box for expressing a fluorescent protein in seed endosperm; 2) A functional fragment that can inhibit the cell wall invertase (InCW2) specifically expressed in corn endosperm.
  • the foreign T-DNA vector contains at least the following two expression boxes in addition to the fertility recovery gene of male nuclear sterility: 1) a functional fragment that can inhibit the expression of the corn herbicide resistance gene CYP81A9; 2) A functional fragment of cell wall invertase (InCW2) that can inhibit the specific expression of maize endosperm.
  • the expression frame of male nuclear sterility recovery gene of the present invention is composed of promoter, fertility recovery gene and terminator functional connection, and the functional protein expressed in the expression frame supplements the loss of mutation in nuclear sterility maize Male fertility function gene, which can restore the male fertility of nuclear-sterile corn.
  • a gene expression frame capable of restoring the fertility of ms45 nuclear sterile corn (ms45 / ms45), that is, an Ms45 gene expression frame is preferred.
  • the Ms45 gene needs to be functionally linked to a promoter and terminator capable of initiating its proper expression.
  • These promoters and terminators can have multiple choices. The more preferred choice is the natural promoter and terminator of these genes.
  • promoters and terminators that can drive proper expression in plants.
  • the appropriate promoter for expression level and expression space-time can be selected as required.
  • the promoter 5126 disclosed in US Patent 5,689,049 can drive anther-specific expression and is one of the preferred promoters for the Ms45 gene.
  • the expression frame for expressing fluorescent protein in seed endosperm of the present invention is functionally connected by a promoter, a fluorescent protein gene and a terminator.
  • the fluorescent protein genes of this expression box can be red fluorescent protein (red fluorescent protein), cyan fluorescent protein (cyan fluorescent protein), yellow fluorescent protein (yellow fluorescent protein), green fluorescent protein (green fluorescent protein), cyanine Prime p1 gene (anthocyanin p1gene) and so on.
  • the expression of these fluorescent protein genes in the endosperm allows the obtained transgenic seeds to be distinguished from seeds that do not express these genes by simple physical methods.
  • the promoter of this expression box is a promoter capable of driving high expression in maize endosperm.
  • a preferred promoter is a promoter of endosperm storage protein of corn or rice.
  • the GluA-1, GluA-2, GluA-3, GluB-3, GluB-5, and GluC promoters of rice (Quetal., JExp.Bot.59: 2417-2424; Xu et al., Plant Cell, Tissue and Organ Culture, 128: 125–132), the ⁇ -zein promoter of corn (Joshi et al., Physiol Mol Biol Plants 21: 35–42), etc.
  • Some constitutive promoters can also promote higher expression of genes in endosperm.
  • the signal peptides of rice or corn endosperm storage proteins can be used to fuse with these fluorescent proteins, so that these proteins can be properly stored and accumulated after expression.
  • the terminator used to construct the expression cassette may be the 35S terminator of CaMV or other terminators that are functional in maize.
  • the sequence of SEQ ID No. 5 is an expression box capable of expressing red fluorescent protein in endosperm.
  • the functional fragment of the cell wall invertase (InCW2) gene expression that inhibits the specific expression of corn endosperm according to the present invention can be constructed by different principles.
  • a method of interfering InCW2 expression with double-stranded RNA is preferred.
  • the specific construction method of the suppression expression box is as follows: 1) Construct a DNA fragment whose two ends are a DNA fragment from InCW2 and an inverted repeat sequence fragment of this fragment. Add any other DNA fragment between these two DNA fragments, preferably a partial intron sequence of the InCW2 gene. Because there are two fragments of InCW2 gene sequence repeated in reverse in this fragment, the RNA obtained by transcribing this DNA fragment can form a double-stranded RNA (dsRNA) of hairpin structure.
  • dsRNA double-stranded RNA
  • the inverted repeat sequence fragment is functionally linked to a promoter at the 5 'end and functionally linked to a terminator at the 3' end to form a DNA functional fragment.
  • the promoter of this functional fragment may be a promoter capable of starting constitutive expression or a promoter specifically promoting expression in the endosperm.
  • the preferred promoter is the natural promoter of the InCW2 gene; the terminator may be a terminator function in maize. Different types of terminators, including the natural terminator of the InCW2 gene.
  • the DNA sequence used to construct this inhibitory expression fragment is from InCW2 (SEQ ID NO: 8), and its length and position can be different. The inhibitory effect can be determined by experiment, and a construction fragment with a relatively high inhibitory efficiency can be selected. SEQ ID No.
  • Gene silencing technology is a mature technology (Agrawal et al., Microbiol Mol Biol Rev. 67: 657–685).
  • Antisense RNA method this method is to let transgenic plants produce a Antisense RNA of the target gene to suppress the expression of the corresponding gene;
  • Artificial miRNA method this method designs an artificial miRNA that can suppress the expression of the target gene (Schwab et al. Plant 18, 1121–1133).
  • the functional fragment for inhibiting the expression of the corn herbicide resistance gene CYP78A9 according to the present invention can be constructed by different principles. Taking the method of double-stranded RNA interfering with the expression of CYP78A9 as an example, it is achieved by silencing the plant's endogenous herbicide resistance gene, which consists of a "constitutive promoter-hairpin RNA-terminator".
  • the specific construction method of the CYP81A9 gene RNA interference functional fragment is as follows: 1) Construct a DNA fragment whose two ends are a DNA fragment from CYP78A9 and an inverted repeat sequence fragment of this fragment. In between these two DNA fragments is added any other DNA fragment, preferably an intron sequence from the CYP78A9 gene.
  • the RNA obtained by transcribing this DNA fragment can form a double-stranded RNA (dsRNA) with a hairpin structure.
  • the inverted repeat sequence fragment is functionally linked to a promoter at the 5 'end and functionally linked to a terminator at the 3' end to form a DNA functional fragment.
  • the promoter of this functional fragment may be a promoter capable of constitutive expression, such as the 35S promoter of CaMV; the terminator may be a terminator commonly used in maize transgenic research, such as the 35S terminator of CaMV.
  • the CYP78A9 gene sequence used to construct this expression-suppressing fragment was derived from SEQ ID NO: 9. The length and sequence of the dsRNA obtained by transcription of this fragment can be different, and its inhibitory effect can be measured through experiments, and a construction fragment with a relatively high inhibition efficiency can be selected.
  • the method of inhibiting the expression of corn herbicide resistance gene CYP78A91 can also be: 1) antisense RNA method, this method is to let transgenic plants produce antisense RNA of target gene CYP78A9 to inhibit the expression of CYP78A9 gene; 2) artificial miRNA method (Schwab et al .Plant Cell 18,1121–1133). Using this method, an artificial miRNA functional fragment capable of inhibiting the expression of the target gene CYP78A9 can be designed.
  • transgenic corn can cause the transgenic corn to be sensitive to the herbicides nicosulfuron, bendazon and other herbicides, so that these transgenic corns can be selectively killed by the herbicides.
  • the exogenous T-DNA plasmid of the transgenic maize nuclear male sterility maintenance line may also include the following expression boxes:
  • the expression cassette of ⁇ -amylase expressed in pollen ie, the expression cassette that causes sterility of the pollen containing the above expression cassette. This is achieved by introducing a gene that inactivates pollen and consists of a "promoter- ⁇ -amylase-terminator".
  • the preferred expression box nucleotide sequence is SEQ ID. NO: 4 (wherein the 13bp-2053bp is pPg47 start 2054bp-2278bp is the signal peptide sequence of the amyloid of the BRITTLE1 (BT1) gene; 2279bp-3838bp is the coding region of the amylase gene; 3839bp-4039bp is the terminator).
  • the promoter of this expression box may be the PG47 and ZM13 promoters (Hamilton et al., Plant Mol. Biol. 38: 663-669).
  • the ⁇ -amylase of this expression frame may be the ⁇ -amylase of Bacillus stearothermophilus (gb: A24549), or the maize amylase gene (gb: ADH43281.1), or it may be an ⁇ -amylase from other sources.
  • the signal peptide of ⁇ -amylase was excised and then replaced with the signal peptide (Sullivan et al, Plant 3: 3: 1337–1348) of the amyloid from the BRITTLE1 (BT1) gene.
  • the products expressed in this expression box can cause corn pollen to lack starch and fail to germinate, which in turn leads to male sterility. In this way, it can be achieved that the foreign DNA of the transgenic corn remains in a heterozygous state, rather than a homozygous state.
  • the marker expression box (ie the herbicide resistance gene expression box). Conferring resistance to one or more herbicides in the maintainer line is achieved by introducing a herbicide tolerance gene, which consists of a "promoter-herbicide resistance gene-terminator".
