WO2017049833A1 - 一种利用CalS5基因突变创制光温敏不育系的方法及其应用 - Google Patents

一种利用CalS5基因突变创制光温敏不育系的方法及其应用 Download PDF

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WO2017049833A1
WO2017049833A1 PCT/CN2016/073287 CN2016073287W WO2017049833A1 WO 2017049833 A1 WO2017049833 A1 WO 2017049833A1 CN 2016073287 W CN2016073287 W CN 2016073287W WO 2017049833 A1 WO2017049833 A1 WO 2017049833A1
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plant
cals5
synthase
seq
tannin
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English (en)
French (fr)
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杨仲南
许特
朱骏
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上海师范大学
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
    • 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)
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/04—Plant cells or tissues

Definitions

  • the invention belongs to the field of agriculture and biotechnology, and particularly relates to a method for creating a photothermophilic sterile line by using a mutation of the CalS5 gene and an application thereof.
  • cytoplasmic male sterility In agricultural production, male sterile lines occupy a huge advantage in hybrid seed production and agricultural production.
  • Male sterility is often divided into two types: cytoplasmic male sterility (CMS) and nuclear male sterility (GMS).
  • CMS cytoplasmic male sterility
  • GMS nuclear male sterility
  • the establishment of a three-line hybrid system relies on cytoplasmic male sterility.
  • cytoplasmic male sterility has its own defects: First, the quality of cytoplasmic male sterility plants is generally poor; secondly, the combined yield stimulation potential of three-line hybrid rice is getting smaller and smaller; again, due to wild-type male sterility The cytoplasm is single, and once cytoplasmic male sterility is lost or some kind of devastating pests and diseases occur, it will cause huge losses.
  • the photothermophilic male sterile line has both the sterile line and the maintainer line.
  • the two-line method is not limited by the recovery relationship, that is, the nuclear sterility can be crossed with a large number of conventional varieties, the group is free, and it is easier to obtain the hybrid advantage with excellent traits, and fundamentally solve the male traits in the three lines.
  • the problem of cytoplasmic simplification In recent years, the application of two-line hybrid rice in agricultural production in China has become more and more extensive.
  • the rice photoperiod-sensitive male sterile line is controlled by a single gene recessive locus; studies have shown that the infertility traits of Nongken 58S and Pei'ai 64S are controlled by the same genetic locus, and both temperature and illumination will affect the locus. .
  • Arabidopsis thaliana (A. thaliana) has a small genome, rapid growth cycle and a large number
  • the mutant library has unparalleled advantages, and becomes a model plant in the field of botany and biology. In addition, it can be cultured in a narrow space where temperature, light, and the like are strictly controlled.
  • Some Arabidopsis sterile mutants such as the PEAMT gene mutant t365 and the GA/IAA biosynthesis-blocked ms33 mutant, were found to be thermo-sensitive phenotypes.
  • Tannin synthase is a family of proteins present in different species and plays an important role in the synthesis of tannins. In the plant kingdom, it has been reported that some species of tannin synthase are involved in the reproductive development of plants, such as a temporary cell wall mechanical barrier in the comfrey, preventing cell fusion (Waterkeyn et al. 1962); Glycogen is provided on the outer wall (Larson Lewis et al. 1962) and the like.
  • the conventional ways to discover new germplasm of male sterile plants include: discovery of natural male sterile original strains, artificial mutagenesis and continuous backcross nuclear replacement.
  • the early wild-type and horse-type male sterile cytoplasm in China was the discovery of the original male sterile male plants; the Xinjiang New Zealand sterile cotton cultivated by Xinjiang Academy of Agricultural Sciences was induced by 60CO ⁇ -rays.
  • traditional breeding methods have many difficulties in screening and cultivating ideal male sterile lines.
  • the male sterile line has limited germplasm resources, the transfer cycle is too long, the hybridization is not compatible, and the combination breeding is limited.
  • there is no simple and convenient plant sterility line in the field for plant breeding so there is an urgent need for a simple and convenient plant sterility breeding technique.
  • the present invention provides a method for creating a photothermophilic male sterile line by using a mutation of CalS5 gene and an application thereof, thereby solving the problem that the current nuclear infertility photo-sensitivity genetic locus is small and the seed purity is not high. problem.
  • a first aspect of the present invention provides a method for cultivating a plant sterile line, comprising the steps of: reducing expression or activity of a tannin synthase associated with pollen development in said plant plant, said tannin synthase being CalS5 Protein or its homologous protein.
  • the enamel synthase is involved in the synthesis of enamel in the tetrad of the plant pollen development process.
  • the enamel synthase is specifically expressed in a cell, tissue or organ of a plant inflorescence or anther.
  • the cell or tissue comprises: microspores, microspore mother cells or a combination thereof.
  • the anthocyanin synthase specificity table during anther development Da As a preferred embodiment of the present invention, the anthocyanin synthase specificity table during anther development Da.
  • the anther development period includes a pre-anther formation stage (-3-0 days), an anther formation stage, and a post anther formation stage (1-5 days), wherein 0 days refers to the formation of anthers.
  • -3-0 days is the starting point of the anther formation day
  • the third day, the first day and the fifth day are the starting point of the anther formation day, and the first day and the fifth day are calculated backward.
  • the enamel synthase is specifically expressed in the seventh stage of anther development.
  • the enamel synthase reaches the highest expression peak upon completion of pollen meiosis.
  • the "reducing the expression or activity of a tannin synthase associated with pollen development in the plant plant” satisfies the following conditions:
  • A1 is the enzymatic activity of the tannin synthase related to pollen development in the plant;
  • A0 is the enzymatic activity of the same tannin synthase in the wild type of the same type of plant.
