WO2015035951A1 - 雄性核不育基因及其突变体在杂交育种上的应用 - Google Patents

雄性核不育基因及其突变体在杂交育种上的应用 Download PDF

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WO2015035951A1
WO2015035951A1 PCT/CN2014/086505 CN2014086505W WO2015035951A1 WO 2015035951 A1 WO2015035951 A1 WO 2015035951A1 CN 2014086505 W CN2014086505 W CN 2014086505W WO 2015035951 A1 WO2015035951 A1 WO 2015035951A1
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plant
gene
seq
nucleotide sequence
sequence
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French (fr)
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周君莉
汪颖
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兴旺投资有限公司
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Priority to US14/917,477 priority patent/US10117390B2/en
Publication of WO2015035951A1 publication Critical patent/WO2015035951A1/zh
Priority to US16/142,800 priority patent/US10856481B2/en

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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C12N15/8289—Male sterility
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
    • A01H1/022—Genic fertility modification, e.g. apomixis
    • A01H1/023—Male sterility
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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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
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10—Seeds
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
    • A01H6/4684—Zea mays [maize]
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/823—Reproductive tissue-specific promoters
    • C12N15/8231—Male-specific, e.g. anther, tapetum, pollen

Definitions

  • the invention belongs to the field of biotechnology, and particularly relates to crop cross breeding methods, including sterile line breeding and hybrid seed preparation, and more particularly to the maize ZmFL1 gene or its allele and mutant plants produced by the gene variation.
  • Maize is China's main food crop, and plays a huge role in feed and bioenergy. It has become the crop with the largest planting area and the largest total output in China. Heterosis can significantly increase crop yield, resistance and quality. Maize is one of the early hybrids to use heterosis. In 1924, the first corn hybrid was born. The main bottleneck in the utilization of maize heterosis is the maternal emasculation during the seed production process. Commercial breeding mainly uses two methods to de-male: artificial emasculation and mechanical emasculation.
  • male sterile seeding can not only avoid the problem of the purity of the seed due to the incomplete male emasculation, but also replace the mechanical emasculation and reduce the cost of seed production.
  • the most important thing to use this technology is to obtain the sterility trait.
  • a male sterile line that is completely, stable, and easy to find.
  • Maize cytoplasmic male sterility is susceptible to leaf spot disease and it is difficult to obtain a restorer line, while nuclear male sterile line can overcome leaf spot disease and easily find a restorer line. Therefore, the study of strengthening maize nuclear male sterile mutants and their control genes is of great significance for maize cross breeding and production.
  • the present invention provides a corn pollen development gene and a male sterile line produced based on the mutation of the gene, which is stable in fertility and is not affected by environmental conditions.
  • the gene and the sterile line produced by the mutation of the gene provide the necessary components for constructing a novel hybrid breeding system.
  • the invention includes a fertility-related gene and its nucleotide and protein sequences, and also includes the use of the gene in regulating male fertility of plants.
  • any of the methods described below can be used with the corresponding nucleotide sequences provided by the present invention, for example, introducing a mutant sequence of the fertility gene into a plant to cause male sterility of the plant, Mutation of the endogenous sequence of the plant, introduction of the antisense sequence of the sequence into the plant, use of a hairpin form, or attachment to other nucleotide sequences to modulate the phenotype of the plant, or is known to those skilled in the art Any of a variety of methods that affect the male fertility of the plant.
  • the present invention provides a male sterile restorer gene and a male sterile mutant material of the gene, and the use of the gene and its mutant material in breeding.
  • the invention provides a fertility restorer gene FL1, the nucleotide sequence of which is selected from one of the following group of sequences:
  • the maize ZmFL1 gene has the nucleotide sequence shown in SEQ ID NO: 1 or 2, the encoded amino acid sequence is set forth in SEQ ID NO: 3; and the rice OsFL1 gene has the nucleoside as shown in SEQ ID NO: 6.
  • the Arabidopsis AtFL1 gene has a nucleotide sequence as shown in SEQ ID NO: 10, and the encoded amino acid sequence is shown in SEQ ID NO: 11.
  • the fertility restorer gene FL1 of the present invention further comprises a homologous nucleotide sequence of SEQ ID NO: 1, 2, 6, 8 or 10 and has the same fertility regulating function.
  • the highly homologous functionally equivalent sequence includes a DNA sequence capable of hybridizing under stringent conditions to a DNA having the sequence set forth in SEQ ID NO: 1, 2, 6, 8, or 10.
  • stringent conditions are well known and include, for example, hybridization at 60 ° C for 12-16 hours in a hybridization solution containing 400 mM NaCl, 40 mM PIPES (pH 6.4) and 1 mM EDTA, followed by 0.1 at 65 °C. Wash the SDS, and 0.1% SSC wash solution for 15-60 minutes.
  • the functional equivalent sequence further comprises at least 90%, 95% of the sequence set forth in SEQ ID NO: 1, 2, 6, 8, or 10. 96%, 97%, 98%, or 99% sequence identity, and a DNA sequence with a fertility regulatory function, can be isolated from any plant.
  • the percentage of sequence identity can be obtained by well-known bioinformatics algorithms, including Myers and Miller algorithm (Bioinformatics, 4(1): 11-17, 1988), and Needleman-Wunsch global alignment method (J. Mol. Biol., 48(3): 443-53, 1970), Smith-Waterman local alignment (J. Mol.
  • the invention also provides an expression cassette comprising the DNA sequence of the fertility restoring gene disclosed herein, one of the sequences selected from the group consisting of:
  • the present invention also provides a plant male sterility mutant by mutating a plant endogenous SEQ ID NO: 1, 2, 6, 8 or 10 gene, Or mutating the nucleotide sequence of a gene that is highly homologous to the plant, causing the plant to lose the process of male fertility.
  • the "mutation” includes, but is not limited to, a method such as a mutation caused by a physical or chemical method, and the chemical method includes mutagenesis caused by treatment with a mutagen such as EMS, and the mutation may also be a point mutation.
  • It may also be a DNA deletion or insertion mutation, or may be a method by gene silencing means such as RNAi or by site-directed mutagenesis, and the method of site-directed mutagenesis includes, but is not limited to, ZFN site-directed mutagenesis method, TALEN site-directed mutagenesis method, and/or Site-directed mutagenesis methods such as CRISPR/Cas9. .
  • the maize male-sterile mutant zmfl1 provided by the present invention contains a mutant male-sterile gene caused by Mutator insertion, and the present invention finds the gene caused by two Mutator insertions in the maize fertility gene ZmFL1.
  • Mutations, which cause the male sterility phenotype of maize have Mutator insertion sites of chr1:80,964,768 (MU1) and chr1:80,963,850 (MU3), respectively, and the early termination of gene expression due to Mutator insertion, unable to encode functional proteins .
  • the present invention also provides a promoter pZmFL1 capable of initiating gene expression, the nucleotide sequence of which is set forth in SEQ ID NO: 4 or SEQ ID NO: 5.
  • SEQ ID NO: 4 or SEQ ID NO: 5 was ligated to the reporter gene GUS, and the vector was constructed to transform rice, and the GUS expression activity and expression pattern in the transgenic plants were detected and analyzed by using the roots, stems, leaves and flowers of the transgenic plants. GUS staining analysis revealed that the pZmFL1 promoter drives the GUS gene. It is expressed in plant anthers, specifically in the late stage of pollen development.
  • the SEQ ID NO: 4 or 5 promoter provided by the present invention is an anther-specific expression promoter.
  • the plant anther-specific expression promoter pZmFL1 provided by the present invention comprises the nucleotide sequence shown by SEQ ID NO: 4 or 5 in the Sequence Listing or comprises the nucleotide sequence set forth in SEQ ID NO: 4 or 5. a nucleotide sequence having more than 90% similarity, or comprising 100 and more than 100 consecutive nucleotide fragments derived from the sequence of SEQ ID NO: 4 or 5, and driving a nucleoside operably linked to the promoter
  • the expression of acid sequences in plant anthers Expression vectors, transgenic cell lines, host bacteria and the like containing the above sequences are all within the scope of the present invention. Primer pairs that amplify any of the nucleotide fragments of the SEQ ID NO: 4 or 5 promoter disclosed herein are also within the scope of the invention.
  • the promoter nucleotide sequences provided by the present invention can also be used to isolate corresponding sequences from other plants other than maize, especially homologously cloned from other monocots. Based on the sequence homology between these corresponding sequences and the promoter sequences listed herein, or homology to the present promoter gene, techniques such as PCR, hybridization, and the like are used to identify and isolate these corresponding sequences. Accordingly, corresponding fragments isolated according to their sequence similarity to the SEQ ID NO: 4 or 5 promoter sequences (or fragments thereof) set forth herein are also included in the embodiments.
  • the promoter region of this embodiment can be isolated from any plant including, but not limited to, Brassica, corn, wheat, sorghum, two genus, white mustard, castor, sesame, cottonseed, linseed, soybean, and virgin Mustard, Bean, Peanut, Alfalfa, Oat, Rapeseed, Barley, Oat, Rye, Millet, Pestle, Tritical, Single Wheat, Spelt, Durum, Flax, Gramma grass, rubbing grass, false scorpion, fescue, perennial wheat straw, sugar cane, cranberry, papaya, banana, safflower, oil palm, cantaloupe, apple, cucumber, sarcophagus, sword Orchid, chrysanthemum, lily family, cotton, alfalfa, sunflower, canola, sugar beet, coffee, yam, ornamental plants and pines.