  • the herbicide resistance gene expression box is the glufosinate tolerance gene bar expression box, and the sequence is shown in SEQ ID No. 2 (where 1bp-2002bp is the pUbi promoter; 2052bp-2600bp is the bar gene coding region; 2610bp- 2947bp is rbcs terminator).
  • the glyphosate-resistant gene expression cassette or the glufosinate-resistant expression cassette is a commonly used selection marker expression cassette.
  • the promoter used is preferably a constitutive promoter, such as the cauliflower mosaic virus (CaMV) 35S promoter p35S (Odell, Joan T., Ferenc Nagy, and Nam-Hai Chua, 1985, Nature, 810-812), Actin1 of rice Promoter pAct1 (McElroy D, Zhang W, Cao J, et al., 1990, The Plant Plant Cell, 2: 163-171.), Maize Ubiquitin promoter pUbi (Christensen A H, Sharrock R R, Quail P H, 1992 , Plantmolecularbiology, 18: 675-689) and its promoters with similar expression patterns; the herbicide resistance gene may be the glyphosate resistance gene CP4-EPSPS (Mons
  • the exogenous T-DNA vector of the present invention includes the following expression boxes: 1) Ms45 expression box; 2) red fluorescent protein expression box; 3) CYP78A9 gene expression-inhibiting functional fragment; 4) ⁇ -amylase expressed in pollen The expression box; 5) Select the marker expression box.
  • the other T-DNA may be an expression box containing the following expression boxes: 1) Ms45 expression box; 2) InCW2 suppression expression functional fragment; 3) CYP78A9 gene suppression expression functional fragment; 4) ⁇ -amylase expression box expressed in pollen; 5) Select the marker expression box.
  • T-DNA may include the following expression boxes: 1) Ms45 expression box; 2) red fluorescent protein expression box; 3) InCW2 inhibitory expression functional fragment; 4) expression box of ⁇ -amylase expressed in pollen; 5 ) Glufosinate resistance gene expression box.
  • T-DNAs containing these expression boxes and functional fragments that can achieve the purpose of the present invention can also be constructed by general molecular biology methods. Moreover, the difference in the direction and order of these expression boxes and functional fragments does not affect the realization of the purpose of the present invention.
  • the invention also provides a plant cell containing the T-DNA vector in the transgenic maize nuclear male sterility maintenance line.
  • the DNA construct containing the above several expression cassettes is introduced into maize by Agrobacterium-mediated methods or other methods.
  • the genetic transformation of maize is a mature technology, which can be realized by ordinary technicians in this field.
  • Related transformation methods have been published publicly for many times, such as Yadava et al., Front. Plant Sci. 7: 1949.
  • the transformants obtained by transformation need to be further selected to obtain the transformants required to achieve the object of the present invention.
  • the corn transformant into which the foreign gene provided by the present invention has been introduced can integrate the foreign gene into the nuclear male sterility corn genome by backcrossing.
  • the male sterile heterozygous genotype Ms45 / ms45 plants were used for multiple backcrosses with the selected transformant plants, thereby introducing the exogenous MS45 restorer gene into the male homozygous sterile mutant ms45 to obtain the genotype Ms45 /- , ms45 / ms45 plants. Due to the presence of the wild-type MS45 gene in the introduced foreign gene, the transgenic plants (Ms45 /-, ms45 / ms45) appeared fertile.
  • transgenic heterozygous plants (genotype Ms45 /-, ms45 / ms45) are selfed, two progeny will be produced, one is male sterile grain (ms45 / ms45) which does not contain exogenous transgenic sequence, the sterile grain Can be restored to fertility by any wild type plant (Ms45 / Ms45), can be used as a sterile line (mother) during seed production; the other is to restore the male fertility of grain (Ms45 /-, ms45 / ms45 ), Can be used as a restorer line to prepare the next season sterile line seeds.
  • the main technical purpose of the present invention is to ensure that male-fertile corn (ms45 / ms45) is not mixed with transgenic corn (Ms45 /-, ms45 / ms45) containing male genes whose foreign fertility is restored.
  • the key technical measure of the present invention is to adopt at least two methods for removing transgenic corn, which greatly reduces the possibility of transgenic corn (Ms45 /-, ms45 / ms45) in which male fertility is restored in male-sterile corn.
  • the invention provides an application of the transgenic maize nuclear male sterility maintenance line in preparing non-transgenic nuclear sterility seeds.
  • the application method is: the transgenic corn nuclear male sterility maintenance line and homozygous recessive sterility Lines are planted at intervals, and non-transgenic nuclear sterile seeds are selected by one of the following methods:
  • transgenic male fertile grains can be removed by fluorescent recognition, and the male sterile seeds obtained are further smaller than the grains. (Eg, at least 30% less than the average weight) seeds. Potentially mixed transgenic seeds (male fertile) can be further removed. In this way, through the selection of two traits, male fertile corn plants are further excluded, and the purity of hybrid corn is improved.
  • the present invention uses at least two of the above three methods to distinguish between transgenic and non-transgenic methods, to further reduce the possibility of relying on only one method of discrimination (or selection) error.
  • FIG. 1 Schematic diagram of three T-DNA structures (TWS-HC, TWS-HS and TWS-CS) containing male fertility recovery gene Ms45 and grain selection gene.
  • LB T-DNA left border
  • RB T-DNA right border
  • pPg47-Amy ⁇ -amylase gene expression box expressed in pollen
  • p5126-MS45 Ms45 (wild type) gene expression box
  • pGt1-red Red fluorescent protein expression box specifically expressed in endosperm
  • CYP81A9-RNAi produces functional fragments of dsRNA to inhibit CYP81A9 gene expression
  • InCW2-RNAi produces functional fragments of dsRNA to inhibit InCW2 gene expression
  • pUbi-Bar glufosinate Gene expression box
  • bar glufosinate-resistant gene coding sequence.
  • the glufosinate resistance gene is used as a selection gene for maize transformation.
  • p35S CaMV 35S promoter; 35sT: CaMV 35S terminator.
  • the direction of the arrow is 5 'to 3', that is, the order of promoter, gene and terminator.
  • nsf1 nucleotide sequence such as SEQ ID No. 1
  • dsRNA double-stranded RNA
  • the restriction enzyme site XhoI is set on both sides of the sequence. The 3 'end of this fragment is connected to the CaMV 35S promoter, and the 5' end is connected to the CaMV 35S terminator to form a functional fragment that can inhibit the expression of the herbicide resistance gene CYP81A9.
  • nucleotide sequence is shown in SEQ ID No: 10 Shown (where 1bp-781bp is the 35S promoter of CaMV; 788bp-2428bp is a functional double-stranded RNA fragment capable of targeting the CYP81A9 gene; 2436bp-2619bp is the 35S terminator of CaMV), named nsf1-RNAi ( p35S-CYP81A9-RNAi-35sT).
  • glufosinate tolerance gene bar expression box ie, herbicide resistance gene expression box.
  • the nucleotide sequence is shown in SEQ ID No. 2, named pUbi-Bar, which contains the promoter, bar gene and terminator (where 1bp-2002bp is the pUbi promoter; 2052bp-2600bp is the bar gene coding region; 2610bp-2947bp is the rbcs terminator).
  • EcoRI and HpaI restriction sites were set in sequence at the 5 'end of the sequence, and EcoRI restriction sites were set at the 3' end.
  • the expression box of the fertility restoring gene Ms45 (that is, the fertility restoring restorer expression box), the sequence is shown in SEQ ID No. 3 (where the 7bp-511bp is the p5126 promoter; the 512bp-2059bp is the Ms45 gene Coding region; the 2060bp-2462bp Ms45 terminator), named p5126-Ms45.
  • This sequence includes the promoter, Ms45 coding sequence and terminator, and HpaI restriction sites are set at both ends of the sequence.
  • the 5 'end of the sequence is provided with HpaI restriction sites, and the 3' end is provided with KpnI and HpaI restriction sites in sequence.
  • amylase expression box specifically expressed in pollen ie, the expression box leading to pollen sterility containing the above expression box
  • the sequence is shown in SEQ ID No. 4, named pPg47-Amy, including the promoter, amylase Protein coding sequence and terminator (wherein 13bp-2053bp is pPg47 promoter; 2054bp-2278bp is the signal peptide sequence of the amyloid of BRITTLE1 (BT1) gene; 2279bp-3838bp is the coding region of amylase gene; 3839bp-4039bp Is a terminator). KpnI and HpaI restriction sites were placed in sequence at the 5 'end of the sequence, and HindIII restriction sites were placed at the 3' end.
  • Red fluorescent protein gene expression cassettes specifically expressed in maize seeds even if the maintainer seeds have significantly different physical traits than the sterile seeds.