  • the wild type amino acid sequence of CalS5 is selected from any one or more of the group consisting of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. .4, SEQ ID NO. 5, SEQ ID NO. 6.
  • the expression or activity of the tannin synthase can be reduced by any of the following means:
  • the partial or complete deletion of the polynucleotide encoding the protein is a vector using a chromosomal gene insertion, and the polynucleotide encoding the endogenous target protein is replaced with a polynucleotide deleted by the marker gene or a partial nucleotide sequence.
  • the length of the "partial" deletion varies depending on the type of polynucleotide, and may be 1-300 bp, preferably It is 1-100 bp, more preferably 1-5 bp, wherein bp is a base pair.
  • the expression control sequence can be modified by any of the following means:
  • the expression control sequence comprises a sequence encoding a promoter sequence, an operator sequence, a ribosome binding site, and a sequence that controls transcription and translation termination.
  • the polynucleotide sequence on the chromosome can be modified in any of the following ways to reduce the activity of the protein:
  • the method of "reducing the activity of a tannin synthase associated with pollen development in a plant plant” comprises: lowering the expression level of a gene encoding a tannin synthase.
  • E1 is an expression level of a tannin synthase associated with pollen development in the plant plant
  • E0 is the expression level of the same tannin synthase in the wild type of the same type of plant
  • A1 is an enzymatic activity of a tannin synthase associated with pollen development in the plant plant
  • A0 is the enzymatic activity of the same tannin synthase in wild-type plants of the same type.
  • the "reducing the tannin synthase activity in the plant is achieved by one or a combination of gene mutation, gene knockout, gene disruption, and RNA interference.
  • the gene encoding the enamel synthase is the CalS5 gene or a homologous gene thereof.
  • the CalS5 gene is capable of encoding SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. Shows the amino acid sequence.
  • polynucleotide sequence encoding the enamel synthase is selected from the group consisting of: SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12.
  • polynucleotide sequence encoding SEQ ID NO. 1 is SEQ ID NO.
  • polynucleotide sequence encoding SEQ ID NO. 2 is SEQ ID NO.
  • polynucleotide sequence encoding SEQ ID NO. 3 is SEQ ID NO.
  • polynucleotide sequence encoding SEQ ID NO. 4 is SEQ ID NO.
  • polynucleotide sequence encoding SEQ ID NO. 5 is SEQ ID NO.
  • polynucleotide sequence encoding SEQ ID NO. 6 is SEQ ID NO.
  • the "reducing the expression or activity of a tannin synthase associated with pollen development in a plant plant” is achieved by any of the following means:
  • the plant includes crops, forestry plants, flowers, and the like; preferably includes Gramineae, Leguminosae sp., Brassicaceae, and Arabidopsis (Arabidopsis) plants, more preferably including rice, corn, sorghum, wheat, soybean or Arabidopsis.
  • crops, forestry plants, flowers, and the like preferably includes Gramineae, Leguminosae sp., Brassicaceae, and Arabidopsis (Arabidopsis) plants, more preferably including rice, corn, sorghum, wheat, soybean or Arabidopsis.
  • the tannin synthase related to pollen development is not particularly limited and may be derived from any plant species, and representative plants include, but are not limited to, rice (gene number: BGIOSGA021782, and Arabidopsis thaliana) Orthologous CalS5 protein homology is 73%), millet (gene number: Si008368m.g, homology with Arabidopsis orthologous CalS5 protein is 77%), sorghum (gene number: Sb10g005550, and Arabidopsis thaliana) Orthologous CalS5 protein homology is 81%), wheat (gene number: Traes_7BS_170A2F4BB, 75% homology to Arabidopsis orthologic CalS5 protein), maize (gene number: GRMZM2G353905_T01, direct with Arabidopsis thaliana) The source CalS5 protein has a homology of 81%).
  • a second aspect of the invention provides a use of a gene encoding a tannin synthase associated with pollen development for cultivating a plant sterile line or for preparing a reagent or kit for cultivating a plant sterile line .
  • the coding gene is a CalS5 gene or a homologous gene thereof.
  • the CalS5 gene is capable of encoding SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. Shows the amino acid sequence.
  • a third aspect of the present invention provides a method for converting a plant from a sterile to a fertile, comprising the steps of reducing the rate of development of the pollen.
  • the plant is a plant having a decreased expression level of a tannin synthase associated with pollen development.
  • the plant is a plant sterility line cultivated according to the method of the first aspect of the invention.
  • the "reducing the rate of development of pollen” is achieved by reducing the ambient temperature at which the plant grows and/or reducing the illumination time of the plant.
  • the ambient temperature for reducing plant growth comprises controlling the ambient temperature (average temperature) to 18-23 ° C, such as 19 ° C, 20 ° C or 22 ° C, and the like.
  • the time to reduce the ambient temperature of plant growth includes the anther formation stage, the pollen maturation stage, and the flowering pollination stage or 2 weeks before and after.
  • the growth temperature of the plant is started to decrease when the plant is twitched or headed, and after 7-14 days of low temperature cultivation, the normal temperature is restored.
  • a fourth aspect of the present invention provides a plant breeding method comprising the steps of:
  • Step 1 Maintain plant infertility
  • Step 2 Convert the plant from infertility to fertility
  • Step 3 Maintain plant fertility and breed.
  • maintaining plant sterility is a plant sterility line maintained by the method of the first aspect provided by the present invention.
  • the method of converting a plant from infertility to fertility is according to the method of the third aspect provided by the present invention. Plants are converted from infertile to fertile.
  • a fifth aspect of the present invention provides a plant cell in which expression of a quinoid synthase associated with pollen development is reduced in a plant developed from the plant cell.