  • a “promoter” as used herein refers to a DNA regulatory region that typically comprises a TATA box that directs RNA polymerase II to initiate RNA synthesis at a suitable transcription initiation site for a particular coding sequence. Promoters may also contain additional recognition sequences, which are typically located upstream or 5' of the TATA box, commonly referred to as upstream promoter elements, which function to regulate transcription efficiency. It will be appreciated by those skilled in the art that while nucleotide sequences for the promoter regions disclosed herein have been identified, other regulatory elements for isolating and identifying upstream regions of the TATA box of a particular promoter region identified in the present invention are also Within the scope of the invention.
  • the promoter regions disclosed herein are generally further defined as comprising upstream regulatory elements, such as those elements, enhancers, and the like, for regulating tissue expression and temporal expression functions of the coding sequences.
  • upstream regulatory elements such as those elements, enhancers, and the like
  • promoter elements that enable expression in a target tissue can be identified and isolated for use with other core promoters to verify the preferential expression of male tissues.
  • the core promoter refers to the minimal sequence required for initiation of transcription, such as the sequence known as the TATA box, which is commonly found in the promoters of genes encoding proteins.
  • the upstream promoter of the FL2 gene can be associated with its own or from other sources of core promoters. Use together.
  • the core promoter may be any known core promoter, such as the cauliflower mosaic virus 35S or 19S promoter (U.S. Patent No. 5,352,605), the ubiquitin promoter (U.S. Patent No. 5,510,474), the IN2 core promoter ( U.S. Patent No. 5,364,780) or the Scrophularia mosaic virus promoter.
  • the function of the gene promoter can be analyzed by operably linking the promoter sequence to the reporter gene to form a transformable construct, which is then transferred into the plant and passed through the transgenic progeny. Observing the expression of the reporter gene in various tissues and organs of the plant to confirm its expression characteristics; or subcloning the above construct into an expression vector for transient expression experiments, and detecting the function of the promoter or its regulatory region by transient expression experiments
  • the choice of appropriate expression vector for testing the function of the promoter or regulatory region will depend on the host and the method by which the expression vector is introduced into the host, such methods being well known to those of ordinary skill in the art.
  • the regions in the vector include regions that control transcription initiation and control processing. These regions are operably linked to a reporter gene, including the YFP, UidA, GUS gene or luciferase.
  • An expression vector comprising a putative regulatory region located in a genomic fragment can be introduced into a complete tissue, such as a staged pollen, or introduced into a callus for functional verification.
  • the promoter pZmFL1 of the present invention can be ligated to a nucleotide sequence other than the FL1 gene to express other heterologous nucleotide sequences.
  • the promoter nucleotide sequences of the present invention, and fragments and variants thereof, can be assembled together with a heterologous nucleotide sequence in an expression cassette for expression in a plant of interest, more specifically, in a male organ of the plant. expression.
  • the expression cassette has suitable restriction sites for insertion of the promoter and heterologous nucleotide sequences. These expression cassettes can be used to genetically manipulate any plant to achieve the desired corresponding phenotype.
  • the maize pZmFL1 promoter disclosed herein can be used to drive expression of a heterologous nucleotide sequence such that a transformed plant obtains a male sterile phenotype that encodes a carbohydrate degradation Enzyme or modified enzyme, amylase, debranching enzyme and pectinase, more specifically such as a amylase gene, auxin (auxin), rot B, cytotoxin gene, diphtheria toxin, DAM methylase, pro
  • the gemini may be selected from a prokaryotic regulatory system or a dominant male sterility gene.
  • nucleic acid as referred to in the invention operably linked downstream of a promoter of the invention can be a structure operably linked to a promoter disclosed herein.
  • the invention also provides an expression cassette comprising:
  • the expression cassette of the present invention comprises the promoter of the present invention SEQ ID NO: 4 or 5 in the transcription direction of 5'-3', operatively A nucleic acid, optionally a transcriptional and translational termination region (eg, a transcription termination element or a polyadenylation signal) ligated downstream of the promoter of the invention, SEQ ID NO: 4 or 5.
  • the expression cassette of the present invention may further comprise an origin of replication required for replication in bacteria (for example, an ORI region from pBR322 or P15A ori), an element required for Agrobacterium T-DNA transfer (for example, the left border of T-DNA) And / or right border).
  • an enhancer an intron, a multiple cloning site, an operator, a repressor binding site, a transcription factor binding site, and the like.
  • exemplary enhancers include enhancer elements from the CaMV 35S promoter, the octopine synthase gene, the rice actin I gene, the corn alcohol dehydrogenase gene, the maize dwarf I gene, the TMV ⁇ element, and the promoter of yeast.
  • Viral leader sequences can also be employed as a prodigy with enhancer utility, such as leader sequences from tobacco mosaic virus (TMV), maize chlorotic spot virus (MCMV), and alfalfa mosaic virus (AMV).
  • Exemplary plant introns include introns from Adh 1, bronze 1, actin 1, actin 2, and sucrose synthase introns.
  • a nucleic acid as referred to in the present invention operably linked downstream of the promoter of the present invention, SEQ ID NO: 4 or 5, wherein said "nucleic acid” may be operably linked to a promoter disclosed herein.
  • the fertility regulatory gene SEQ ID NO: 1, 2, 6, 8, or 10 provided by the present invention can be constructed downstream of the promoter SEQ ID NO: 4 or 5, thereby driving the fertility regulatory gene in anther Specific expression in SEQ ID NO: or by constructing a DNA vector which can be silenced by SEQ ID NO: 4 or 5 and which can silence the gene of SEQ ID NO: 1, 2, 6, 8, or 10, thereby obtaining SEQ ID NO: Male sterile mutant of 1, 2, 6, 8 or 10 genes.
  • any of the above nucleic acid sequences can be operably linked to the SEQ ID NO: 4 or 5 promoter sequence of the present invention and expressed in plants.
  • the anther-specific expression promoter provided by the invention can be used for the specific expression of a foreign gene in anther, thereby avoiding the adverse effects of the sustained expression of the foreign gene in other tissues of the plant, and can also be used for plant pollen.
  • Functional analysis and identification of genes related to growth and development can be used for the creation of male sterile lines and restorer lines; and can be applied to pollen abortion experiments to avoid biosafety problems caused by plant transgenic drift or pollen escape, The creation of plant male sterile lines and restorer lines is of great significance.
  • the expression cassette provided by the invention can be inserted into a plasmid, cosmid, yeast artificial chromosome, bacterial artificial chromosome or any other vector suitable for transformation into a host cell.
  • Preferred host cells are bacterial cells, especially bacterial cells for cloning or storing polynucleotides, or for transforming plant cells, such as Escherichia coli, Agrobacterium tumefaciens and Agrobacterium rhizogenes.
  • the expression cassette or vector can be inserted into the genome of the transformed plant cell. Insertions can be either positioned or randomly inserted.
  • the insertion is achieved by, for example, homologous recombination.
  • the expression cassette or vector can be kept extrachromosomally.
  • the expression cassette or vector of the present invention may be present in the nucleus or leaf of a plant cell In chloroplasts, mitochondria and/or plastids.
  • the expression cassette or vector of the invention is inserted into the chromosomal DNA of the plant cell nucleus.
  • the present invention provides a method of producing a plant comprising:
  • the plants obtained in the step (4) are propagated to obtain progeny.
  • the transgenic plants of the invention are prepared using transformation methods known to those skilled in the art of plant biotechnology. Any method can be used to transform a recombinant expression vector into a plant cell to produce a transgenic plant of the invention. Transformation methods can include direct and indirect transformation methods. Suitable direct methods include polyethylene glycol-induced DNA uptake, liposome-mediated transformation, introduction using a gene gun, electroporation, and microinjection, and the like. In a particular embodiment of the invention, the invention employs an Agrobacterium-based transformation technique (see Horsch RB et al. (1985) Science 225: 1229; White FF, Vectors for Gene Transfer in Higher Plants, Transgenic Plants, Vol. 1 , Engineering and Utilization, Academic Press, 1993, pp.
  • Agrobacterium-based transformation technique see Horsch RB et al. (1985) Science 225: 1229; White FF, Vectors for Gene Transfer in Higher Plants, Transgenic Plants, Vol. 1 , Engineering and Utilization, Academic Press, 1993,
  • Agrobacterium strains (eg, Agrobacterium tumefaciens or Agrobacterium rhizogenes) comprise a plasmid (Ti or Ri plasmid) and a T-DNA element, which are transferred to plants after transfection with Agrobacterium, and the T-DNA is integrated Into the genome of plant cells.
  • the T-DNA can be located on the Ri-plasmid or Ti-plasmid, or independently in a so-called binary vector.
  • Agrobacterium-mediated transformation methods are described, for example.
  • Agrobacterium-mediated transformation is most suitable for dicotyledons, but is also suitable for monocots.
  • the transformation of Agrobacterium to plants is described, for example. Transformation can result in transient or stable transformation and expression.
  • the nucleotide sequences of the present invention can be inserted into any of the plants and plant cells that fall into these broad categories, they are particularly suitable for use in crop plant cells.
  • the present invention also encompasses the use of the disclosed FL1 gene and its promoter.
  • the FL1 gene provided by the present invention or a promoter thereof can be used to achieve FL1 or other similar fertility-related gene mutations. The reproduction and maintenance of the obtained male sterile lines.
  • the above-mentioned male sterile line is propagated and maintained, and the homozygous recessive nuclear male sterility mutant is used as a transforming receptor material, and the three closely related target genes are transformed into the sterile mutant recipient plant.