  • the sequence is shown in SEQ ID No. 5 (where 7bp-845bp is Gt1 promoter; 846bp-917bp is Gt1 protein signal peptide; 918bp-1622bp is red fluorescent protein gene coding region; 1639bp-1825bp is terminator) It is shown that the name pGt1-Red includes the promoter, protein coding sequence and terminator. HindIII restriction sites were set at both ends of the sequence.
  • RNA interference functional fragments that suppress the expression of the InCW2 gene ie, functional fragments that make the seeds of the maintainer lines have a significant difference visible to the naked eye compared to the seeds of the sterile lines.
  • the sequence is shown in SEQ ID No. 6, named InCW2-RNAi, and HindIII sites are set at both ends of the sequence.
  • the anti-hygromycin gene hptII included in the pCambia1300 vector was replaced with the CYP81A9 gene RNAi fragment (SEQ ID No. 1) to obtain the vector pCambia1300-nsf1- RNAi. Then use EcoRI to cut the synthetic pUbi-Bar (SEQ ID No. 2) into pCambia1300-nsf1-RNAi to obtain the vector pCambia1300-nsf1-RNAi-pUbi-bar. Then use the cleavage site HpaI to join the p5126-Ms45 fragment (SEQ ID No.
  • the synthetic pGt1-red (SEQ ID No. 5) was connected to the vector pCambia1300-nsf1-RNAi-pUbi-bar-p5126-Ms45-pPg47-Amy to obtain the vector pCambia1300-nsf1-RNAi-pUbi -bar-p5126-Ms45-pPg47-Amy-pGt1-red, this vector is named: TWS-HC, the structure of the T-DNA sequence in this vector is shown in Figure 1.
  • the pGt1-red fragment in the vector pCambia1300-nsf1-RNAi-pUbi-bar-p5126-Ms45-pPg47-Amy-pGt1-red can be replaced with the artificially synthesized InCW2-RNAi fragment (SEQ ID No. 6).
  • the vector pCambia1300-nsf1-RNAi-pUbi-bar-p5126-Ms45-pPg47-Amy-InCW2-RNAi was finally obtained.
  • This vector was named TWS-HS.
  • the structure of the T-DNA sequence in this vector is shown in FIG.
  • the XhoI restriction site was used to replace the hygromycin resistance gene hptII contained in it with the bar gene (SEQ ID No. 7) fragment to obtain the vector pCambia1300-p35S-bar.
  • the pPg47-Amy fragment (SEQ ID No. 4) was ligated into the vector pCambia1300-p35S-bar to obtain the pCambia1300-p35S-bar-pPg47-Amy vector. Then, the p5126-Ms45 fragment (SEQ ID No.
  • TWS-CS the structure of the T-DNA sequence in this vector is shown in Figure 1.
  • T-DNA plasmid was transferred into Agrobacterium LB4404 by electrotransfer method, and positive clones were selected by YEP solid medium containing 15 ⁇ g / ml tetracycline and 50 ⁇ g / mL kanamycin, and preserved for bacteria Plant transformation down.
  • the transformation method of corn has been relatively mature. For example, Frame and the like describe the method of transforming corn with Agrobacterium (Frame et al., (2002) Plant Physiol, 129: 13-22) or (Yadava et al., Front. Plant Sci. 7: 1949).
  • Agrobacterium containing vectors TWS-HC, TWS-HS and TWS-CS ie, Agrobacterium containing T-DNA vector
  • the receptor used in the transformation of maize is the hybrid F1 generation of inbred lines HiIIA and HiIIB. Ears of corn 8-12 days after pollination. Collect all immature embryos (size 1.0-1.5 mm).
  • Agrobacterium containing the T-DNA vector was co-cultured with immature embryos for 2-3 days (22 ° C). Transfer immature embryos to the callus induction medium (the medium contains 200 mg / L Timentin for killing Agrobacterium), and culture in the dark at 28 ° C for 10-14 days. All the calli were transferred to the selection medium with a final concentration of 2 mM glufosinate (Frame et al., (2002) Plant Physiol, 129: 13-22) and cultured in the dark at 28 ° C for 2-3 weeks. All tissues were transferred to fresh selection medium containing a final concentration of 2mM glufosinate and incubated in the dark at 28 ° C for 2-3 weeks.
  • the selected transformants must all be male fertile in the homozygous ms45 sterile background (ms45 / ms45), that is, the transformants have the ability to recover from male sterility.
  • the method is: the pollen of male sterile heterozygosity (ms45 / Ms45, expressed as fertile) is received on the corn cobs of transgenic plants, and the transgenic corn with the background of Ms45 / ms45 (Ms45 (exogenous) /-, ms45 / Ms45), self.
  • the transgenic maize with background ms45 / ms45 was selected from self-breeding offspring, and pollen activity was detected. If the activity of pollen is normal, it means that the transformant gene can restore male fertility.
  • the selection of transgenic corn can be achieved by spraying glyphosate (1 times the field recommended dose), and only the transgenic corn plants can survive. Choosing the genotype of ms45 can be achieved by molecular marker technology.
  • the transformants with excellent characteristics are selected according to the characteristics of the transformed T-DNA.
  • the transformants obtained with the vector TWS-HC are mainly selected based on the following: 1) The transgenic seeds have obvious red fluorescence; 2) The transgenic corn can be killed by nicosulfuron at twice the field dose.
  • the transformants obtained using the vector TWS-CS are mainly selected based on the following: 1) Transgenic seeds have obvious red fluorescence; 2) Transgenic corn kernels are significantly smaller than those of the same genetic background that do not contain foreign genes. Transformants that are 50% or more smaller are preferred.
  • the transformants obtained using the vector TWS-HS the main basis for selection is to simultaneously satisfy: 1) Transgenic corn kernels are significantly smaller than those of the same genetic background that do not contain foreign genes, and are preferably 50% or more smaller transformants; 2 ) The transgenic corn can be killed by nicosulfuron at twice the field dose.
  • ms45 homozygous recessive mutants as the female parent, cross with different elite inbred lines such as Zheng 58.
  • the obtained F1 continued to be backcrossed with Zheng 58.
  • Genotype analysis was performed on the obtained BC1 population, and the plants identified as heterozygous at Ms45 continued to be backcrossed with Zheng 58.
  • the Ms45 locus was screened for heterozygosity by molecular markers, and the other plants with Zheng 58 at other loci were selfed, thereby obtaining the ms45 homozygous recessive inbred line Zheng 58 (ms45 / ms45).
  • This inbred line can be used as a sterile line.
  • the progeny Zheng 58 (heterozygous ms45 / Ms45) backcrossed 5 times in Example 4 was crossed with the transgenic plants selected in Example 3, and the progeny with recessive ms45 and foreign genes were selected, and then implemented In Example 4, the offspring of Zheng 58 (heterozygous ms45 / Ms45), which was backcrossed 5 times, were used as recurrent parents for 5 generations of backcrossing. Single plants with a recessive ms45 and foreign genes similar to Zheng 58 were selected.
  • Example 6 Improve the purity of male sterile seeds
  • the infertility mother prepared by the transgenic male sterility maintenance line obtained by the vector TWS-HC can remove most of the transgenic male fertile grains by fluorescence recognition, and the male sterile male grows to the period of 2-8 leaves. Further spraying twice the recommended dosage of nicosulfuron in the field, the transgenic plants (male fertile) that may be mixed can be killed and removed. This further eliminates male fertile corn plants and further improves the purity of hybrid corn.
  • the infertile mothers prepared by the transgenic male sterility maintenance line obtained by the vector TWS-CS can remove most of the transgenic male fertile grains by fluorescence recognition.
  • the obtained male sterile seeds further remove seeds with smaller grains (more than 30% smaller than the average weight of male sterile seeds).
  • Potentially mixed transgenic seeds male fertility can be further removed. This further eliminates male fertile corn plants and further improves the purity of hybrid corn.
  • the infertile mothers prepared by the transgenic male sterile maintainer line obtained by the vector TWS-HS exclude most of the transgenic male fertile grains by removing small seeds (at least 30% less than the average weight of male sterile seeds).
  • the recommended dosage of nicosulfuron is recommended.
  • Potentially mixed transgenic plants male fertile
  • Example 7 Large-scale multiplication of male sterile inbred lines using male sterile maintainer lines (ms45 / ms45)
  • the male sterility maintenance line Zheng 58 (Ms45 /-, ms45 / ms45) in Example 5 was planted at intervals with the male sterility line Zheng 58 (ms45 / ms45).
  • the active pollen produced by the male sterility maintenance line Zheng 58 that supplies pollen is non-transgenic pollen (transgenic pollen cannot germinate)
  • the seeds obtained from the plants of the male sterile line Zheng 58 are still non-transgenic Male sterile seeds.