  • the reduction satisfies the following conditions:
  • A1 is the enzymatic activity of the tannin synthase related to pollen development in the plant;
  • A0 is the enzymatic activity of the same tannin synthase in the wild type of the same type of plant.
  • the enamel synthase is a CalS5 protein or a homologous protein thereof.
  • CalS5 protein and “CalS5 polypeptide” refer to a protein or polypeptide having the amino acid sequence of CalS5, such as SEQ ID NOS. 1-6, and the CalS5 protein includes a wild type CalS5 protein and a mutant CalS5 protein, unless otherwise specified.
  • the tannin synthase of the present invention comprises, but is not limited to, the amino acid sequence shown in SEQ ID NO. 1-6, and the amino acid sequence of the protein may differ depending on the plant species or variety.
  • the CalS5 protein may be a mutant protein or an artificial variant thereof, as long as it helps to cultivate a plant sterile line by reducing the activity of the protein, the amino acid sequence of the mutant protein and its artificial variant is in SEQ ID One or more positions of the amino acid sequence set forth in NO. 1-6 comprise substitutions, deletions, insertions, additions or inversions of one or more amino acids.
  • the "plurality” described in the present invention varies depending on the position or type of the three-dimensional structure of the amino acid residue in the protein, and is preferably 2-20, more preferably 2-10, more preferably 2-5. Furthermore, substitutions, deletions, insertions, additions or inversions of amino acids include substitutions, deletions, insertions, additions or inversions of amino acids resulting from artificial variants or natural mutations, depending on the individual or species of the plant.
  • isolated means that the substance is separated from its original environment, and if it is a natural substance, its original environment is the natural environment, for example, a polynucleotide in a natural state in a living cell and The polypeptide is not isolated and purified, and is isolated and purified if the same polynucleotide or polypeptide is separated from other substances from the natural state.
  • isolated CalS5 protein or polypeptide as used herein means substantially free of phase with CalS5 protein.
  • Those skilled in the art will be able to purify CalS5 proteins in plants such as rice using standard protein purification techniques. Basically, a pure polypeptide produces a single major band on a non-reducing polyacrylamide gel.
  • polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, etc., preferably a recombinant polypeptide.
  • polypeptides of the present invention may be naturally purified products, may also be products of chemical synthesis, or may be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques;
  • the polypeptide may be a glycosylated or non-glycosylated polypeptide according to the host used in the recombinant technique; the polypeptide of the invention may also include an initial methionine residue.
  • the CalS5 protein of the present invention also includes fragments, derivatives and analogs of the CalS5 protein.
  • fragment refers to a polypeptide which retains the same biological function or activity as the CalS5 protein of the present invention.
  • polypeptide fragments, derivatives or analogs of the invention may be:
  • a polypeptide having one or more conservative or non-conservative amino acid residues substituted and preferably a polypeptide in which a conservative amino acid residue is substituted, the substituted amino acid residue may be encoded by a genetic code;
  • a polypeptide formed by fusing an additional amino acid sequence to the polypeptide sequence such as a leader sequence or a secretory sequence or a sequence or proprotein sequence or fusion protein used to purify the polypeptide.
  • the "CalS5 protein” or “CalS5 polypeptide” sequence is set forth in SEQ ID NO.
  • the "CalS5 protein” or “CalS5 polypeptide” further comprises variants of the sequence of SEQ ID NO. 1-6 that are functionally identical to the CalS5 protein, including but not limited to one Or a plurality, usually from 1 to 50, preferably from 1 to 30, more preferably from 1 to 20, more preferably from 1 to 10, amino acid deletions, insertions and/or substitutions, and at the carbon end and/or Or one or several amino acids are added to the nitrogen terminal, usually within 20, preferably within 10, more preferably within 5.
  • amino acids having similar or similar properties usually not It will alter the function of the protein, and adding one or several amino acids at the carbon end and/or the nitrogen end will usually not change the function of the protein.
  • the "CalS5 protein” or “CalS5 polypeptide” also includes active fragments and active derivatives of the CalS5 protein or polypeptide.
  • the variant form of the CalS5 polypeptide comprises: a homologous sequence, a conservative variant, an allelic variant, a natural mutant, an induced mutant, and can hybridize to the CalS5 protein DNA under high or low stringency conditions.
  • the protein encoded by DNA is not limited to: a conservative variant, an allelic variant, a natural mutant, an induced mutant.
  • the invention also provides other polypeptides, such as fusion proteins comprising a CalS5 protein or a fragment thereof.
  • the present invention also encompasses soluble fragments of the CalS5 protein.
  • the fragment has at least about 10 contiguous amino acids of the CalS5 protein sequence, typically at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, most Preferably, there are at least about 100 contiguous amino acids.
  • modifications described herein generally do not alter the primary structure, which includes primarily chemically derived forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro; and includes glycosylation. Modified forms also include sequences having phosphorylated amino acid residues, such as phosphotyrosine, phosphoserine, and phosphothreonine; and polypeptides modified to increase their antiproteolytic properties or optimize solubility properties.
  • the "CalS5 conserved variant polypeptide” means up to 10, preferably up to 8, more preferably up to 5, most preferably compared to the amino acid sequence shown by SEQ ID NO.: 1-6.
  • a polypeptide is formed by replacing at most three amino acids with amino acids of similar or similar nature.
  • These conservative variant polypeptides are preferably produced by amino acid substitution according to Table 1.