  • the three target genes are a fertility restorer gene, a pollen inactivating gene, and a selection marker gene, respectively.
  • the fertility restoration gene can restore the transformation of infertility by sports, and the pollen inactivating gene can inactivate the pollen containing the transformed foreign gene, that is, lose the ability to inseign, and screen the gene. It can be used for the sorting of genetically modified seeds and non-transgenic seeds.
  • the sorted non-transgenic seeds are used as hybrids for the production of sterile lines, and the transgenic seeds are used as a source of maintenance to continuously and stably produce sterile lines.
  • the maize nuclear recessive sterility sterility zmfl1/zmfl1 mutant can be used as a transforming receptor material, and the three closely related target genes can be transformed into the sterile line: among them, fertility
  • the restorer gene ZmFL1 can restore the transformation to sports;
  • the pollen inactivating gene Zm-PA can inactivate the pollen containing the foreign gene, ie lose the ability to insemination;
  • the fluorescent color-selection gene RFP(r) is used for transgenic seeds and non-GM seeds. Sorting, sorted non-GM seeds are used as hybrid lines for the production of sterile lines, and transgenic seeds are used as a source of maintenance lines to continuously produce sterile lines. Because the technology uses biotechnology to produce non-GM products, it solves the bottleneck problem in the corn hybrid seed production process.
  • the present invention Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a corn pollen development gene and a male sterile line produced based on the mutation of the gene, and the sterile line is stable and free from the environment. Conditional effects, Mutator insertions at both sites found on this gene sequence can result in stable male sterility mutations.
  • the gene and the sterile line produced by the mutation of the gene provide resources for maize cross breeding, and also provide the necessary components for constructing a third-generation hybrid breeding system.
  • the male sterile line produced by the mutation is used to produce hybrid seeds. It is of great significance to break through and improve the existing "three-line” and "two-line” hybrid technology.
  • Figure 1 shows the comparison of the anthers of the wild-type individual plants with the zmfl1 sterile mutants in the loose powder and mature stages.
  • Figure 2 is a comparison of anther maturity stages of wild type individuals and zmfl1 infertile mutants (scanning electron microscopy results).
  • Fig. 3 shows the results of semi-thin section analysis of anthers of wild type individuals and zmfl1 infertile mutants.
  • Figure 4 shows the pollen development process of wild-type individuals and zmfl1 sterile mutants by DAPI staining.
  • Fig. 5 is a view showing the development of the austenite and pollen grains of a wild type single plant and a zmfl1 sterile mutant by transmission electron microscopy.
  • Fig. 6 is a structural analysis of mononuclear microspores to binuclear pollen grains of pollen of wild type single plants and zmfl1 sterile mutants.
  • Figure 7 is a map clone of a male sterile mutant gene.
  • Figure 8 is the structure of a candidate gene for a sterile mutant.
  • Figure 9 shows the expression of GUS in the tissues and organs of rice by the pZmFL1 promoter, in which A is the root and B is the stem;
  • C is the leaf
  • D-I is the staining of rice flower in each period.
  • Figure 10 is a sequence alignment of predicted proteins of the ZmFL1 gene with predicted homologous proteins in the rice, sorghum, and Arabidopsis genomes.
  • Mutator is one of the most active and mutagenic transposons found in plants, with its high positive-positive mutation rate, tendency to insert gene-rich regions and low-copy sequence regions. It plays an important role in the study of maize functional genes and the construction of mutant libraries.
  • the mutant library was constructed using the Mutator9 material and the domestic excellent inbred ensemble 31. The two were crossed to obtain M 1 , and M 1 was planted and selfed to obtain M 2 seeds. M 1 is divided into single ear harvesting, threshing, as a strain. Each plant has 20 M2 seeds, which were planted on the campus of Hunan Agricultural University in March 2009. Plant 2 rows, 10 per line, single seeding. One of the 1000 strains was found to have three sterile plants, nine fertile plants, and sisters, and the sterile line was named zmfl1.
  • Example 3 Physical stability analysis of zmfl1 male sterile mutation
  • Example 2 The zmfl1 sterile single plant in Example 1 was crossed with the fertile single plant, and the infertile parents and isolated populations obtained from three different ecological sites in Sanya, Hainan, Changsha and Beijing were investigated in the loose powder period. All the single plant fertility of the population can obtain the infertile phenotype consistent with the parent, indicating that the sterile phenotype controlled by the fertility gene is not affected by temperature and light conditions. Specifically, the fertility performance of the parents of the parental plants at different times and at different locations is shown in Table 2. The fertility performance of the isolated population at different times and at different locations is shown in Table 3:
  • the mononuclear microspores of the pollen grains of wild type individuals and zmfl1 infertile mutants were analyzed.
  • the outer wall of pollen grains of wild type plants formed obvious outer wall outer layer, outer wall inner layer and columnar layer (Fig. 6, A).
  • Infertile mutants form only the inner layer of the outer wall and a small amount of sporopollen substance (Fig. 6, B); the late stage of mononuclear microspores to the binuclear pollen grain stage, the outer layer of pollen grains of the wild type single plant Thickening (Fig. 6, C), the pollen grain outer wall layer of the sterile mutant body still has the outer layer inner layer and the sporopollen substance filled thereon, and no obvious three-layer outer wall is formed (Fig. 6, D).
  • the zmfl1 sterile mutant was used as the female parent, and the wild type inbred line Zheng 58 was crossed, and the F1 generation was selfed to construct the F2 population.
  • the corn loose powder period whether the anthers were exposed, the anther color and the presence or absence of pollen were used as the breeding phenotypic identification criteria, and the infertile mutant phenotypes in the F2 population were selected for preliminary localization and fine mapping, and a sterile mutant phenotype was screened. 2757 strains.
  • the target gene is defined between the markers S1 and S11 by preliminary positioning (Fig. 7).
  • the phenotypes of the exchanged plants on both sides of the target gene were combined and genotyped with the developed markers, and the results were found to be labeled S2, S3, S4, S5 and the target gene.
  • the number of exchanged plants was reduced to 29, 25, 16 and 9 respectively, and the exchanged plants between S10, S9, S8 and the target gene were reduced to 14 plants, 13 and 11 plants, respectively.
  • the strains are different; the exchange rate between S, S6, S7 and the target gene is 0.
  • the target gene was finally locked between S5 and S8, and there were 9 exchanged plants and 11 exchanged plants, respectively.
  • the actual physical distance defined by these two markers is approximately 300Kb ( Figure 7).
  • the allele of the male fertility mutant gene was obtained from the MAIZEGDB application, and the equament was found to have a Mutator3 sequence insertion at the first exon of chm1:80,963,850 of ZmFL1, and the phenotype was consistent with the genotype.
  • the two promoter fragments were obtained by maize genome amplification, one promoter was 875 bp in length, and its nucleotide sequence was as shown in SEQ ID NO: 4; the length of one promoter It is 2500 bp and its nucleotide sequence is shown in SEQ ID NO: 5.
  • the two fragments were ligated to GUS to obtain a promoter function to identify a plant expression vector. It was transformed into wild type rice callus by Agrobacterium-mediated method, and 12 and 18 transgenic rice plants were obtained by screening and regeneration, respectively.
  • the expression pattern of pZmFL1 promoter was analyzed by analyzing the activity of B-galactosidase.
  • the GUS staining analysis was carried out in the roots, stems, leaves and flowers of transgenic plants. It was found that the pZmFL1 promoter drives GUS mainly in anthers, more specifically. The expression of GUS staining in the late stage of pollen development is shown in Figure 9.
  • Example 8 Sequence alignment of predicted proteins of the ZmFL1 gene with predicted homologous proteins in the rice, sorghum, and Arabidopsis genomes
  • the full-length sequence of the protein encoded by the maize ZmFL1 gene was searched in the protein database using the protein blast tool, and the predicted homologous proteins in the rice, sorghum, and Arabidopsis genomes were obtained, and these protein sequences were aligned. Analysis showed that the homologous proteins from different plants have very similar conserved sequences, and the homology between them is very high (Fig. 10), indicating that the protein is biologically conserved during the development of male organs of plant flowers. Very important role.
  • the genomic nucleotide sequence of the homologous gene OsFL1 in rice is shown in SEQ ID NO: 6, and the encoded amino acid sequence is shown in SEQ ID NO: 7; the genomic nucleotide of the homologous gene SbFL1 in sorghum The sequence is set forth in SEQ ID NO: 8, the encoded amino acid sequence is set forth in SEQ ID NO: 9; the genomic nucleotide sequence of the homologous gene AtFL1 in Arabidopsis is shown in SEQ ID NO: 10, The encoded amino acid sequence is set forth in SEQ ID NO:11.
  • the ZmFL1 gene can be used in a new generation of cross breeding technology.
  • the core idea of this technology is to transform the tightly linked three target genes into infertile mutants by using the maize nuclear recessive male sterility mutant as a transforming receptor material.
  • the fertility restoration gene can make the transformation be restored by sports.
  • the pollen inactivating gene can inactivate the pollen containing the foreign gene, that is, lose the ability to insemination, and the screening gene can be used for the sorting of the transgenic seed and the non-GM seed.
  • the sorted non-transgenic seeds are sterile lines, and the transgenic seeds are used as maintainer lines.
  • the sterile line By maintaining a pollination hybridization to the sterile line, the sterile line can be sturdy, thereby breeding the sterile line.
  • the maintainer system can be continuously reproduced through self-intersection. Because the technology uses biotechnology to produce non-GMO products, it solves the problem of artificial or mechanical emasculation during corn hybrid seed production, eliminating the need for manual emasculation or mechanical emasculation, and can provide higher quality and purity seeds to growers. , saving labor costs.