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Abstract

本发明公开了一种转基因玉米核雄性不育保持系及应用，所述保持系是将外源T-DNA载体导入玉米基因组构建而成，导入的外源T-DNA中除了雄性核不育的育性恢复基因以外，还包含如下3种功能片段中至少2个功能片段：1)在种子胚乳中表达一种荧光蛋白质的表达框；2)一种可以抑制玉米抗除草剂基因CYP81A9表达的DNA片段；3)一种可以抑制玉米胚乳特异表达的细胞壁转化酶InCW2表达的DNA片段。本发明同时利用上述三种区分转基因与非转基因方法中至少2种方法，进一步降低仅仅依靠一种区分(或选择)方法可能导致的错误。

Description

一种转基因玉米核雄性不育保持系及应用 (一)技术领域
本发明涉及一种转基因玉米核雄性不育保持系及其应用。
(二)背景技术
杂交优势的利用给粮食的增产最重要的技术之一。玉米生产中通常通过2个自交系杂交制备杂交种子。杂交种子比较自交种子在生产上更加具有产量、抗性等方面的优势。在生产上目前大部分的杂交种子的生产仍然通过人工或者机械去雄的方法，成本高，工作量大。而且在生产杂交种的过程中由于环境的变化可能导致去雄完成后植株又雄穗化，或去雄不完全，从而导致母本自交授粉，影响杂交种子纯度。
雄性不育系统提供了简单高效的制备杂交玉米种子的手段。比较成熟的杂交技术体系是基于核-质互作雄性不育(CMS)自交系的三系杂交制种体系。该技术包括3个主要材料：1)不育系：雄性不育材料；2)保持系：可以为不育系提供花粉，使不育系的后代仍为不育系；3)恢复系：可以恢复不育系的育性。不育系与恢复系杂交产生F1，即用于农业生产的杂交种。更具体的说，核-质互作不育型，表现为核-质互作遗传。不但需要细胞质有不育基因S，而且需要细胞核里有纯合的不育基因(rf/rf)，二者同时存在，方能使植株表现为雄性不育。CMS三系杂交制种体系存在恢复系比较难找、技术体系复杂等缺陷，在玉米制种上推广面积仍然比较小。
利用核基因隐性不育基因是玉米杂交制种的另外一个可能的技术路线。控制雄性不育的核基因隐性纯合(ms/ms)植株才会表现为雄性不育，由于雄性不育植株无法自交，因此只能通过杂合植株(Ms/ms)与其杂交，才会得到雄性不育植株(ms/ms)。但是在同一果穗上雄性不育籽粒(ms/ms)与可育的杂合籽粒(Ms/ms)同时存在，无法简单通过物理方法分辨。
目前一种利用隐性核不育基因制备杂交玉米种子的新方法已经报道(Wu et al.,Plant Biotechnology,14:1046-1054；An et al.,Molecular Plant 12,343-359；Wu et al.,Shukla et al.,Molecular Biotechnology 59,445-457；美国专利7,612,251)。这种方法利用转基因的不育保持系制备非转基因的核雄性不育玉米种子。这个方法的技术要点是：转基因玉米保持系是在一个隐性核不育纯合系(如ms45/ms45)中导入了包含如下表达框的外源DNA：1)一个正常野生型的雄性育性基因(例如，Ms45)，其功能是互补导致雄性核不育基因(如ms45)从而挽救育性；2)一个种子颜色的标记基因(荧光蛋白质基因)；3)一个在花粉中表达的α‐淀粉酶基因，导致转基因花粉因为没有淀粉而不能萌发。这个技术体系中纯合隐性不育基因(如ms45/ms45)被转基因导入的野生型基因(如MS45)互补而表现为雄性可育。同时由于转基因的花粉因其表达的α-淀粉酶而不能萌发，因此外源基因是不能纯合的。不带外源DNA的50％花粉(非转基因)能够成功授粉于纯合的核性不育(如ms45/ms45)的雌配子，从而获得雄性不育玉米种子。这些种子可以作为玉米杂交制种的母本。由于导入的外源DNA包含一个在胚乳中表达的荧光蛋白质基因，包含有外源 DNA并且可育的种子因此可以与不包含外源DNA的雄性不育种子可以利用荧光特征而区分。这种技术可以简化玉米杂交制种的程序，降低成本。
中国发明专利CN102960234B披露了另外一种利用转基因的不育保持系制备非转基因的核不育玉米的方法。该方法的特点是利用种子大小而不是种子荧光蛋白质的荧光区分雄性不育种子和雄性可育种子。其技术方案是构建了干扰玉米胚乳特异表达的细胞壁转化酶(endosperm-specific cell wall invertase，InCW2)基因(Cheng et al.Plant Cell 8:971–983)的干扰表达框，将该基因沉默。InCW2沉默后玉米籽粒变小。这种技术区别雄性不育种子和雄性可育种子的方法非常简单。
但是无论是利用表达荧光蛋白质还是利用玉米籽粒大小作为区分雄性不育和雄性可育的种子的方法仍然可能在一定程度存在不足。区分雄性不育和可育种子的错误都可以导致杂交种子的纯度降低，并且将转基因玉米传播到食物链(雄性不育玉米是非转基因玉米)。为了提高制种纯度，本发明提供了同时利用2种选择性状，通过2次筛选以获得纯度更高的雄性不育种子的技术路线。本发明的方法比现有的方法更加能够提高雄性不育系玉米的纯度并且防止转基因恢复系进入大田生产体系。特别是，本发明创造性地将转基因玉米的选择性杀灭技术应用于提高雄性不育系玉米的纯度。在玉米中通过RNA干扰抑制耐除草剂基因CYP81A9的表达可以导致转基因玉米对烟嘧磺隆等除草剂敏感。这些转基因玉米可以被被烟嘧磺隆等除草剂选择性杀灭(Li,Jing,et al.，2013，Plos One 8(12):e81645)。
(三)发明内容
本发明的目的是提供一种转基因玉米核雄性不育保持系的构建与应用。这种转基因保持系的种子的特点是利用2种或多种与雄性不育系种子区别的标记，能够进一步降低和防止转基因保持系种子混入商业杂交种的风险。本发明综合利用了包括种子荧光颜色标记、种子形状大小标记技术以及除草剂敏感性标记(转基因玉米可以被选择性杀灭)，而以前的技术仅仅利用一个标记，如荧光蛋白质标记(Wu et al.,Plant Biotechnology,14:1046-1054；美国专利7,612,251),或者控制种子大小的标记(中国专利CN102960234B)。
本发明需要利用雄性核不育玉米材料，表1例举了部分目前已经报道的雄性核不育玉米材料，它们控制的基因已经被克隆或可以被克隆。已知不育基因的雄性不育材料也可以通过基因编辑的方法获得核雄性不育的玉米材料。例如，通过基因编辑的方法可以获得ms45和ms26的雄性不育突变体(Svitashev et al.,Nature Communications 7,13274)。本发明以表1中的一个雄性不育突变体ms45为例，提供一种利用隐性核不育玉米制备杂交种子的方法。下文通过说明和阐述提供了更为详细的描述，但这并非意欲对本发明的范围加以限制。
本发明采用的技术方案是：
本发明提供一种转基因玉米核雄性不育保持系，所述转基因玉米保持系是将外源T-DNA载体导入玉米基因组构建而成，所述外源T-DNA载体中除了雄性核不育的育性恢复基因Ms45表达框(即不育系育性恢复表达框)以外，还包含如下3种功能片段中至少2个：1)在种子胚乳中表 达荧光蛋白的表达框(即使得保持系种子与不育系种子相比具有肉眼可见的颜色上的显著差异的表达框)；2)一种可以抑制玉米抗除草剂基因CYP81A9(nsf1)表达的功能片段(即植物内源抗除草剂基因的沉默或敲除表达框)，3)一种可以抑制玉米胚乳特异表达的细胞壁转化酶(endosperm-specific cell wall invertase/InCW2)表达的功能片段(即使得保持系种子与不育系种子相比具有肉眼可见的形状上的显著差异的表达框)。
进一步，优选所述外源T-DNA载体中除了雄性核不育的育性恢复基因以外，还包含如下2个表达框：1)在种子胚乳中表达一种荧光蛋白质的表达框；2)一种可以抑制玉米抗除草剂基因CYP81A9表达的功能片段。
进一步，优选所述外源T-DNA载体中除了雄性核不育的育性恢复基因以外，还包含至少如下2个表达框：1)在种子胚乳中表达一种荧光蛋白质的表达框；2)一种可以抑制玉米胚乳特异表达的细胞壁转化酶(InCW2)的功能片段。
进一步，优选所述外源T-DNA载体中除了雄性核不育的育性恢复基因以外，还包含至少如下2个表达框：1)一种可以抑制玉米抗除草剂基因CYP81A9表达的功能片段；2)一种可以抑制玉米胚乳特异表达的细胞壁转化酶(InCW2)的功能片段。