  • Gly(G) Pro Ala Ala His(H) Asn, Gln, Lys, Arg Arg Ile(I) Leu, Val, Met, Ala, Phe Leu Leu(L) Ile, Val, Met, Ala, Phe Ile Lys(K) Arg, Gln, Asn Arg Met(M) Leu, Phe, Ile Leu Phe(F) Leu, Val, Ile, Ala, Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr, Phe Tyr Tyr(Y) Trp, Phe, Thr, Ser Phe Val(V) Ile, Leu, Met, Phe, Ala Leu
  • said representative substitution refers to a base of the present invention which is capable of substituting the first residue
  • said preferred substitution is the most preferred base of the present invention which is capable of substituting the first residue
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • the DNA form includes cDNA, genomic DNA or synthetic DNA; the DNA may be single-stranded or double-stranded; the DNA may be a coding strand or a non-coding strand; the coding region encoding the mature polypeptide may be The coding region sequences shown in SEQ ID NOS. 7-12 are identical or degenerate variants.
  • degenerate variant means, in the present invention, a nucleic acid sequence which encodes a protein having SEQ ID NOS. 1-6 but differs from the coding region sequence shown in SEQ ID NO. 7-12. .
  • polynucleotide encoding the mature polypeptide of SEQ ID NO. 1-6 is selected from any one of the following:
  • the coding sequence of the CalS5 polypeptide is selected from the group consisting of:
  • a "polynucleotide encoding a polypeptide” may be a polynucleotide comprising the polypeptide, or may be a polynucleotide further comprising an additional coding and/or non-coding sequence.
  • the invention also relates to variants of said polynucleotides, said fragments or analogs and derivatives of polypeptides or polypeptides having the same amino acid sequence as the invention, which variants may be naturally occurring Allelic variants or non-naturally occurring variants.
  • nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is a replacement for a polynucleotide that can be substituted with one or more nucleotides. , deletion or insertion, but does not substantially alter the function of the polypeptide it encodes.
  • the invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the invention also relates to polynucleotides that hybridize to the polynucleotides of the invention under stringent conditions.
  • stringent conditions means: (1) hybridization and elution at a lower ionic strength and a higher temperature, such as 0.2 x SSC, 0.1% SDS, 60 ° C; or (2) hybridization a denaturant such as 50% (v/v) formamide, 0.1% calf serum/0.1% phenanthrene 400, 42 ° C, etc.; or (3) at least 90% identity between the two sequences, More preferably, hybridization occurs more than 95%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide represented by SEQ ID NO.
  • the present invention also relates to a nucleic acid fragment which hybridizes to the above sequence, said "nucleic acid fragment" having a length of at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, Most preferably, it contains at least 100 nucleotides.
  • the nucleic acid fragments can be used in nucleic acid amplification techniques, such as PCR, to identify and/or isolate polynucleotides encoding CalS5 proteins.
  • the full-length nucleotide sequence of the CalS5 protein of the present invention or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • primers can be designed according to the disclosed nucleotide sequences, particularly open reading frame sequences, and can be prepared using commercially available cDNA libraries or conventional methods known to those skilled in the art.
  • As a template amplification is performed to obtain the relevant sequence. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order.
  • the sequence is cloned into a vector, transferred to a cell, and the relevant sequence is isolated from the proliferated host cell by a conventional method.
  • the artificial synthesis method is especially suitable for the case where the length of the fragment is short, and usually, a plurality of small fragments are synthesized first, and then connected to obtain a long-length fragment.
  • the invention also relates to vectors comprising the polynucleotides and encoding with the vectors or CalS5 proteins A host cell genetically engineered, and a method of producing a polypeptide of the invention by recombinant techniques.
  • the recombinant rice CalS5 protein is expressed or produced by the polynucleotide sequence of the present invention by conventional recombinant DNA technology (Science, 1984; 224: 1431). Generally speaking, the following steps are included:
  • a polynucleotide sequence encoding the CalS5 protein can be inserted into a recombinant expression vector.
  • recombinant expression vector refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus or other vector well known in the art.
  • the recombinant expression vector is any plasmid and vector that can be replicated and stabilized in a host.
  • An important feature of the expression vector is that it typically contains an origin of replication, a promoter, a marker gene, and a translational control element.
  • Methods well known to those skilled in the art can be used to construct expression vectors containing the CalS5 protein-encoding DNA sequence and appropriate transcription/translation control signals. Such methods well known to those skilled in the art include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombinant DNA techniques, and the like.
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis.
  • the expression vector further comprises a ribosome binding site for translation initiation and a transcription terminator, preferably comprising one or more selectable marker genes to provide for selection transformation.
  • a ribosome binding site for translation initiation and a transcription terminator preferably comprising one or more selectable marker genes to provide for selection transformation.
  • the phenotypic traits of the host cell such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences, can be used to transform appropriate host cells to enable expression of the protein.
  • the host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell. Representative examples are: Escherichia coli, Streptomyces, Agrobacterium, and the like.
  • an enhancer sequence When a polynucleotide of the present invention is expressed in a higher eukaryotic cell, insertion of an enhancer sequence into the vector will enhance transcription.
  • An enhancer is a cis-acting factor of DNA, usually about 10 to 300 base pairs, acting on a promoter to enhance transcription of the gene.
  • Recombinant DNA can be transformed into host cells using conventional techniques well known to those skilled in the art.
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase, treated with the CaCl 2 method, using methods well known in the art.
  • Another method is to use MgCl 2 , and the conversion can also be carried out by electroporation.
  • the host cell is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.
  • the transformed plant may be subjected to a method such as Agrobacterium transformation or gene gun transformation, such as a leaf disc method.
  • a method such as Agrobacterium transformation or gene gun transformation, such as a leaf disc method.
  • Agrobacterium transformation or gene gun transformation such as a leaf disc method.
  • plants can be regenerated by conventional methods to obtain plants with altered tolerance.
  • the obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture may be selected from various conventional media depending on the host cell used.