  • a plant expression vector was constructed using the maize ZmFL1 gene.
  • the expression vector comprises three expression cassettes of Zm-AA1 (pollen inactivating gene), ZmFL1 (fertility restorer gene) and RFP (red fluorescent color selection gene).
  • ZmFL1 fertility restorer gene can restore transformation of sports
  • Zm-AA1 pollen inactivation Genes can inactivate pollen containing foreign genes, ie, lose fertility
  • RFP screening genes can be used for sorting of transgenic seeds and non-transgenic seeds, and sorted non-transgenic seeds are sterile lines, while transgenic seeds are used. Keep the system. Thus, a new generation of hybrid breeding technology system was established.
  • the inventors constructed an expression vector using the maize ZmFL1 gene. Before constructing the plant expression vector of maize, the inventors first separately transformed the three expression cassettes of pollen inactivating gene Zm-PA, fertility restoring gene ZmFL1 and screening gene RFP(r) in the expression vector, and further The function of each expression cassette was verified. The results showed that each expression cassette was able to work well when it was transformed into corn alone, achieving the desired design effect.
  • the inventors constructed a maize expression vector of a new generation of cross breeding technology by assembling the following DNA elements:
  • ZmFL1 gene expression cassette consisting of the target gene ZmFL1 and its promoter and terminator, wherein the promoter sequence of the ZmFL1 gene is shown in SEQ ID NO: 5, and the terminator sequence thereof is shown in SEQ ID NO: 16, ZmFL1
  • SEQ ID NO: 15 The genomic DNA sequence of the gene from the initiation codon to the stop codon is as shown in SEQ ID NO: 15, and the amino acid sequence of the protein encoded by the nucleotide sequence is shown in SEQ ID NO:
  • Gene expression cassette PG47: ZM-BT1: ZM-PA: IN2-1 the open reading frame of the target gene ZM-PA (the nucleotide sequence of which is shown in SEQ ID NO: 17) is ligated to the promoter PG47 ( Its nucleotide sequence is shown in SEQ ID NO: 18), the transit peptide ZM-BT1 (the nucleotide sequence of which is shown in SEQ ID NO: 19), and the terminator IN2-1 (the nucleotide sequence thereof Upstream of SEQ ID NO: 20).
  • the constructed expression vector described above was subjected to maize transformation to obtain a positive transgenic maize plant.
  • transgenic maize plants Analysis of the above-mentioned single-copy transgenic maize (containing homozygous zmfl1 recessive sterile locus) plants showed no significant morphological differences between transgenic plants and non-transgenic control plants. However, pollen fertility is significantly different. The transgenic plant material was tested for pollen dyeability, and the wild type corn was tested for pollen dyeability.
  • the method used is as follows: in the flowering stage of maize, a single plant is randomly selected from the transgenic corn plants and the wild type control plants, and each plant takes a spikelet of the tassel, and then a small flower is taken from the spikelet, and the small flower is taken from the small flower.
  • the pollen staining rate of the transgenic maize plants was analyzed.
  • ZmFL1 gene can restore the fertility of male sterile mutant receptors
  • RFP gene can achieve the expected pollen inactivation function and seed screening markers, respectively.

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Abstract

提供玉米杂交育种方法，包括不育系繁殖和杂交种子制备，更具体地提供植物FL1基因或其等位基因以及由该基因变异所产生的突变体植株。

Description

雄性核不育基因及其突变体在杂交育种上的应用 技术领域
本发明属于生物技术领域，具体涉及作物杂交育种方法，包括不育系繁殖和杂交种子制备，更具体地涉及玉米ZmFL1基因或其等位基因以及由该基因变异所产生的突变体植株。
技术背景
玉米是我国主要粮食作物，同时在饲用和生物能源中发挥巨大作用，现已成为我国种植面积最大、总产最多的作物。杂种优势能大幅提高作物产量、抗性和品质，玉米是较早利用杂种优势的作物之一，1924年第一个玉米杂交种诞生。玉米杂种优势利用的主要瓶颈是制种过程中母本去雄。商业育种中主要采用两种方式去雄：人工去雄和机械去雄。但两种去雄方式均存在不足：人工去雄不彻底，容易导致种子纯度下降，同时大幅度地提高成本；机械去雄要求上部叶片稀疏的植株结构和平整且面积较大的地块，但我国玉米品种以紧凑型为主，甘肃等西北制种地难以实现整块连片，因此无法采用机械去雄。同时，玉米杂交种生产中存在着常用育种的亲本遗传基础差异不够大，并因此影响到主要育种目标如高产、稳产、抗逆、早熟等的尽快实现。采用雄性不育制种，既能避免由于母本去雄不彻底导致的种子纯度下降的问题，又能取代机械去雄，降低种子生产成本；采用该技术制种最为重要的是获得不育性状彻底、稳定且容易找到恢复系的雄性不育系。玉米细胞质雄性不育易感染叶斑病且难以获得恢复系，而核雄性不育系能克服叶斑病，易于找到恢复系。因此，加强玉米核雄性不育突变体及其控制基因的研究，对玉米杂交育种和生产具有重大意义。