本发明所述雄性核不育的育性恢复基因表达框由启动子、育性恢复基因、终止子功能性连接组成，该表达框所表达的功能蛋白质回补了核不育玉米中突变失去的雄性育性功能基因，从而可以恢复核不育玉米的雄性育性。本发明优选能够恢复ms45核不育玉米(ms45/ms45)育性的基因表达框，即Ms45基因表达框。Ms45基因需要与能够启动其适当表达的启动子和终止子功能性连接，这些启动子和终止子可以有多重选择，比较优先的选择是这些基因的天然启动子和终止子。能够驱动在植物中适当表达的启动子和终止子已经有大量文献资料。根据需要可以选择表达水平和表达时空合适的启动子。例如，美国专利5,689,049披露的启动子5126可以驱动花药(anther)特异表达，是启动Ms45基因的优选启动子之一。
表1：由核基因引起的雄性不育突变体
Figure PCTCN2019115675-appb-000001
Figure PCTCN2019115675-appb-000002
本发明所述在种子胚乳中表达荧光蛋白质的表达框是由启动子、荧光蛋白质基因和终止子功能性连接而成。这个表达框的荧光蛋白质基因可以是红色荧光蛋白质(red fluorescent gene)、青色荧光蛋白(cyan fluorescent protein gene)、黄色荧光蛋白(yellow fluorescent protein gene)、绿色荧光蛋白(green fluorescent protein gene)、花青素p1基因(anthocyanin p1gene)等。这些荧光蛋白基因在胚乳中的表达，使获得的转基因种子可以通过简单的物理方法与没有表达这些基因的种子区分开来。这个表达框的启动子是能够驱动在玉米胚乳中高表达的启动子。优选的启动子是玉米或水稻的胚乳储存蛋白质的启动子。例如水稻的GluA-1、GluA-2、GluA-3、GluB-3、GluB-5和GluC启动子(Qu et al.,J Exp Bot.59:2417–2424；Xu et al.,Plant Cell,Tissue and Organ Culture,128:125–132)，玉米的α-zein启动子(Joshi et al.,Physiol Mol Biol Plants 21:35–42)等。一些组成型启动子也可以启动基因在胚乳中较高地表达。水稻或者玉米的胚乳储存蛋白质的信号肽可以用来与这些荧光蛋白质融合，使这些蛋白质表达以后能够适当存储和积累。这些启动子、编码带有信号肽的荧光蛋白质的核苷酸序列以及终止子的核苷酸序列，通过功能性连接而形成完整 的荧光蛋白质的表达框。用于构建表达框的终止子可以是CaMV的35S终止子，也可以是其它在玉米中具有功能的终止子。例如，SEQ ID No.5的序列是一个能够在胚乳中表达红色荧光蛋白质的表达框。
本发明所述抑制玉米胚乳特异表达的细胞壁转化酶(endosperm-specific cell wall invertase，InCW2)基因表达的功能片段可以通过不同的原理构建。本发明优选以双链RNA干扰InCW2表达的方法。抑制表达框的具体构建方法如下：1)构建一个DNA片段，其二端是分别来自InCW2的一个DNA片段以及这个片段的反向重复序列片段。在这二个DNA片段中间加入一个其他任何DNA片段，优选是InCW2基因的部分内含子序列。由于这个片段中存在2个反向重复InCW2基因序列片段，转录这个DNA片段获得的RNA就可以形成发夹结构的双链RNA(dsRNA)。这个反向重复序列片段的5’端与一个启动子功能性连接，3’端与一个终止子功能性连接，形成一个DNA功能片段。这个功能片段的启动子可以是能够启动组成型表达的启动子或者启动胚乳中特异性表达的启动子，优选启动子是InCW2基因的天然启动子；终止子可以是在玉米中具有终止子功能的不同种类的终止子，包括InCW2基因的天然终止子。构建这个抑制表达片段使用的DNA序列来自InCW2(SEQ ID NO:8)，其长度和位置可以不同，抑制效果可以通过实验测定，选择抑制效率比较高的构建片段。SEQ ID No.6(其中第7bp-950bp为InCW2启动子；第977bp-1930bp为能够形成靶向InCW2基因的双链RNA功能片段；第1931bp-2150bp为CaMV的35S终止子)提供了一种能够干扰InCW2表达的功能片段的核苷酸序列，这个功能片段能够导致玉米种子变小。
基因沉默技术是一个成熟的技术(Agrawal et al.,Microbiol Mol Biol Rev.67:657–685)，抑制InCW2基因表达的方法还有：1)反义RNA方法，这个方法是让转基因植物产生一个靶标基因的反义RNA以抑制对应基因的表达；2)人工miRNA方法，这个方法设计一个能够抑制靶标基因表达的人工miRNA(Schwab et al.Plant Cell 18,1121–1133)。
本发明所述抑制玉米抗除草剂基因CYP78A9表达的功能片段可以通过不同的原理构建。以双链RNA干扰CYP78A9表达的方法为例，通过沉默植物内源耐除草剂基因来实现，由“组成型启动子-发夹RNA-终止子”构成。CYP81A9基因RNA干扰功能片段具体构建方法如下：1)构建一个DNA片段，其二端是分别来自CYP78A9的一个DNA片段以及这个片段的反向重复序列片段。在这二个DNA片段中间加入一个其他任何DNA片段，优选是来自CYP78A9基因的内含子序列。由于这个片段中存在2个反向重复CYP78A9基因序列片段，转录这个DNA片段获得的RNA就可以形成发夹结构的双链RNA(dsRNA)。这个反向重复序列片段的5’端与一个启动子功能性连接，3’端与一个终止子功能性连接，形成一个DNA功能片段。这个功能片段的启动子可以是能够启动组成型表达的启动子，如CaMV的35S启动子；终止子可以是在玉米中转基因研究中常用的终止子，如CaMV的35S终止子。构建这个抑制表达片段使用的CYP78A9基因序列来自SEQ ID NO:9。这个片段转录获得的dsRNA的长度和序列可以不同，其抑制效果可以通过实验进行测定，选择抑制效率比较高的构建片段。
抑制玉米抗除草剂基因CYP78A91表达的方法还可以：1)反义RNA方法，这个方法是让转基因植物产生靶标基因CYP78A9的反义RNA以抑制CYP78A9基因的表达；2)人工miRNA方法(Schwab et al.Plant Cell 18,1121–1133)。利用这个方法可以设计一个能够抑制靶标基因CYP78A9表达的人工miRNA功能片段。
转基因玉米中包含抑制玉米抗除草剂基因CYP78A9表达的功能片段可以导致转基因玉米对除草剂烟嘧磺隆、苯达松等除草剂敏感，从而这些转基因玉米可以被除草剂选择性地杀灭。
本发明所述转基因玉米核雄性不育保持系的外源T-DNA质粒中还可以同时包括以下的表达框：
1)在花粉中表达的α-淀粉酶的表达框(即导致含有上述表达框的花粉不育的表达框)。通过导入一个使花粉失活的基因来实现，由“启动子-α-淀粉酶-终止子”构成，优选表达框核苷酸序列为SEQ ID.NO：4(其中第13bp-2053bp为pPg47启动子；第2054bp-2278bp为BRITTLE1(BT1)基因的淀粉体的信号肽序列；第2279bp-3838bp为淀粉酶基因编码区；第3839bp-4039bp为终止子)所示。这个表达框的启动子可以是PG47和ZM13启动子(Hamilton et al.,Plant Mol.Biol.38:663-669)。这个表达框的α-淀粉酶可以是Bacillus stearothermophilus的α-淀粉酶(gb：A24549)，或者玉米的淀粉酶基因(gb:ADH43281.1)，也可以是其它来源的α-淀粉酶。将α-淀粉酶的信号肽切除，然后用来自BRITTLE1(BT1)基因的淀粉体的信号肽(Sullivan et al,Plant Cell 3:1337–1348)替换。这个表达框所表达的产物可以导致玉米花粉缺乏淀粉而不能萌发，进而导致雄性不育。这样就可以实现转基因玉米的外源DNA保持是杂合状态，而不是纯合状态。