  • the cultivation is carried out under conditions suitable for the growth of the host cell.
  • the selected promoter is induced by a suitable method, such as temperature conversion or chemical induction, and the cells are cultured for a further period of time.
  • a part or all of the polynucleotide of the present invention can be immobilized as a probe on a microarray or a DNA chip, also referred to as a "gene chip", for analyzing differential expression analysis of genes in tissues.
  • Transcripts of CalS5 protein can also be detected by RNA-polymerase chain reaction (RT-PCR) in vitro amplification using CalS5 protein-specific primers.
  • the main advantage of the present invention is that it is first discovered that for certain plant sterility lines, the regulation of the plant's fertility by regulating the expression or activity of CalS5 protein associated with pollen development in plant plants, thereby achieving infertility and Controllable conversion between fertility.
  • the application of plant sterility lines in agricultural breeding has been developed, which greatly simplifies the breeding methods of plant sterility lines.
  • Figure 1 shows the abnormal wall pattern of pollen caused by defects in the enamel wall.
  • Figure 1-1 is a schematic diagram showing the insertion position of the T-DNA of the CalS5 mutant.
  • FIG 1-2 shows the RT-PCR analysis of the CalS5 mutant
  • Figure 1-3 shows real-time quantitative PCR expression of three allelic mutants of CalS5
  • Figure 1-4 shows the loss or reduction of the enamel wall of the CalS5 allelic mutant, Alexander staining and pollen wall pattern
  • Figure 2 shows the effect of temperature on the physical fitness of male sterile CalS5 mutation.
  • Figure 2-1 shows the fertility of CalS5 mutant plants under low temperature and short light conditions.
  • Figure 2-2 shows the number of fertile seeds recovered from CalS5 mutant plants.
  • Figure 3-1 shows the staining of the quaternary wall of the mutant tetrad
  • Figure 3-2 shows the scanning electron microscopic observation of pollen development.
  • Figure 3-3 shows the semi-thin section of the anther development of the CalS5 mutant
  • Figure 4 is a transmission electron microscopic analysis of the recovered pollen development stage of mutants.
  • Figure 5 shows the effect of ambient temperature on the pollen development rate of Arabidopsis thaliana
  • Figure 5-1 shows the time during which the anthers complete their development under different conditions.
  • Figure 5-2 shows the diameter division of microspores produced under different conditions.
  • Figure 5-3 shows the microspore meiosis process under different conditions.
  • FIG. 6 is a diagram showing the fertility recovery of different mutants of CalS5.
  • Figure 7 shows the fertility of CalS5 under high temperature conditions.
  • Figure 7-1 shows the fertility of CalS5 mutant plants under high temperature conditions.
  • Figure 7-2 shows the pollen produced by CalS5 mutant plants under high temperature conditions.
  • a is wild type
  • b is CalS5-6
  • c is CalS5-2
  • d is CalS5-5
  • Arabidopsis thaliana is a Col ecotype. Seeds were pre-germinated on 0.1% agarose medium for 72 h at 4 °C. The material was then cultured in vermiculite under the conditions of: room temperature 24 ° C, light culture for 16 h / dark culture for 8 h (normal conditions) until convulsions. Thereafter, the sputum strain was transferred to different conditions to continue the culture.
  • Low temperature culture (L) conditions are: light incubator, 18 ° C or 21 ° C; general light (N) culture conditions: 24 ° C, light culture 16 h / dark culture 8 h; short light culture (S) conditions: 24 ° C, light culture 8h/ Dark culture for 16h.
  • Plant materials were taken with a Nikon digital camera (D-7000). For Alexander staining and DAPI staining, see Alex & Er, 1969; Ross et al., 1996. For semi-thin sections, the flower buds were fixed at different developmental stages and embedded in Spurr epoxy resin. 1 ⁇ m sections were made using a Powertome XL (RMC Products, Arlington, Arizona, USA) microtome and stained with toluidine blue. Anther slice was taken using an Olympus DX51 digital camera (Olympus, Japan). Fresh stamens and pollen grains were wrapped in 8 nm gold particles for scanning electron microscopy and observed using a JSM-840 microscope (JEOL, Japan).
  • the Arabidopsis flower was fixed on the ice in the fixing solution.
  • the calyx material was further embedded in a resin (Hard Plus' Embedding Resin, Unite Kingdom). Ultrathin sections (50-70 nm) were observed using a JEM-1230 transmission electron microscope (JEOL, Japan).
  • RNA can be extracted from mature soil cultured Arabidopsis plant flower tissue using Trizol reagent (Invitrogen, USA).
  • Trizol reagent (Invitrogen, USA).
  • Poly-dT (12-18) was used as a primer; MMLV reverse transcriptase and corresponding reagents inverted 5 ⁇ g of RNA into the first cDNA strand (transcription at 42 ° C for 60 min), and the synthesized cDNA strand was used as a PCR template.
  • Quantitative RT-PCR was performed by ABI PRISM 7300 system (Applied Biosystems, USA) using SYBR Green I master mix (Toyobo, Japan) with ⁇ -Tubulin as a control.
  • the program parameters of quantitative RT-PCR were: 95 ° C for 5 min, 94 ° C, 10 s denaturation for 40 cycles, and 60 ° C annealing for 1 min.
  • the present invention inserts three allelic mutants into the T-DNA mutant library of the Arabidopsis Col ecotype, CalS5-6, CalS5-2 and CalS5-5 (Fig. 1-1), and examines Arabidopsis thaliana. Fertility of the CalS5 mutant.