为了解决目前玉米杂交种育种方法中存在的缺陷，如人工去雄、杂交品种资源的局限性、制种技术复杂、制种成本高等技术瓶颈，人们正在尝试新的杂交育种技术，新型的杂交育种技术充分利用隐性核基因控制的雄性不育基因，构建育性稳定不受环境影响的不育系。主要技术优点包括：第一，省去人工去雄或机械去雄步骤，可以提供更高质量及纯度的种子给种植者；第二，所利用的隐性核不育基因适用于绝大多数品种，使杂种优势资源利用大幅提高，解决杂种优势的资源利用问题；第三，简化通过杂交繁殖不育系的过程。本发明即提供了一种玉米花粉发育基因以及基于该基因突变所产生的雄性不育系，该不育系的育性稳定、不受环境条件影响。该基因以及该基因突变产生的不育系为构建新型杂交育种体系提供了必要的元件。
发明内容
本文提到的所有参考文献都通过引用并入本文。
除非有相反指明，本文所用的所有技术和科学术语都具有与本发明所属领域普通技术 人员通常所理解的相同的含义。除非有相反指明，本文所使用的或提到的技术是本领域普通技术人员公知的标准技术。材料、方法和例子仅作阐述用，而非加以限制。
本发明包括一种育性相关基因及其核苷酸和蛋白序列，还包括通过操作该基因在调控植株雄性生育力中的应用。非限制性地举例而言，下文描述的任何方法都可与本发明所提供的相应核苷酸序列一起使用，例如，将所述育性基因的突变体序列引入植株以导致植株雄性不育、使植株内源序列突变、向植株中引入该序列的反义序列、使用发卡形式、或将其与其它核苷酸序列连接起来调控植株的表型，或者是本领域技术人员已知的可用于影响植株的雄性生育力的多种方法中的任一方法。
本发明提供了一个雄性不育恢复基因及该基因的雄性不育突变体材料，及所述基因及其突变体材料在育种中的应用。
在本发明的第一方面，本发明提供了一种育性恢复基因FL1，其核苷酸序列选自下列组的序列之一：
a)具有SEQ ID NO：1或SEQ ID NO:2所示的核苷酸序列；
b)具有SEQ ID NO：6所示的核苷酸序列；
c)具有SEQ ID NO：8所示的核苷酸序列；
d)具有SEQ ID NO：10所示的核苷酸序列；
e)在严格条件下能够与(a)-(d)之任一所述序列的DNA杂交的DNA序列；
f)与(a)-(d)之任一所述序列互补的DNA序列。
其中玉米ZmFL1基因具有如SEQ ID NO:1或2所示的核苷酸序列，其编码的氨基酸序列如SEQ ID NO:3所示；水稻OsFL1基因具有如SEQ ID NO:6所示的核苷酸序列，其编码的氨基酸序列如SEQ ID NO:7所示；高粱SvFL1基因具有如SEQ ID NO:8所示的核苷酸序列，其编码的氨基酸序列如SEQ ID NO:9所示；拟南芥AtFL1基因具有如SEQ ID NO:10所示的核苷酸序列，其编码的氨基酸序列如SEQ ID NO:11所示。
本领域技术人员应该知晓，本发明所述的育性恢复基因FL1还包括与核苷酸序列SEQIDNO：1、2、6、8或10高度同源，并且具有同样的育性调控功能的高度同源的功能等价体序列。所述高度同源的功能等价体序列包括在严格条件下能够与具有SEQ ID NO：1、2、6、8或10所示的序列的DNA杂交的DNA序列。本文中使用的“严格条件”是公知的，包括诸如在含400mM NaCl、40mM PIPES(pH6.4)和1mM EDTA的杂交液中于60℃杂交12-16小时，然后在65℃下用含0.1SDS、和0.1％SSC的洗涤液洗涤15-60分钟。
功能等价体序列还包括与SEQ ID NO：1、2、6、8或10所示序列有至少90％、95％、 96％、97％、98％、或99％序列同一性，且具有育性调控功能的DNA序列，可以从任何植物中分离获得。其中，序列同一性的百分比可以通过公知的生物信息学算法来获得，包括Myers和Miller算法(Bioinformatics，4(1)：11-17，1988)、Needleman-Wunsch全局比对法(J.Mol.Biol.，48(3)：443-53，1970)、Smith-Waterman局部比对法(J.Mol.Biol.，147：195-197，1981)、Pearson和Lipman相似性搜索法(PNAS，85(8)：2444-2448，1988)、Karlin和Altschul的算法(Altschul等，J.Mol.Biol.，215(3)：403-410，1990；PNAS，90：5873-5877，1993)。这对于本领域技术人员来说是熟悉的。
在本发明的第二方面，本发明还提供了一种表达盒，所述表达盒含有本发明所公开的育性恢复基因的DNA序列，选自下列组的序列之一：
(a)具有SEQ ID NO：1、2、6、8或10所示的序列；
(b)在严格条件下能够与(a)所述序列的DNA杂交的DNA序列；
(c)与(a)所述序列有至少90％(优选为至少95％)序列同一性，且具有相同功能的DNA序列；和
(d)与(a)-(c)之任一所述序列互补的DNA序列。
在本发明的第三方面，本发明还提供了一种植物雄性不育突变体，所述雄性不育突变体通过突变植物内源的SEQ ID NO：1、2、6、8或10基因，或突变与其高度同源的基因的核苷酸序列，使该植物体丧失雄性育性的过程。所述“突变”包括但不限于以下方法，如用物理或化学的方法所导致的基因突变，化学方法包括用EMS等诱变剂处理所导致的诱变，所述突变还可以是点突变，也可以是DNA缺失或插入突变，也可以是通过RNAi等基因沉默手段或者通过基因定点突变的方法，所述基因定点突变的方法包括但不限于ZFN定点突变方法、TALEN定点突变方法、和/或CRISPR/Cas9等定点突变方法。。
具体地，本发明所提供的玉米雄性不育突变体zmfl1，其含有Mutator插入造成的突变的雄性不育基因，本发明在该玉米育性基因ZmFL1中分别发现了两个Mutator插入造成的该基因突变，均造成玉米的雄性不育表型，其Mutator插入位点分别为chr1:80,964,768(MU1)和chr1:80,963,850(MU3)，并由于Mutator插入造成了基因表达的提前终止，从而无法编码功能蛋白。
在本发明的第四方面，本发明还提供了一种能启动基因表达的启动子pZmFL1，其核苷酸序列如SEQ ID NO:4或SEQ ID NO:5所示。将SEQ ID NO:4或SEQ ID NO:5与报告基因GUS相连，构建载体转化水稻，检测分析转基因植株中的GUS表达活性和表达模式，通过在转基因植株的根、茎、叶和花中都进行GUS染色分析，结果发现pZmFL1启动子驱动GUS基因在 植物花药中表达，具体的在花粉发育后期表达。说明本发明所提供的SEQ ID NO:4或5启动子是一个花药特异性表达的启动子。
本发明所提供的植物花药特异表达启动子pZmFL1，含有序列表中SEQ ID NO:4或5所示的核苷酸序列，或包含与SEQ ID NO:4或5中所列核苷酸序列具有90％以上相似性的核苷酸序列，或包含来源于SEQ ID NO:4或5序列上的100个及100以上连续的核苷酸片段，并且可以驱动与该启动子操作性连接的核苷酸序列在植物花药中的表达。含有上述序列的表达载体、转基因细胞系以及宿主菌等均属于本发明的保护范围。扩增本发明所公开的SEQ ID NO:4或5启动子的任一核苷酸片段的引物对也在本发明的保护范围之内。
本发明所提供的启动子核苷酸序列还可用于从玉米以外的其它植物中分离相应序列，尤其是从其他单子叶植物中进行同源克隆。根据这些相应序列与本文所列启动子序列间的序列同源性，或与本启动子基因的同源性，使用如PCR、杂交等技术来鉴别分离这些相应序列。因此，根据它们与本文所列的SEQ ID NO:4或5启动子序列(或其片段)间的序列相似性而分离的相应片段，也包括在实施方案中。本实施方案的启动子区域可从任何植物中分离，包括但不限于芸苔属、玉米、小麦、高粱、两节荠属、白芥、蓖麻子、芝麻、棉籽、亚麻子、大豆、拟南芥属、菜豆属、花生、苜蓿、燕麦、油菜籽、大麦、燕麦、黑麦(Rye)、粟、蜀黍、小黑麦、单粒小麦、斯佩尔特小麦(Spelt)、双粒小麦、亚麻、格兰马草(Gramma grass)、摩擦禾、假蜀黍、羊茅、多年生麦草、甘蔗、红莓苔子、番木瓜、香蕉、红花、油棕、香瓜、苹果、黄瓜、石斛、剑兰、菊花、百合科、棉花、桉、向日葵、芸苔、甜菜、咖啡、薯蓣、观赏植物和松类等。
本发明所述的“启动子”是指一种DNA调控区域，其通常包含能指导RNA聚合酶II在特定编码序列的合适转录起始位点起始RNA合成的TATA盒。启动子还可包含其它识别序列，这些识别序列通常位于TATA盒的上游或5’端，通常被称为上游启动子元件，起调控转录效率的作用。本领域技术人员应该知晓，虽然已经鉴定了针对本发明公开的启动子区域的核苷酸序列，但是分离和鉴定处于本发明鉴定的特定启动子区域的TATA盒上游区域的其它调控元件也在本发明的范围内。因此，本文公开的启动子区域通常被进一步界定为包含上游调控元件，例如用于调控编码序列的组织表达性和时间表达功能的那些元件、增强子等。以相同的方式，可以鉴定、分离出使得能在目标组织(例如雄性组织)中进行表达的启动子元件，将其与其它核心启动子一起使用，以验证雄性组织优先的表达。核心启动子指起始转录所需的最小限度的序列，例如被称为TATA盒的序列，这是编码蛋白质的基因的启动子通常都具有的。因此，可选地，FL2基因的上游启动子可与其自身的或来自其它来源的核心启动子关 联使用。
核心启动子可以是任何一种已知的核心启动子，例如花椰菜花叶病毒35S或19S启动子(美国专利No.5,352,605)、泛素启动子(美国专利No.5,510,474)、IN2核心启动子(美国专利No.5,364,780)或玄参花叶病毒启动子。
所述基因启动子的功能可以通过以下方法进行分析：将启动子序列与报告基因可操作性连接，形成可转化的构建体，再将该构建体转入植株中，在获得转基因后代中，通过观察报告基因在植物各个组织器官中的表达情况来确认其表达特性；或者将上述构建体亚克隆进用于瞬时表达实验的表达载体，通过瞬时表达实验来检测启动子或其调控区的功能