2)选择标记表达框(即除草剂抗性基因表达框)。赋予保持系一种或多种除草剂抗性，通过导入一个耐除草剂基因来实现，由“启动子-抗除草剂基因-终止子”构成。优选除草剂抗性基因表达框为草铵膦耐受基因bar表达框，序列如SEQ ID No.2(其中第1bp-2002bp为pUbi启动子；第2052bp-2600bp为bar基因编码区；第2610bp-2947bp为rbcs终止子)所示。抗草甘膦基因表达框或者抗草铵膦表达框是常用的选择标记表达框。例如利用草甘膦作为筛选化合物的方法已有详细描述(Howe et al.,Molecular Breeding 10:153–164；Zhou et al.,Plant Cell Reports 15,159-163)。采用的启动子优选组成型启动子，如花椰菜花叶病毒(CaMV)35S启动子p35S(Odell,Joan T.,Ferenc Nagy,and Nam-Hai Chua,1985,Nature,810-812)、水稻的Actin1启动子pAct1(McElroy D,Zhang W,Cao J,et al.,1990,The Plant Cell,2:163-171.)、玉米Ubiquitin启动子pUbi(Christensen A H,Sharrock R A,Quail P H,1992,Plant molecular biology,18:675-689)及其具有类似表达模式的启动子；抗除草剂基因可以是抗草甘膦基因CP4-EPSPS(孟山都公司)、G10(中国专利：2011100093290)，抗草铵膦基因bar(Thompson C J,Movva N R,Tizard R,et al,1987，The EMBO journal,6(9):2519.)及其具有类似功能的基因。
进一步，本发明所述外源T-DNA载体包含如下表达框：1)Ms45表达框；2)红色荧光蛋白质表达框；3)CYP78A9基因抑制表达功能片段；4)花粉中表达的α-淀粉酶的表达框；5)选择标记表达框。
另一种T-DNA可以是包含如下表达框：1)Ms45表达框；2)InCW2抑制表达功能片段；3)CYP78A9基因抑制表达功能片段；4)花粉中表达的α-淀粉酶的表达框；5)选择标记表达框。
还有一种T-DNA可以是包含如下表达框：1)Ms45表达框；2)红色荧光蛋白质表达框；3)InCW2抑制表达功能片段；4)花粉中表达的α-淀粉酶的表达框；5)抗草铵膦基因表达框。
能够实现本发明目的的包含这些表达框和功能片段的其它T-DNA同样可以通过一般分子生物学方法构建。并且，这些表达框和功能片段的方向和次序的不同并不能影响实现本发明的目的。
本发明还提供一种含所述转基因玉米核雄性不育保持系中T-DNA载体的植物细胞。
将包含上述几个表达框的DNA构建体通过农杆菌介导的方法或者其它方法导入到玉米。玉米的遗传转化是成熟的技术，这个领域的一般技术人员就可以实现。相关转化方法已经多次公开发表，例如Yadava et al.,Front.Plant Sci.7:1949。转化获得的转化体需要进一步选择以获得实现本发明目的所需要的转化体。
导入了本发明提供的外源基因的玉米转化体可以通过回交转育将外源基因整合到核雄性不育的玉米基因组中。利用雄性不育杂合基因型Ms45/ms45植株与筛选获得的转化体植株进行多次回交，从而将包含外源MS45恢复基因导入到雄性纯合不育突变体ms45中，获得基因型Ms45/-,ms45/ms45的植株。由于导入的外源基因中野生型MS45基因的存在，转基因植株(Ms45/-,ms45/ms45)表现为可育。
当转基因杂合体植株(基因型Ms45/-,ms45/ms45)自交时，会产生两种后代，一种为不含有外源转基因序列的雄性不育籽粒(ms45/ms45)，该不育籽粒可以被任意一种野生型植株(Ms45/Ms45)恢复育性，在制种过程中可作为不育系(母本)；另一种为籽粒雄性育性被恢复(Ms45/-,ms45/ms45)，可以作为恢复系制备下一季不育系种子。
本发明的主要技术目的是确保雄性不育玉米(ms45/ms45)中没有混杂包含外源基因的雄性育性被恢复的转基因玉米(Ms45/-,ms45/ms45)。而本发明的关键技术措施是采用了至少二种清除转基因玉米的方法，大幅度降低雄性不育玉米中混杂雄性育性被恢复的转基因玉米(Ms45/-,ms45/ms45)的可能性。
本发明提供一种所述转基因玉米核雄性不育保持系在制备非转基因核不育种子中的应用，所述应用的方法为：将转基因玉米核雄性不育保持系与纯合隐性不育系间隔种植，通过下列方法之一筛选非转基因核不育种子：
(1)当导入的外源基因包含荧光蛋白质和抑制CYP78A9表达功能片段的转基因玉米时：通过荧光识别去除绝大部分的转基因雄性可育籽粒，获得的雄性不育种子在制种过程中，在雄性不育母本生长到2-8叶时期进一步喷洒足够剂量的烟嘧磺隆和/或苯达松杀灭混杂的转基因玉米。这样就进一步排除了转基因雄性可育的玉米株，进一步提高杂交玉米的纯度。
(2)当导入的外源基因包含荧光蛋白质和抑制InCW2表达功能片段的转基因玉米时：通过荧光识别就可以去除绝大部分的转基因雄性可育籽粒，获得的雄性不育种子进一步去除籽粒比较小(如，至少比平均重量小30％以上)的种子。可能混杂的转基因种子(雄性可育)就可以被进一 步去除。这样通过2个性状的选择，进一步排除了雄性可育的玉米株，提高杂交玉米的纯度。
(3)当导入的外源基因包含抑制CYP78A9表达功能片段和抑制InCW2表达功能片段的转基因玉米时：通过去除小种子(至少比平均重量小30％以上)排除绝大部分的转基因雄性可育籽粒，获得的雄性不育种子在制种过程中，在雄性不育母本生长到2-8叶时期进一步喷洒足够剂量的烟嘧磺隆和/或苯达松杀灭混杂的转基因玉米。
与现有的技术相比，本发明的优点要体现在：本发明同时利用上述三种区分转基因与非转基因方法中至少2种方法，进一步降低仅仅依靠一种区分(或选择)方法可能导致的错误。
(四)附图说明
图1.含有雄性生育力恢复基因Ms45及籽粒选择基因的三种T-DNA结构示意图(TWS-HC，TWS-HS和TWS-CS)。LB：T-DNA左边界；RB：T-DNA右边界；pPg47-Amy：在花粉中表达的α-淀粉酶基因表达框；p5126-MS45：Ms45(野生型)基因表达框；pGt1-red：在胚乳中特异性表达的红色荧光蛋白质表达框；CYP81A9-RNAi:产生dsRNA抑制CYP81A9基因表达的功能片段；InCW2-RNAi：产生dsRNA抑制InCW2基因的表达的功能片段；pUbi-Bar：抗草铵膦基因表达框；bar：抗草铵膦基因编码序列。抗草铵膦基因作为玉米转化的选择基因。p35S：CaMV的35S启动子；35sT：CaMV的35S终止子。箭头方向是5’到3’，即启动子、基因和终止子的顺序。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：
在不背离本发明的精神和实质的情况下，对本发明方法、步骤或条件所做的修改或替换均属于本发明的范围。本文使用的DNA及其载体的构建方法是分子生物学一般的方法。大部分DNA片段可以根据提供的核苷酸序列人工合成，简化构建过程。
实施例1、T-DNA载体构建
1、为了构建转化玉米的T-DNA载体，分别合成或者构建以下DNA片段：
(1)合成包含来自CYP81A9(nsf1)的反向重复DNA序列片段(核苷酸序列如SEQ ID No.1)。该片段转录为RNA时能够形成双链RNA(dsRNA)，能够干扰CYP81A9的表达。序列两边都设置了限制性内切酶位点XhoI。这个片段的3’端与CaMV的35S启动子连接，5’端与CaMV的35S终止子连接，形成一个可以抑制抗除草剂基因CYP81A9表达的功能片段，核苷酸序列如SEQ ID No:10所示(其中第1bp-781bp为CaMV的35S启动子；第788bp-2428bp为能够形成靶向CYP81A9基因的双链RNA功能片段；第2436bp-2619bp为CaMV的35S终止子)，命名为nsf1-RNAi(p35S-CYP81A9-RNAi-35sT)。
(2)合成抗草铵膦基因编码序列，序列如SEQ ID No.7所示，命名bar，序列两端分别设置XhoI限制性酶切位点。
(3)合成草铵膦耐受基因bar表达框(即除草剂抗性基因表达框)DNA片段。核苷酸序列如SEQ ID No.2所示，命名为pUbi-Bar，包含了启动子，bar基因和终止子(其中第1bp-2002bp 为pUbi启动子；第2052bp-2600bp为bar基因编码区；第2610bp-2947bp为rbcs终止子)。序列5’端依次设置EcoRI和HpaI限制性酶切位点，3’端设置EcoRI限制性酶切位点。