  • the CalS5 mutant was cultured at room temperature until twitching, and it was transferred to 18 °C for continuous culture, and the subsequent pods recovered fertility (as shown in Figure 2-1), and the number of seeds was large ( Figure 2-2) Show). Under the same low temperature conditions, the fertility of wild-type plants was not affected (as shown in Figure 2-1). Alexander staining showed that the pollen of the mutant was stained purple (as shown in Figure 2-1) under low temperature conditions, the same as the wild type, and there was no or only less fertile pollen in the mutant anther under normal conditions. . This indicates that low temperature can compensate for the developmental defects of male gametophytes in CalS5 mutants.
  • the CalS5 mutants were cultured at 18 ° C, 20 ° C, and 23 ° C, respectively, and the fertility recovery was observed.
  • the results showed that: CalS5- at 18 ° C 2
  • the mutants recovered all fertility, and the fertility decreased to 85% and 48% at 20 ° C and 23 ° C, respectively, and higher temperatures resulted in complete infertility (as shown in Figures 6 and 7). It is indicated that the low temperature can restore the fertility of the CalS5 mutant, the temperature is increased, the fertility is reduced, and even the fertility is completely lost.
  • FIG. 3-1 shows that the fertility was restored compared with the wild type plants.
  • the enamel wall of the CalS5-2 mutant plant still failed to form.
  • Scanning electron microscopy (TEM) of Figure 3-2 shows that the wild type plants and the mature anthers of the fertile CalS5-2 plants contain many pollen, but the outer wall structure is still abnormal.
  • Figure 3-3 shows the semi-thin section of the anther development stage 11 of CalS5-2 incubated at 23 ° C and 18 ° C, respectively. The results showed that there was a clear red cell content in the CalS5-2 mutant grown at 18 °C. The cell contents of the CalS5-2 mutant grown at 23 °C leaked into the drug chamber.
  • Fig. 4 is a transmission electron microscopic analysis of the developmental stage of the mutant which restores fertility, and the results show that the low temperature mutant cannot form the outer wall of the pollen like the normal temperature.
  • stage 6 and 7 microspore mother cells undergo meiosis to form tetrads; subsequently, microspores are released from the tetrads and gradually form trinuclear pollen grains with normal pollen walls.
  • stage 6 and 7 microspore mother cells undergo meiosis to form tetrads; subsequently, microspores are released from the tetrads and gradually form trinuclear pollen grains with normal pollen walls.
  • the normal temperature CalS5 mutant no developmental differences between the mutant and the wild type were observed until the sixth stage of anther development, indicating that the mutant male gametophyte meiosis was not affected.
  • the semi-quantitative RT-PCR technique was used to detect the relative expression of genes related to the primary wall of pollen in the context of CalS5 mutants. The results showed that the genes involved in the synthesis of primary outer wall were normal in the CalS5 background, and the synthesis of the primary outer wall was not affected. 4).
  • the present invention detects CalS5 transcription and protein levels in wild-type and mutant flower buds at different temperatures. Quantitative PCR assay showed that there was no significant difference in transcriptional level between mutant and wild-type CalS5 at room temperature (23 ° C) and low temperature (18 ° C). These results indicate that temperature does not produce induced expression of CalS5.
  • the present invention investigates the speed of pollen development at different temperatures, and according to the growth process of the flower buds, the diameter of the microspores of the flower buds is counted (Fig. 5-2), and the microspores are reduced according to the pollen development process. Counting process statistics ( Figure 5-3).
  • the pollen development process is divided into three periods: the unnuclear stage (uninucleate stage), the double-core flower The bicellular stage and the tricellular stage.
  • the statistical results show that the microspores start from the tetrad stage, and the diameter at room temperature is about 14 ⁇ m. After two days after the first mitosis, the diameter of the microspores in the binuclear phase is about doubled. When mature pollen was formed, the pollen diameter increased to 21 ⁇ m.
  • the invention counts the growth rate of microspores at different temperatures at different temperatures, and the results show that as the temperature increases, the growth rate of microspores increases.
  • DAPI staining results showed that the meiosis process was normal at different temperatures, but accelerated with increasing temperature. The results indicate that the slow-growing growth and development time is an important reason to compensate for the microspores of CalS5.
  • the CalS5-2 mutant is cultured under normal temperature (23 ° C), normal illumination (16 h light / 8 h dark) to the convulsion stage, and then placed in short daylight (8 h light / 16 h dark) for 5 days to examine The fertility restoration of CalS5 microspore developmental defects was restored by sunshine conditions. The results showed that the fertility of the mutant was restored (as shown in Figure 2). Alexander staining experiments showed that there was no pollen in the mutant anther under normal light conditions, but there was a certain amount of pollen formation under short daylight conditions (as shown in Figure 2). These results indicate that the fertility recovery mechanism of CalS5 under short-day conditions is similar to that of low-temperature treatment.
  • Figures 6 and 7 show the seed amount of the CalS5 mutant under high temperature conditions. At high temperature (28 ° C), the number of seeds of the weak allelic mutant of CalS5 cultured under normal light was significantly reduced, and the temperature increased, the number of pollen in the mutant plants decreased, and at 28 ° C, there was almost no pollen in the mutant plants. It is indicated that under this condition, the fertility of the CalS5 mutant is seriously degraded.
  • the ambient temperature plays a more important role in the recovery of fertility relative to the photoperiod.