用来测试启动子或调控区域功能的适当表达载体的选择将取决于宿主和将该表达载体引入宿主的方法，这类方法是本领域普通技术人员所熟知的。对于真核生物，在载体中的区域包括控制转录起始和控制加工的区域。这些区域被可操作地连接到报告基因，所述报告基因包括YFP、UidA、GUS基因或荧光素酶。包含位于基因组片段中的推定调控区的表达载体可以被引入完整的组织，例如阶段性花粉，或引入愈伤组织，以进行功能验证。
此外，本发明的启动子pZmFL1可与并非FL1基因的核苷酸序列相连，以表达其它异源核苷酸序列。本发明的启动子核苷酸序列及其片段和变体可与异源核苷酸序列一起组装在一个表达盒中，用于在目的植株中表达，更具体地，在该植株的雄性器官中表达。所述表达盒有合适的限制性酶切位点，用于插入所述启动子和异源核苷酸序列。这些表达盒可用于对任何植株进行遗传操作，以获得想要的相应表型。
本发明所公开的玉米pZmFL1启动子,可用于驱动下列异源核苷酸序列的表达,以使转化的植株获得雄性不育的表型，所述异源核苷酸序列可编码促使碳水化合物降解的酶或修饰酶、淀粉酶、脱支酶和果胶酶，更具体的如a淀粉酶基因、茁长素(auxin),rot B、细胞毒素基因、白喉毒素、DAM甲基化酶、亲和素，或者可选自原核调控系统，还可以是显性的雄性不育基因。
在某些实施方式中，本发明中所提到的可操作地连接在本发明启动子下游的核酸，其中所述的“核酸”可以是操作性连接于本文所公开的启动子之上的结构基因、调节基因、结构基因的反义基因、调节基因的反义基因或者能够干扰内源基因表达的小RNA。
在本发明的第五方面，本发明还提供了一种表达盒，其包含：
(a)本发明第四方面的启动子SEQ ID NO:4或5；和
(b)核酸，其可操作地连接在本发明的启动子SEQ ID NO:4或5下游。
本发明的表达盒在5’-3’的转录方向上包含本发明的启动子SEQ ID NO:4或5、可操作地 连接在本发明的启动子SEQ ID NO:4或5的下游的核酸、任选的转录和翻译的终止区(如，转录终止元件或多聚腺苷酸化信号)。本发明的表达盒还可以包括，在细菌中复制所需的复制起点(例如，来自pBR322或P15A ori的ORI区)，土壤杆菌T-DNA转移所需要的元件(例如，T-DNA的左边界和/或右边界)。本发明表达盒可以包含的其他成分包括，增强子、内含子、多克隆位点、操纵基因、阻遏物结合位点、转录因子结合位点等。示例性的增强子包括来自CaMV 35S启动子、章鱼碱合酶基因、稻肌动蛋白I基因、玉米醇脱氢酶基因、玉米矮缩I基因、TMVΩ元件和酵母的启动子的增强子元件。也可以采用病毒前导序列来作为具有增强子效用的远见，如来自烟草花叶病毒(TMV)、玉米褪绿斑点病毒(MCMV)和苜蓿花叶病毒(AMV)的前导序列等。示例性的植物内含子包括来自Adh 1、bronze 1、肌动蛋白1、肌动蛋白2的内含子、以及蔗糖合酶内含子。
本发明中所提到的可操作地连接在本发明的启动子SEQ ID NO:4或5下游的核酸，其中所述的“核酸”可以是操作性连接于本文所公开的启动子之上的结构基因、调节基因、结构基因的反义基因、调节基因的反义基因或者能够干扰内源基因表达的小RNA。
具体地，可以将本发明所提供的育性调控基因SEQ ID NO：1、2、6、8或10构建到启动子SEQ ID NO:4或5的下游，从而驱动该育性调控基因在花药中的特异表达，或是通过RNAi的技术原理，构建由SEQ ID NO:4或5启动的可以沉默SEQ ID NO:1、2、6、8或10基因的DNA载体，从而获得SEQ ID NO:1、2、6、8或10基因的雄性不育突变体。
由上可见，可将上述任何核酸序列操作性连接到本发明中的SEQ ID NO:4或5启动子序列上，并在植物体中进行表达。
本发明的所提供的花药特异表达启动子可用于外源基因在花药中的特异性表达，从而避免该外源基因在植物其他组织中持续表达所带来的不利影响，还可以用于植物花粉生长发育相关基因的功能分析和鉴定；可用于雄性不育系和恢复系的创建；并可应用于花粉败育实验中，从而避免由植物转基因漂移或花粉逃逸所带来的生物安全问题，对植物雄性不育系和恢复系的创造具有重要意义。
在本发明的第六方面，本发明所提供的表达盒可被插入质粒、粘粒、酵母人工染色体、细菌人工染色体或其他适合转化进宿主细胞中的任何载体中。优选的宿主细胞是细菌细胞，尤其是用于克隆或储存多核苷酸、或用于转化植物细胞的细菌细胞，例如大肠杆菌、根瘤土壤杆菌和毛根土壤杆菌。当宿主细胞是植物细胞时，表达盒或载体可被插入被转化的植物细胞的基因组中。插入可以是定位的或随机的插入。优选地，插入通过诸如同源重组来实现。另外，表达盒或载体可保持在染色体外。本发明的表达盒或载体可存在于植物细胞的核、叶 绿体、线粒体和/或质体中。优选地，本发明的表达盒或载体被插入植物细胞核的染色体DNA中。
在第七方面，本发明提供了一种植物的生产方法，其包括：
(1)构建本发明第二方面或第五方面所提供的表达盒；
(2)将步骤(1)获得的表达盒导入植物细胞；
(3)再生出转基因植物；和
(4)选择出转基因植物；并且
(5)任选地，增殖步骤(4)获得的植物以获得后代。
本发明的转基因植物使用植物生物技术领域技术人员已知的转化方法制备。任何方法可被用于将重组表达载体转化进植物细胞中，以产生本发明的转基因植物。转化方法可包括直接和间接的转化方法。合适的直接方法包括聚乙二醇诱导的DNA摄入、脂质体介导的转化、使用基因枪导入、电穿孔、以及显微注射，等。在本发明的具体实施方式中，本发明使用了基于土壤杆菌的转化技术(可参见Horsch RB等(1985)Science 225：1229；White FF，Vectors for Gene Transfer in Higher Plants，Transgenic Plants，第1卷，Engineering and Utilization，Academic Press，1993，pp.15-38；Jenes B等.Techniques for Gene Transfer，Transgenic Plants，第1卷，Engineering and Utilization，Academic Press，1993，pp.128-143，等)。土壤杆菌菌株(例如根瘤土壤杆菌或毛根土壤杆菌)包含质粒(Ti或Ri质粒)和T-DNA元件，所述质粒和元件在用土壤杆菌转染后被转移至植物，而T-DNA被整合进植物细胞的基因组中。T-DNA可位于Ri-质粒或Ti-质粒上，或独立地包含在所谓的双元载体中。土壤杆菌介导的转化方法描述于例如中。土壤杆菌介导的转化最适合双子叶植物，但是也适合单子叶植物。土壤杆菌对植物的转化描述于例如中。转化可导致瞬时或稳定的转化和表达。尽管本发明的核苷酸序列可被插入落入这些广泛种类中的任何植物和植物细胞中，但是其尤其适用于作物植物细胞。
本发明还包括所公开的FL1基因及其启动子的应用，在某些应用的实施方式中，可以应用本发明所提供的FL1基因或其启动子来实现FL1或其他类似育性相关基因突变所获得的雄性不育系的繁殖和保持。
具体地，上述雄性不育系的繁殖和保持，是指以纯合隐性核雄性不育突变体为转化受体材料，将紧密连锁的3个目标基因转化至该不育突变体受体植株中。所述3个目标基因分别是育性恢复基因、花粉失活基因和筛选标记基因。其中，育性恢复基因可使不育的转化受体育性恢复，花粉失活基因可使含有转化的外源基因的花粉失活，即失去授精能力，筛选基因 可以用于转基因种子和非转基因种子的分拣，分拣出的非转基因种子用作不育系生产杂交种，转基因种子用作保持系来源源不断地、稳定地生产不育系。
更具体地，根据本发明的一个实施例，可以以玉米核隐性不育zmfl1/zmfl1突变体为转化受体材料，将紧密连锁的3个目标基因转化至该不育系：其中，育性恢复基因ZmFL1可使转化受体育性恢复；花粉失活基因Zm-PA可使含有外源基因的花粉失活，即失去授精能力；荧光色选基因RFP(r)用于转基因种子和非转基因种子的分拣，分拣出的非转基因种子用作不育系生产杂交种，转基因种子用作保持系来源源不断地稳定地生产不育系。由于该技术利用生物技术生产非转基因产品，解决了玉米杂交制种过程中面临的瓶颈问题。
与现有技术相比，本发明具有如下的有益效果：本发明提供了一种玉米花粉发育基因以及基于该基因突变所产生的雄性不育系，该不育系的育性稳定、不受环境条件影响，在该基因序列上发现的两个位点的Mutator插入均能造成稳定的雄性不育突变。该基因以及该基因突变产生的不育系为玉米杂交育种提供了资源，也为构建第三代杂交育种体系提供了必要的元件，该基因突变产生的雄性不育系，用来生产杂交种子，对于突破并改良现有的“三系”和“两系”杂交技术有重要意义。
附图说明
图1为野生型单株与zmfl1不育突变体单株的散粉期和成熟期花药比较。
图2为野生型单株和zmfl1不育突变体单株的花药成熟期比较(扫描电镜结果)。
图3为对野生型单株和zmfl1不育突变体单株的花药进行半薄切片分析结果。
图4为用DAPI染色观察野生型单株和zmfl1不育突变体单株的花粉发育过程。
图5为利用透射电镜观察野生型单株和zmfl1不育突变体单株的乌氏体和花粉粒壁发育。
图6为野生型单株和zmfl1不育突变体单株的花粉的单核小孢子期至双核花粉粒期结构分析。
图7为雄性不育突变体基因的图位克隆。
图8为不育突变体候选基因的结构。
图9为pZmFL1启动子驱动GUS在水稻各组织器官中的表达情况，其中A为根；B为茎；
C为叶片；D-I为水稻花各个时期的染色情况。
图10为ZmFL1基因编码的蛋白与水稻、高粱、拟南芥基因组中预测的同源蛋白的序列比对。
具体实施方式
下面对本发明的实施例作详细说明，本实施例在以本发明技术方案为前提下进行实施，给出了详细的实施方式和具体的操作过程，但本发明的保护范围不限于下述的实施例。