(3)育性恢复基因Ms45的表达框(即不育系育性恢复表达框)，序列如SEQ ID No.3所示(其中第7bp-511bp为p5126启动子；第512bp-2059bp为Ms45基因编码区；第2060bp-2462bp为Ms45终止子)，命名p5126-Ms45。这个序列包括了启动子、Ms45编码序列和终止子，序列两端分别设置HpaI限制性酶切位点。序列5’端设置HpaI限制性酶切位点，3’端依次设置KpnI和HpaI限制性酶切位点。
(4)花粉中特异表达的淀粉酶表达框(即导致含有上述表达框的花粉不育的表达框)，序列如SEQ ID No.4所示，命名pPg47-Amy，包括了启动子、淀粉酶蛋白质编码序列和终止子(其中第13bp-2053bp为pPg47启动子；第2054bp-2278bp为BRITTLE1(BT1)基因的淀粉体的信号肽序列；第2279bp-3838bp为淀粉酶基因编码区；第3839bp-4039bp为终止子)。序列5’端依次设置KpnI和HpaI限制性酶切位点，3’端设置HindIII限制性酶切位点。
(5)玉米种子特异表达的红色荧光蛋白基因表达框(即使保持系种子与不育系种子相比具有物理性状显著差异的表达框)。序列如SEQ ID No.5(其中第7bp-845bp为Gt1启动子；第846bp-917bp为Gt1蛋白信号肽；第918bp-1622bp为红色荧光蛋白基因基因编码区；第1639bp-1825bp为终止子)所示，命名pGt1-Red，包括了启动子、蛋白质编码序列和终止子。序列两端分别设置HindIII限制性酶切位点。
(6)抑制InCW2基因表达的RNA干扰功能片段(即使得保持系种子与不育系种子相比具有肉眼可见的显著差异的功能片段)。序列如SEQ ID No.6所示，命名为InCW2-RNAi，序列两端分别设置HindIII位点。
2、构建包含pGt1-Red和nsf1-RNAi功能片段的T-DNA载体
以双元载体pCambia1300为基础，利用XhoI酶切位点，把pCambia1300载体中自带的的抗潮霉素基因hptII置换为CYP81A9基因RNAi片段(SEQ ID No.1)，获得载体pCambia1300-nsf 1-RNAi。然后利用酶切位点EcoRI把人工合成的pUbi-Bar(SEQ ID No.2)克隆到pCambia1300-nsf1-RNAi中，获得载体pCambia1300-nsf1-RNAi-pUbi-bar。再利用酶切位点HpaI把p5126-Ms45片段(SEQ ID No.3)连入载体pCambia1300-nsf1-RNAinsf1-RNAi-pUbi-bar中，获得载体pCambia1300-nsf1-RNAi-pUbi-bar-p5126-Ms45。然后，利用KpnI和HindIII，把pPg47-Amy片段(SEQ ID No.4)连入载体pCambia1300-nsf1-RNAi-pUbi-bar-p5126-Ms45中，获得载体pCambia1300-nsf1-RNAi-pUbi-bar-p5126-Ms45-pPg47-Amy载体。最后，通过HindII，把人工合成的pGt1-red(SEQ ID No.5)连入载体pCambia1300-nsf1-RNAi-pUbi-bar-p5126-Ms45-pPg47-Amy中，获得载体pCambia1300-nsf1-RNAi-pUbi-bar-p5126-Ms45-pPg47-Amy-pGt1-red，这个载体命名为：TWS-HC，该载体中的T-DNA序列的结构如图1所示。
3、构建包含nsf1-RNAi和InCW2-RNAi功能片段的T-DNA载体
将载体pCambia1300-nsf1-RNAi-pUbi-bar-p5126-Ms45-pPg47-Amy-pGt1-red中的pGt1-red片 段替换成人工合成的InCW2-RNAi片段(SEQ ID No.6)即可。最终获得载体pCambia1300-nsf1-RNAi-pUbi-bar-p5126-Ms45-pPg47-Amy-InCW2-RNAi，这个载体命名为TWS-HS，该载体中的T-DNA序列的结构如图1所示。
4、构建包含InCW2RNAi和pGt1-Red功能片段的T-DNA载体
以双元载体pCambia1300为基础，利用XhoI酶切位点，把其中自带的抗潮霉素基因hptII置换为bar基因(SEQ ID No.7)片段，获得pCambia1300-p35S-bar的载体。利用KpnI和HindIII，把pPg47-Amy片段(SEQ ID No.4)连入载体pCambia1300-p35S-bar，获得pCambia1300-p35S-bar-pPg47-Amy载体。然后，再利用酶切位点HpaI把p5126-Ms45片段(SEQ ID No.3)连入载体pCambia1300-p35S-bar--pPg47-Amy中，获得载体pCambia1300-p35S-bar-p5126-Ms45-pPg47-Amy。最后，通过HindIII，把人工合成的pGt1-red(SEQ ID No.5)和InCW2-RNAi(SEQ ID No.6)连入载体pCambia1300-p35S-bar-pPg47-Amy-p5126-Ms45中，获得载体pCambia1300-p35S-bar-pPg47-Amylase-p5126-Ms45-pGt1-red-InCW2-RNAi，这个载体命名为：TWS-CS，该载体中的T-DNA序列的结构如图1所示。
最后，通过电转的方法把上述T-DNA质粒转入农杆菌LB4404中，通过含有15μg/ml四环素和50μg/mL的卡那霉素的YEP固体培养基筛选出阳性克隆，并保菌，用于接下来的植物转化。
实施例2、玉米转化
玉米的转化方法已经比较成熟，例如Frame等描述了利用农杆菌转化玉米的方法(Frame et al.,(2002)Plant Physiol,129:13-22)或(Yadava et al.,Front.Plant Sci.7:1949)。分别取含载体TWS-HC、TWS-HS和TWS-CS的农杆菌(即含T-DNA载体的农杆菌)划板，挑单菌落接种，准备转化用农杆菌。玉米转化中所用受体为自交系HiIIA和HiIIB的杂交F1代。取授粉后8-12天的玉米穗。收集所有的未成熟胚(大小为1.0-1.5mm)。将含有T-DNA载体的农杆菌与未成熟胚共培养2-3天(22℃)。转移未成熟胚到愈伤诱导培养基上(培养基中含有200mg/L的Timentin，用于杀灭农杆菌)，28℃暗培养10-14天。将所有的愈伤转到带有终浓度2mM草铵膦的筛选培养基上(Frame et al.,(2002)Plant Physiol,129:13-22)，28℃暗培养2-3周。转移所有的组织到新鲜含终浓度2mM草铵膦的筛选培养基上，28℃暗培养2-3周。然后，转移所有筛选后成活的胚性组织到再生培养基上，28℃暗培养10-14天，每皿一个株系。转移胚性组织到新鲜的再生培养基上，26℃光照培养10-14天。转移所有发育完全的植株到生根培养基上，26℃光照培养直到根发育完全，然后移植到温室中单株培养，检测转基因玉米的抗除草剂能力。用稀释200倍的拜耳的18％的草铵膦水剂喷洒，7天后叶片发黄，枯死的为阴性；和喷清水对照长势一样的为阳性植株，分别获得用载体TWS-HC、TWS-HS和TWS-CS转化获得的转基因玉米品系。
实施例3 转化体选择
对来自实施例2的转基因植株进行评估，选择同时满足如下3个条件的转化体：1)能够挽救 纯合不育背景下(ms45/ms45)玉米的育性；2)目的基因控制的种子荧光、大小或除草剂敏感性等性状显著；3)转基因的花粉不能萌发。
首先，选择的转化体都必须在纯合的ms45不育背景下(ms45/ms45)表现为雄性可育，即转化体具有雄性不育的恢复能力。方法是：将雄性不育杂合子(ms45/Ms45,表现为可育)的花粉受到转基因植株的玉米棒子上，在后代中选择背景是Ms45/ms45的转基因玉米(Ms45(外源)/-,ms45/Ms45)，自交。从自交后代中选择背景是ms45/ms45的转基因玉米(Ms45(外源)/-,ms45/ms45)，检测花粉的活性。如果花粉的活性正常，说明该转化体基因能够恢复雄性的育性能力。选择转基因玉米可以通过喷洒草甘膦(1倍大田建议剂量)实现，只有转基因玉米株才能成活。选择ms45的基因型可以通过分子标记技术实现。
其次，根据转化的T-DNA特征选择性状优良的转化体。