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Abstract

本发明提供了一种利用CalS5基因突变创制光温敏不育系的方法，通过降低所述植物植株中与花粉发育相关的胼胝质合成酶的表达来实现。本发明通过调控植株中与花粉发育相关的CalS5蛋白的表达或活性，来调控所述植株的育性，实现了不育与可育之间的转换，简化了植物不育系的育种方法。

Description

一种利用CalS5基因突变创制光温敏不育系的方法及其应用 技术领域
本发明属于农业和生物技术领域，尤其涉及一种利用CalS5基因突变创制光温敏不育系的方法及其应用。
背景技术
农业生产中，雄性不育系在杂交制种、提高农业产量中占据巨大优势。雄性不育往往被分为细胞质雄性不育(CMS)和细胞核雄性不育(GMS)两种。三系杂交体系的建立即依赖于细胞质雄性不育。但是，细胞质雄性不育有其自身的缺陷：首先，细胞质雄性不育植株的品质普遍较差；其次，三系杂交水稻的组合增产潜力越来越小；再次，由于野败型的雄性不育细胞质单一，一旦细胞质雄性不育丧失或某种毁灭性病虫害发生，那么就会造成巨大的损失。
随着细胞核雄性不育中光温敏条件性雄性不育的发现，两系法杂交水稻应运而生。相对于三系杂交法，光温敏不育系兼有不育系和保持系两种状态。与三系法相比，两系法不受恢保关系的限制，即细胞核不育可以与大量常规品种杂交，配组自由，更容易获得性状优良的杂交优势，从根本上解决三系中雄性不育细胞质单一化的问题。近年来，两系杂交水稻在中国农业生产中的应用越来越广泛。
早在1973年石明松在中国湖北从晚粳品种(Oryza sativa ssp.Japonica)农垦58中选育出光敏感不育系，并提出了一系两用的水稻杂交优势利用新途径。随后，以农垦58S(NK58S)为父本，与籼稻杂交获得的培矮64S(PA64S)也在两系杂交中得到广泛应用，只是培矮64S的育性对温度更加敏感。水稻光温敏不育系受单基因隐性位点控制；研究表明，农垦58S与培矮64S的不育性状均受同一个遗传位点控制，且温度和光照均会对该位点产生影响。
至今，共发现十三个水稻光温敏不育系pms1、pms2、pms3、rpms1、rpms2、tms1、tms2、tms3、tms4、tms5、tms6、rtms1和ms-h，分别定位在第7、3、12、8、9、8、7、6、2、2、5、10和第9条染色体上。研究发现，一个突变的小RNA(small RNA)osa-smR5864m，导致pms2以及p/tms2-1(农垦58S和培矮64S)突变体的不育表型。
拟南芥(A.thaliana)因为具有较小的基因组、快速的生长周期以及大量 的突变体库等无可比拟的优势，而在植物学、生物学领域成为模式植物。此外，还能在严格控制温度、光照等条件的狭小空间中培养。研究发现了一些拟南芥不育突变体，如PEAMT基因突变体t365以及GA/IAA生物合成受阻的ms33突变体均为温敏不育表型。
胼胝质合成酶是一类存在于不同物种中家族蛋白，在胼胝质的合成中起重要作用。在植物界，有报道称一些物种的胼胝质合成酶与植物的生殖发育有关，如在紫鸭跖草中作为暂时细胞壁机械屏障，防止细胞融合(Waterkeyn et al.1962)；在百合中为初生外壁提供糖原(Larson Lewis et al.1962)等。
目前，发掘植物雄性不育新种质的常规途径主要包括：对天然雄性不育原始株的发现，人工诱变和连续回交核置换。我国早期的野败型和马协型水稻雄性不育细胞质是对对天然雄性不育原始株的发现；新疆农科院培育的新海不育系棉花是利用海岛棉的杂种经60COγ射线诱变。但是，传统育种方法筛选和培育理想的可转育的雄性不育系存在诸多困难，如雄性不育系种质资源有限，转育周期过长，杂交不亲和，组合选育局限大等。目前，本领域尚缺乏调控方式简单方便的植物不育系用于植物育种过程，因此迫切需要调控方式简单方便的植物不育系培育技术。
发明内容
针对本领域存在的技术问题，本发明提供一种利用CalS5基因突变创制光温敏不育系的方法及其应用，从而解决目前核不育光温敏遗传位点少，制种纯度不高的问题。
本发明的第一个方面在于提供一种培育植物不育系的方法，包括步骤：降低所述植物植株中与花粉发育相关的胼胝质合成酶的表达或活性，所述胼胝质合成酶为CalS5蛋白或其同源蛋白。
作为本发明的一个优选方案，所述胼胝质合成酶参与所述植物花粉发育过程四分体时期的胼胝质合成。
作为本发明的一个优选方案，所述胼胝质合成酶在植物花序或花药的细胞、组织或器官中特异性表达。
作为本发明的一个优选方案，所述细胞或组织包括：小孢子、小孢子母细胞或其组合。
作为本发明的一个优选方案，所述胼胝质合成酶在花药发育期特异性表 达。
作为本发明的一个优选方案，所述花药发育期包括前花药形成阶段(-3-0天)、花药形成阶段和后花药形成阶段(1-5天)，其中，0天指花药形成的第1天，-3天为以花药形成当天为起始点，向前推算的第3天，1天、5天分别为以花药形成当天为起始点，向后推算的第1天和第5天。
作为本发明的一个优选方案，所述胼胝质合成酶在花药发育第7期特异表达。
作为本发明的一个优选方案，所述胼胝质合成酶在花粉减数分裂完成时达到表达最高峰。
作为本发明的一个优选方案，所述“降低所述植物植株中与花粉发育相关的胼胝质合成酶的表达或活性”满足以下条件：
A1/A0＝0-80％，优选为A1/A0＝0-60％，更优选为A1/A0＝0-40％，最优选为A1/A0＝0-30％；
其中，A1为所述植株中与花粉发育相关的胼胝质合成酶的酶活性；A0为野生型同种类型植物植株中相同胼胝质合成酶的酶活性。