实施例1、雄性不育突变体筛选
Mutator是迄今为止在植物中发现的活性最高,诱变能力最强的一类转座子，以其高的正正向突变率、倾向插入基因富含区和低拷贝序列区等独特的遗传特性，使其在玉米功能基因研究以及突变体库构建中发挥重要作用。利用携带Mutator9材料和国内优良自交系综31构建突变体库。将两者杂交获得M1，将M1种植后自交获得M2种子。M1分单穗收获、脱粒，作为一个株系。每个株系的M2种子分出20粒，2009年3月种植于湖南农业大学校园内。种植2行，每行10粒，单粒播种。在1000个株系中发现1个系有3株不育株，9株可育株，姊妹交保种，将该不育系命名为zmfl1。
实施例2、zmfl1雄性不育突变体遗传分析
利用四个玉米自交系分别与实施例1获得的不育单株zmfl1杂交，对四个F1群体在散粉期进行田间育性鉴定，四个F1群体均表现出可育表型；将F1单株自交，分单株收获和播种果穗(F2)，在散粉期对四个F2群体进行田间育性鉴定，四个群体都出现育性分离，且正常株与不育株比例符合孟德尔分离定律3:1分离比(表1)，故可推断其受一对隐性核单基因控制。
表1四个F2群体正常株和不育株分离比例
Figure PCTCN2014086505-appb-000001
实施例3、zmfl1雄性不育突变体育性稳定性分析
将实施例1中的zmfl1不育单株与可育单株姊妹交，并将获得的在海南三亚、湖南长沙和北京三个不同生态点分别播种的不育亲本及分离群体，于散粉期考察群体所有单株育性，均可以获得与亲本一致的不育表型，说明该育性基因控制的不育表型不受温度和光照条件的影响。具体的，亲本的姊妹单株杂交不同时间、不同地点植株育性表现见表2，分离群体不同时间不同地点育性表现见表3：
表2亲本姊妹交不同时间、不同地点育性表现
Figure PCTCN2014086505-appb-000002
Figure PCTCN2014086505-appb-000003
表3分离群体不同时间、不同地点育性表现
Figure PCTCN2014086505-appb-000004
实施例4、zmfl1雄性不育突变体生殖器官表型分析
对可育单株和zmfl1不育单株在花药散粉期进行观察发现，野生型单株的花药(图1，A左)能从内外颖中露出、散粉，不育突变体单株的花药(图1，A右)不能从内外颖中露出、散粉；在花药成熟期进行观察发现，野生型单株的花药(图1，B左)饱满、呈黄色，不育突变体单株的花药(图1，B右)萎蔫、较小且呈红褐色；花粉碘-碘化钾染色观察结果表明，野生型单株的花粉为圆形、黑褐色(图1，C)，而不育突变体单株则只有花药壁和小孢子降解后的残余物质，无花粉粒出现(图1，D)。
成熟期野生型单株和zmfl1不育突变体单株的花药内外表面扫描电镜结果显示：野生型单株的花药的外表面致密(图2，A),不育突变体单株的花药外表面光滑(图2，B)；野生型单株的花药内表面排列着大量乌氏体(图2，C),不育突变体单株的花药内表面光滑，无乌氏体存在(图2，D)。
对野生型单株和zmfl1不育突变体单株的花药进行半薄切片分析，结果表明：四分体时期，野生型单株(图3，A)和不育突变体单株(图3，E)花药和四分体基本无差异；单核 小孢子期，野生型单株(图3，B)的花药绒毡层细胞质浓缩、颜色加深，而不育突变体单株(图3，F)的绒毡层比四分体时期稍微扩大，颜色没有加深，突变体小孢子形状畸形；单核小孢子后期至二核花粉粒期，野生型单株(图3，C)花药绒毡层几乎降解完全，小孢子液泡化并同时进行核分裂形成二核或三核花粉粒，而不育突变体单株(图3，G)绒毡层比野生型大，降解较少，小孢子液泡化程度较轻，小孢子畸形且开始降解；成熟花粉粒期，野生型单株(图3，D)花药花粉粒填满淀粉和脂质，而不育突变体单株(图3，H)花药内只有小孢子降解后的残余物质。
用DAPI染色观察野生型单株和zmfl1不育突变体单株的花粉发育过程：四分体时期，野生型单株的四分体(图4，A)和不育突变体的四分体(图4，E)无差异；单核小孢子前期，不育突变体单株的小孢子(图4，F)相比于野生型单株的小孢子(图4，B)形状畸形；单核小孢子后期，不育突变体单株的小孢子(图4，G)相比于野生型单株的小孢子(图4，C)已开始降解；二核花粉粒期，不育突变体小孢子(图4，H)相比于野生型小孢子(图4,D)已明显降解。
利用透射电镜观察野生型单株和zmfl1不育突变体单株的乌氏体和花粉粒壁发育：四分体时期，野生型绒毡层内表面乌氏体(图5，A箭头)比zmfl1突变体绒毡层内表面乌氏体(图5，B箭头)稍多，体积稍大；单核小孢子前期，野生型绒毡层内表面乌氏体(图5，C箭头)开始累积孢粉素前体物质累积，而zmfl1突变体绒毡层内表面乌氏体(图5，D箭头)似乎已经降解。
对野生型单株和zmfl1不育突变体单株的花粉的单核小孢子期进行分析，野生型单株的花粉粒外壁形成明显外壁外层、外壁内层和柱状层(图6，A),不育突变体单株形成只有外壁内层以及填充在其上的少许孢粉素物质(图6，B)；单核小孢子后期至双核花粉粒期，野生型单株的花粉粒外壁层加厚(图6，C),不育突变体单株的花粉粒外壁层依然有外壁内层以及填充在其上的孢粉素物质，无明显三层外壁形成(图6，D)。
实施例5、zmfl1雄性不育突变体基因的克隆
以zmfl1不育突变体作母本，与野生型自交系郑58杂交，F1代自交构建F2群体。以玉米散粉期花药是否外露、花药颜色以及是否存在花粉为育性表型鉴定标准，挑取F2群体中不育突变表型单株进行初步定位和精细定位，共筛选得到不育突变表型单株2757株。通过初步定位把目标基因限定在标记S1和S11之间(图7)。
根据B73全基因组物理图谱，获得S1和S11两个标记之间的基因组序列，利用此序列来开发新的SSR标记和STS标记。对不育突变体、郑58以及它们组配的F1进行多态性标记 的筛选，最终选择了10对多态性分子标记进行下一步精细定位，它们分别是S、S2、S3、S4、S5、S6、S7、S8、S9和S10(图7)。
针对郑58与不育突变体的F2群体，结合目标基因两侧的交换单株表型，用开发的标记分别对其进行基因型分析，结果发现标记S2、S3、S4、S5和目标基因之间的交换单株分别减少到29株、25株、16株和9株，S10、S9、S8和目标基因之间的交换单株分别减少到14株、13和11株，且两侧交换单株各不相同；S、S6、S7和目标基因之间的交换单株为0。根据交换单株递减规律和三点测验等方法最终把目标基因锁定在S5和S8之间，分别有9个交换单株和11个交换单株。这两个标记界定的实际物理距离约为300Kb(图7)。
对目标基因所在的300kb区间进行基因注释以及候选基因的生物信息学分析，发现该区域内存在六个候选基因(图7)。基因测序发现，在候选基因GRMZM2G434500(Chromosome 1:80,963,525-80,966,109)的第二个外显子chr1:80,964,768处存在Mutator1转座子插入(图8)，且突变表型与基因型相一致，命名为ZmFL1，其基因组DNA序列如SEQ ID NO:1所示；其编码cDNA序列如SEQ ID NO:2所示；其编码的氨基酸序列如SEQ ID NO:3所示。
实施例6、zmfl1雄性不育突变基因的等位突变
从MAIZEGDB申请获得该雄性育性突变基因的等位系，测序发现该等位系在ZmFL1第一个外显子chr1:80,963,850处存在Mutator3序列插入,且表型与基因型相一致。
实施例7、ZmFL1基因的启动子表达载体构建及功能分析
ZmFL1基因的启动子表达载体的构建：通过玉米基因组扩增分别得到两个启动子片段，一个启动子的长度是875bp，其核苷酸序列如SEQ ID NO:4所示；一个启动子的长度是2500bp，其核苷酸序列如SEQ ID NO：5所示。将该两个片段与GUS连接后得到启动子功能鉴定植物表达载体。通过农杆菌介导的方法转化至野生型水稻愈伤组织中，并筛选再生分别得到12和18株转基因水稻植株。通过分析B-半乳糖苷酶的活性分析pZmFL1启动子的表达模式，在转基因的植株的根、茎、叶和花中通过GUS染色分析，发现pZmFL1启动子驱动GUS主要在花药中表达，更具体的在花粉发育的后期表达，其GUS染色结果如图9所示。
实施例8、ZmFL1基因编码的蛋白与水稻、高粱、拟南芥基因组中预测的同源蛋白的序列比对
在NCBI数据库中，利用protein blast工具，对玉米ZmFL1基因编码的蛋白全序列在蛋白数据库中进行查找，得到了水稻、高粱、拟南芥基因组中预测的同源蛋白，将这些蛋白序列进行比对分析，结果显示来自不同植物的同源蛋白都具有非常相似的保守序列，彼此之间同源性很高(图10)，表明该蛋白在植物花的雄性器官发育过程中生物学功能保守，起 着非常重要的作用。其中水稻中的同源基因OsFL1的基因组核苷酸序列如SEQ ID NO:6所示，其编码的氨基酸序列如SEQ ID NO:7所示；在高粱中的同源基因SbFL1的基因组核苷酸序列如SEQ ID NO:8所示，其编码的氨基酸序列如SEQ ID NO:9所示；在拟南芥中的同源基因AtFL1的基因组核苷酸序列如SEQ ID NO:10所示，其编码的氨基酸序列如SEQ ID NO:11所示。
实施例9、ZmFL1基因在新一代杂交育种技术中的应用
ZmFL1基因可以用于新一代杂交育种技术，该技术的核心思想是：以玉米核隐性雄性不育突变体为转化受体材料，通过将紧密连锁的3个目标基因转化至不育突变体中，其中，育性恢复基因可使转化受体育性恢复，花粉失活基因可使含有外源基因的花粉失活，即失去授精能力，筛选基因可以用于转基因种子和非转基因种子的分拣，分拣出的非转基因种子即为不育系，而转基因种子用作保持系。通过保持系给不育系授粉杂交，可以在不育系上结实，由此繁殖不育系。而保持系通过自交可以源源不断地得以繁殖。由于该技术利用生物技术生产非转基因产品，解决了玉米杂交制种过程中面临人工或机械去雄问题，省去人工去雄或机械去雄步骤，可以提供更高质量及纯度的种子给种植者，节约劳动成本。