利用载体TWS-HC获得的转化体，选择的主要依据是同时满足：1)转基因种子具有明显的红色荧光；2)该转基因玉米可以被大田使用剂量1倍的烟嘧磺隆杀灭。
利用载体TWS-CS获得的转化体，选择的主要依据是同时满足：1)转基因种子具有明显的红色荧光；2)转基因玉米籽粒比同样遗传背景的不含有外源基因的籽粒显著小。优选小50％或以上的转化体。
利用载体TWS-HS获得的转化体，选择的主要依据是同时满足：1)转基因玉米籽粒比同样遗传背景的不含有外源基因的籽粒显著小，并优选小50％或以上的转化体；2)该转基因玉米可以被大田使用剂量1倍的烟嘧磺隆杀灭。
最后，通过花粉管萌发试验，选择大约50％的花粉管萌发困难的转化体。
实施例4 将野生型自交系(Ms45Ms45)转变为纯合隐性自交系(ms45/ms45)
以ms45纯合隐性突变体为母本，与不同的优良自交系，如郑58，杂交。获得的F1继续与郑58回交，对获得的BC1群体进行基因型分析，鉴定Ms45位点为杂合的植株继续与郑58回交。如此回交5代后，利用分子标记筛选Ms45位点为杂合，其他位点均为郑58的单株进行自交，从而获得ms45纯合隐性自交系郑58(ms45/ms45)，该自交系即可作为不育系。
实施例5 将外源基因导入ms45纯合隐性自交系
将实施例4中的回交5次的后代郑58(杂合子ms45/Ms45)与实施例3中选择获得的转基因植株杂交，选择带有隐性ms45和外源基因的后代，然后再以实施例4中回交5次的郑58后代(杂合子ms45/Ms45)作为轮回亲本进行5代回交，选择遗传背景与郑58相似、带有隐性ms45和外源基因的单株。如此5代回交以后，选择选择遗传背景与郑58高度相似、带有隐性ms45和外源基因的单株进行自交，并从自交后代中选择含ms45隐性纯合基因的转基因自交系(Ms45/-,ms45/ms45)。
实施例6 提高雄性不育种子的纯度
利用载体TWS-HC获得的转基因雄性不育保持系制备的不育母本，通过荧光识别就可以去除绝大部分的转基因雄性可育籽粒，在雄性不育母本生长到2-8叶时期，进一步喷洒1倍的烟嘧磺隆大田建议使用剂量，可能混杂的转基因植株(雄性可育)就可以被杀灭而去除。这样就进一步排除了雄性可育的玉米株，进一步提高杂交玉米的纯度。
利用载体TWS-CS获得的转基因雄性不育保持系制备的不育母本，通过荧光识别就可以去除绝大部分的转基因雄性可育籽粒。获得的雄性不育种子进一步去除籽粒比较小(比雄性不育种子平均重量小30％以上)的种子。可能混杂的转基因种子(雄性可育)就可以被进一步去除。这样就进一步排除了雄性可育的玉米株，进一步提高杂交玉米的纯度。
利用载体TWS-HS获得的转基因雄性不育保持系制备的不育母本，通过去除小种子(至少比雄性不育种子平均重量小30％以上)排除绝大部分的转基因雄性可育籽粒。获得的雄性不育种子在制种过程中，在不育母本生长到2-8叶时期进一步喷洒1倍的大田建议烟嘧磺隆使用剂量。可能混杂的转基因植株(雄性可育)就可以被杀灭而去除。这样就进一步排除了雄性可育的转基因玉米株，进一步提高杂交玉米的纯度。
实施例7 利用雄性不育保持系大规模扩繁雄性不育自交系(ms45/ms45)
以郑58自交系为例，将实施例5中的雄性不育保持系郑58(Ms45/-,ms45/ms45)与雄性不育系郑58(ms45/ms45)间隔种植，每一行雄性不育保持系郑58(Ms45/-,ms45/ms45)，种5行雄性不育系郑58(ms45/ms45)。由于提供花粉的雄性不育保持系郑58产生的活性花粉都是非转基因花粉(转基因花粉不能够萌发)，从获得雄性不育系郑58(ms45/ms45)植株上获得的种子仍然都是非转基因的雄性不育种子。

Claims (10)

  1. 一种转基因玉米核雄性不育保持系，其特征在于所述保持系是将外源T-DNA载体导入玉米基因组构建而成，导入的外源T-DNA中除了雄性核不育的育性恢复基因以外，还包含如下3种功能片段中至少2个功能片段：1)在种子胚乳中表达一种荧光蛋白质的表达框；2)一种可以抑制玉米抗除草剂基因CYP81A9表达的DNA片段；3)一种可以抑制玉米胚乳特异表达的细胞壁转化酶InCW2表达的DNA片段。
  2. 如权利要求1所述转基因玉米核雄性不育保持系，其特征在于外源T-DNA中除了雄性核不育的育性恢复基因以外，还包含如下2个DNA功能片段：1)在种子胚乳中表达一种荧光蛋白质的表达框；2)一种可以抑制玉米抗除草剂基因CYP81A9表达的DNA片段。
  3. 如权利要求1所述转基因玉米核雄性不育保持系，其特征在于外源T-DNA中除了雄性核不育的育性恢复基因以外，还包含至少如下2个DNA功能片段：1)在种子胚乳中表达一种荧光蛋白质的表达框；2)一种可以抑制玉米胚乳特异表达的细胞壁转化酶InCW2的DNA片段。
  4. 如权利要求1所述转基因玉米核雄性不育保持系，其特征在于外源T-DNA中除了雄性核不育的育性恢复基因以外，还包含至少如下2个DNA功能片段：1)一种可以抑制玉米抗除草剂基因CYP81A9表达的DNA片段；2)一种可以抑制玉米胚乳特异表达的细胞壁转化酶InCW2的DNA片段。
  5. 如权利要求1所述转基因玉米核雄性不育保持系，其特征在于雄性核不育的育性恢复基因核苷酸序列为SEQ ID NO.3所示，在种子胚乳中表达一种荧光蛋白质的表达框核苷酸序列为SEQ ID NO.5所示；所述可以抑制玉米抗除草剂基因CYP81A9表达的DNA片段核苷酸序列为SEQ ID NO.10所示；所述可以抑制玉米胚乳特异表达的细胞壁转化酶InCW2表达的DNA片段核苷酸序列为SEQ ID NO.6所示。
  6. 如权利要求1所述转基因玉米核雄性不育保持系，其特征在于所述保持系还包含选择标记表达框和/或在花粉中表达的α-淀粉酶的表达框，所述选择标记表达框为草铵膦耐受基因表达框。
  7. 如权利要求6所述转基因玉米核雄性不育保持系，其特征在于在花粉中表达的α-淀粉酶的表达框核苷酸序列如SEQ ID No.4所示，所述草铵膦耐受基因的核苷酸序列如SEQ ID No.7示。
  8. 如权利要求6所述转基因玉米核雄性不育保持系，其特征在于外源T-DNA载体由下列之一表达框构成：(1)抑制玉米抗除草剂基因CYP81A9表达的DNA片段、草铵膦耐受基因表达框、雄性核不育的育性恢复基因表达框、在花粉中表达的α-淀粉酶的表达框、在种子胚乳中表达一种荧光蛋白质的表达框；(2)抑制玉米抗除草剂基因CYP81A9表达的DNA片段、草铵膦耐受基因表达框、雄性核不育的育性恢复基因表达框、在花粉中表达的α-淀粉酶的表达框、抑制玉米胚乳特异表达的细胞壁转化酶InCW2表达的DNA片段；(3)雄性核不育的育性恢复基因表达框、在花粉中表达的α-淀粉酶的表达框、在种子胚乳中表达一种荧光蛋白质的表达框、抑制玉米胚乳特异表达的细胞壁转化酶InCW2表达的DNA片段、草铵膦耐受基因表达框。
  9. 一种含权利要求1或6所述转基因玉米核雄性不育保持系中T-DNA载体的植物细胞。
  10. 一种权利要求1所述转基因玉米核雄性不育保持系在制备非转基因核不育种子中的应用，其特征在于所述应用的方法为：将转基因玉米核雄性不育保持系与纯合隐性不育系间隔种植，通过下列方法之一筛选非转基因核不育种子：(1)当保持系T-DNA载体包含在种子胚乳中表达一种荧光蛋白 质的表达框和可以抑制玉米抗除草剂基因CYP81A9表达的DNA片段时，首先利用荧光识别方法去除荧光种子，然后在出苗以后喷洒烟磺隆和/或苯达松进一步杀灭仍然混杂的转基因苗；(2)当保持系T-DNA载体包含在种子胚乳中表达一种荧光蛋白质的表达框和可以抑制玉米胚乳特异表达的细胞壁转化酶InCW2的DNA片段时，同时采用荧光和籽粒显著小的特征清除转基因种子；(3)当保持系T-DNA载体包含可以抑制玉米抗除草剂基因CYP81A9表达的DNA片段和可以抑制玉米胚乳特异表达的细胞壁转化酶InCW2的DNA片段时，首先清除籽粒显著小的种子，然后在出苗以后喷洒烟磺隆和/或苯达松进一步杀灭仍然混杂的转基因苗。
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CN113621724B (zh) * 2020-05-07 2024-05-03 海南波莲水稻基因科技有限公司 一种快速鉴定水稻gat植株的多重pcr检测试剂盒及应用
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