根据以上原理，更具体地，采用玉米ZmFL1基因构建植物表达载体。该表达载体内包含Zm-AA1(花粉失活基因)、ZmFL1(育性恢复基因)和RFP(红色荧光色选基因)三个表达盒。将该表达盒转化玉米zmfl1纯合隐性雄性不育突变体后，获得的植株育性得以恢复，并具有以下特点：ZmFL1育性恢复基因可使转化受体育性恢复，Zm-AA1花粉失活基因可使含有外源基因的花粉失活，即失去授精能力，RFP筛选基因可以用于转基因种子和非转基因种子的分拣，分拣出的非转基因种子即为不育系，而转基因种子用作保持系。由此，建立新一代杂交育种技术体系。
根据以上原理，发明人采用玉米ZmFL1基因构建了表达载体。在构建玉米的植物表达载体之前，发明人首先分别对表达载体内的花粉失活基因Zm-PA、育性恢复基因ZmFL1和筛选基因RFP(r)三个表达盒单独进行了玉米转化，并进一步对各个表达盒的功能进行了验证。结果表明各个表达盒单独转化玉米时，都能够工作良好，达到预期的设计效果。
进一步,发明人通过装配下述DNA元件，构建新一代杂交育种技术的玉米表达载体：
1)以pCAMBIA2300载体为基础；
2)基因表达盒LTP2：RFP(r)-PINII，RFP(r)基因(SEQ ID NO：12)的开放读码框连接于LTP2启动子(SEQ ID NO：13)和PINII终止子(SEQ ID NO：14)之间，重组成RFP(r)的基因表达盒(LTP2：RFP(r)：PINII)；
3)ZmFL1基因表达盒，由目标基因ZmFL1及其启动子和终止子构成，其中ZmFL1基因的启动子序列如SEQ ID NO：5所示，其终止子序列如SEQ ID NO：16所示，ZmFL1基因的从起始密码子到终止密码子的基因组DNA序列如SEQ ID NO：15所示，其核苷酸序列编码的蛋白氨基酸序列如SEQ ID NO：3所示；
4)基因表达盒PG47：ZM-BT1：ZM-PA：IN2-1，目标基因ZM-PA(其核苷酸序列如SEQ ID NO：17所示)的开放读码框连接于启动子PG47(其核苷酸序列如SEQ ID NO：18所示)、转运肽ZM-BT1(其核苷酸序列如SEQ ID NO：19所示)的下游，终止子IN2-1(其核苷酸序列如SEQ ID NO：20所示)的上游。
将构建好的上述表达载体进行玉米转化，获得阳性的转基因玉米植株。
转基因玉米植株的花粉育性检测：对上述所得到的单拷贝转基因玉米(含有纯合的zmfl1隐性不育位点)植株进行分析发现，转基因植株和非转基因对照植株之间没有明显的形态差异，但是花粉育性明显不同。对所述转基因植株材料进行花粉可染率检测，同时对野生型玉米进行花粉可染率检测。
采用的方法为：在玉米开花期，从转基因玉米植株、及其野生型对照植株各随机抽取单株，各株取雄穗的一个小穗，再从小穗中取一朵小花，从小花中取1个花药，置于载玻片中央，滴加一滴1％的I2-KI溶液，用镊子和解剖针释放花粉后，盖上盖玻片，在显微镜下观察、计数可染色花粉数和花粉总数，可以着色为深蓝色的为可育花粉，而不能够着色的为败育花粉。分析转基因玉米植株的花粉可染率，结果显示对照植株的可着色的花粉占98％～100％；而多个随机抽取的转基因植株中，正常花粉(可着色)与败育花粉(不能着色)比例接近1:1，表明所构建的转基因株系可以产生等量的携带外源基因的花粉和不携带外源基因的花粉，即转入的上述玉米表达载体可使转基因株系花粉的50％失活。该结果表明本发明所提供的载体能够达到预期的花粉失活功能。
转基因玉米植株的荧光种子与非荧光种子分离分析：对上述所得到的单拷贝转基因玉米植株(含有纯合的zmfl1隐性不育位点)所结T1代果穗的种子进行荧光分离比例调查，结果表明这些种子均显示1:1分离比，即携带外源基因的荧光种子和不携带外源基因的非荧光种子表现为1:1分离，表明本发明所提供的载体各元件作为整体表达良好，可以实现创制和繁殖不育系的目的；其中，ZmFL1基因可以恢复雄性不育突变体受体的育性，Zm-PA基因和RFP基因的表达可以分别实现预期的花粉失活功能和种子筛选标记功能。

Claims (29)

  1. 一种DNA序列，具有调控植物育性的功能，其特征在于，所述DNA序列选自下列组的序列之一：
    a)具有SEQ ID NO：1或SEQ ID NO:2或SEQ ID NO:15所示的核苷酸序列；
    b)具有SEQ ID NO：6所示的核苷酸序列；
    c)具有SEQ ID NO：8所示的核苷酸序列；
    d)具有SEQ ID NO：10所示的核苷酸序列；
    e)在严格条件下能够与(a)-(d)之任一所述序列的DNA杂交的DNA序列；
    f)与(a)-(e)之任一所述序列互补的DNA序列。
  2. 权利要求1所述的DNA序列，其特征在于所述DNA序列编码的氨基酸序列如SEQ ID NO：3、7、9或11所示。
  3. 一种表达盒，其特征在于所述表达盒包含权利要求1所述的DNA序列。
  4. 一种表达载体，其特征在于所述表达载体包含权利要求3所述的表达盒。
  5. 一种工程菌，其特征在于所述工程菌含有权利要求4所述的表达载体。
  6. 一种基因在植物育性调控中的应用，其特征在于，所述育性调控基因的核苷酸序列选自下列组的序列之一：
    a)具有SEQ ID NO：1或SEQ ID NO:2所示的核苷酸序列；
    b)具有SEQ ID NO：6所示的核苷酸序列；
    c)具有SEQ ID NO：8所示的核苷酸序列；
    d)具有SEQ ID NO：10所示的核苷酸序列；
    e)在严格条件下能够与(a)-(d)之任一所述序列的DNA杂交的DNA序列；
    f)与(a)-(b)之任一所述序列互补的DNA序列。
  7. 权利要求6所述的应用，其中所述的核苷酸序列其编码的氨基酸序列如SEQ ID NO：3、7、9或11所示。
  8. 权利要求6或7所述的应用，其特征在于，通过突变育性调控基因SEQ ID NO:1、2、6、8或10获得雄性不育材料。
  9. 权利要求8所述的应用，其中所述的突变包括在育性调控基因的核苷酸序列上进行取代、缺失或添加一个或几个核苷酸。
  10. 权利要求6或7所述的应用，其特征在于用权利要求1所述的DNA序列恢复由相应的SEQ ID NO::1、2、6、8或10所示基因突变所导致的雄性不育，使雄性不育突变体恢复成可育。
  11. 一种突变体材料的应用，其特征在于所述突变材料是由核苷酸序列的突变所造成，所述核苷酸序列如SEQ ID NO::1、2、6、8或10所示。
  12. 权利要求11所述的应用，其中所述的突变可以是点突变，也可以是DNA缺失或插入突变，也可以是通过RNAi等基因沉默手段产生。
  13. 权利要求11或12所述的应用，包括在育种中的应用。
  14. 权利要求13所述的应用，其中所述的育种是指将突变体植株作为不育系母本，与恢复系杂交，生产杂交种子。
  15. 一种启动子，具有花药特异表达的特性，其特征在于所述启动子的核苷酸序列如SEQ ID NO：4或5所示。
  16. 一种表达盒，其特征在于所述表达盒包含权利要求15所述的启动子序列。
  17. 一种表达载体，其特征在于所述表达载体包含权利要求16所述的表达盒。
  18. 一种工程菌，其特征在于所述工程菌含有权利要求17所述的表达载体。
  19. 一种在植物中表达目的核苷酸序列的方法，所述方法包括向植物体导入DNA构建体，所述DNA构建体含有启动子及操作性连接于所述启动子的目的核苷酸序列，其中所述启动子的核苷酸序列如SEQ ID NO：4或5所示。
  20. 权利要求19所述的方法，其中所述的植物为单子叶植物。
  21. 权利要求20所述的方法，其中所述的单子叶植物为禾本科植物。
  22. 权利要求21所述的方法，其中所述的禾本科植物为水稻、玉米或高粱。
  23. 权利要求22所述的方法，其中所述的目的核苷酸序列对于植物宿主来说可以是内源或是外源的。
  24. 权利要求19所述的方法，其中所述的异源核苷酸序列可以是结构基因、调节基因、结构基因的反义基因、调节基因的反义基因或者能够干扰内源基因表达的小RNA，其在花粉发育晚期的特异性表达可以调节花粉的育性及花粉萌发。
  25. 权利要求15所述的启动子在以下(a)至(d)中任一项中的应用：
    (a)培育植物品种或品系；
    (b)培育授粉受精能力增强的植物品种或品系；
    (c)培育授粉受精能力消弱的植物品种或品系；
    (d)培育雄性不育植物品种或品系。
  26. 权利要求25所述的应用，其特征在于，所述的植物为单子叶植物。
  27. 权利要求26所述的应用，其中所述的单子叶植物为禾本科植物。
  28. 权利要求27所述的方法，其中所述的禾本科植物为水稻、玉米或高粱。
  29. 一种方法，用于保持雄性不育植株的纯合隐性状态，所述方法包括：
    (a)提供第一植株，其包含FL1基因的纯合隐性等位基因，并且其是雄性不育的；
    (b)向第一植株中引入下述构建体，形成第二植株，所述第二植株包含FL1基因的纯合隐性等位基因，所述构建体为半合状态，所述构建体包含：
    (i)第一核苷酸序列，其包含FL1核苷酸序列，当在第一植株中表达时其将恢复雄性生育力；
    (ii)第二核苷酸序列，当其表达时，会抑制所述第二植株中可育雄性配子的形成或功能，具体为玉米α淀粉酶基因Zm-PA；以及
    (c)用所述第二植株的雄性配子使所述第一植株受精，以产生保持了所述第一植株纯合隐性状态的后代。
PCT/CN2014/086505 2013-09-16 2014-09-15 雄性核不育基因及其突变体在杂交育种上的应用 WO2015035951A1 (zh)

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