作为本发明的一个优选方案，所述CalS5的野生型氨基酸序列选自下组中的任意一种或几种：SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6。
作为本发明的一个优选实施例，可通过以下任意一种方式降低所述胼胝质合成酶的表达或活性：
1)编码所述胼胝质合成酶的多核苷酸部分或完全缺失；
2)修饰表达调控序列以降低或抑制编码所述胼胝质合成酶的多核苷酸的表
达；
3)修饰染色体上的序列以降低蛋白的活性；或
4)上述1)-3)中的任意组合。
其中，所述的编码蛋白的多核苷酸部分或完全缺失是利用染色体基因插入的载体，将编码内源性靶蛋白的多核苷酸替换为标记基因或部分核苷酸序列缺失的多核苷酸。
“部分”缺失的长度根据多核苷酸的种类不同，可以为1-300bp，优选 为1-100bp，更优选为1-5bp，其中bp为碱基对。
作为本发明的一个优选实施例，可以通过以下任意一种方式修饰表达调控序列：
1)通过核苷酸序列的缺失、插入、保守、非保守性取代中的一种或几种的组合，在表达调控序列中诱导突变，以进一步降低表达调控序列的活性；
2)将表达调控序列替换成活性更低的序列。
作为本发明的一个优选实施例，所述的表达调控序列包括编码启动子序列、操纵子序列、核糖体结合位点和控制转录与翻译终止的序列。
作为本发明的一个优选实施例，可以通过以下任意一种方式修饰染色体上的多核苷酸序列以降低蛋白的活性：
1)通过核苷酸序列的缺失、插入、保守或非保守性取代中的一种或几种的组合在所述胼胝质合成酶序列中诱导突变，以进一步降低所述胼胝质合成酶的活性；
2)将多核苷酸序列替换成经修饰的序列以便获得更弱的蛋白活性。
作为本发明的一个优选方案，所述的“降低植物植株中与花粉发育相关的胼胝质合成酶的活性”的方法包括：使编码胼胝质合成酶的基因的表达水平下降。
作为本发明的一个优选方案，所述的“使编码胼胝质合成酶的基因的表达水平下降”满足条件：E1/E0＝0-80％，优选为E1/E0＝0-60％，更优选为E1/E0＝0-40％；和/或A1/A0＝0-80％，优选为A1/A0＝0-60％，更优选为A1/A0＝0-40％，最优选为A1/A0＝0-30％；
其中，E1为所述植物植株中与花粉发育相关的胼胝质合成酶的表达水平；
E0为野生型同种类型植物植株中相同胼胝质合成酶的表达水平；
A1为所述植物植株中与花粉发育相关的胼胝质合成酶的酶活性；
A0为野生型同种类型植物植株中相同胼胝质合成酶的酶活性。
作为本发明的一个优选方案，所述的“降低植株中胼胝质合成酶活性是通过基因突变、基因敲除、基因中断、RNA干扰中的一种或几种的结合实现的。
作为本发明的一个优选方案，编码所述胼胝质合成酶的基因为CalS5基因或其同源基因。
作为本发明的一个优选方案，所述CalS5基因能够编码SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6所示氨基酸序列。
其中编码所述胼胝质合成酶的多核苷酸序列选自：SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11和SEQ ID NO.12。
其中，编码SEQ ID NO.1的多核苷酸序列为SEQ ID NO.7。
其中，编码SEQ ID NO.2的多核苷酸序列为SEQ ID NO.8。
其中，编码SEQ ID NO.3的多核苷酸序列为SEQ ID NO.9。
其中，编码SEQ ID NO.4的多核苷酸序列为SEQ ID NO.10。
其中，编码SEQ ID NO.5的多核苷酸序列为SEQ ID NO.11。
其中，编码SEQ ID NO.6的多核苷酸序列为SEQ ID NO.12。
作为本发明的一个优选方案，所述“降低植物植株中与花粉发育相关的胼胝质合成酶的表达或活性”通过以下任意一种方式实现：
1)降低所述植物植株中CalS5基因的表达水平；
2)缺失所述植物植株中CalS5基因；
3)致所述植物植株中CalS5基因突变；或
4)上述1)-3)的任意组合。
作为本发明的一个优选方案，所述的植物包括农作物、林业植物及花卉等；优选地包括禾本科(Gramineae)、豆科(Leguminosae sp.)、十字花科(Brassicaceae)植物以及鼠耳芥属(Arabidopsis)植物，更优选地包括水稻、玉米、高粱、小麦、大豆或拟南芥。
作为本发明的一个优选方案，所述与花粉发育相关的胼胝质合成酶没有特别限定，可以来自任何植物品种，代表性的植物包括但并不限于：水稻(基因号：BGIOSGA021782，与拟南芥直系同源CalS5蛋白同源性为73％)、小米(基因号：Si008368m.g，与拟南芥直系同源CalS5蛋白同源性为77％)、高粱(基因号：Sb10g005550，与拟南芥直系同源CalS5蛋白同源性为81％)、小麦(基因号：Traes_7BS_170A2F4BB，与拟南芥直系同源CalS5蛋白同源性为75％)、玉米(基因号：GRMZM2G353905_T01，与拟南芥直系同源CalS5蛋白同源性为81％)。
本发明的第二个方面在于提供一种编码与花粉发育相关的胼胝质合成酶的基因的用途，所述基因用于培育植物不育系或用于制备培育植物不育系的试剂或试剂盒。
作为本发明的一个优选方案，所述的编码基因为CalS5基因或其同源基因。
作为本发明的一个优选方案，所述CalS5基因能够编码SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6所示氨基酸序列。
本发明的第三个方面在于提供一种将植物从不育转为可育的方法，包括步骤：降低花粉的发育速度。
作为本发明的一个优选方案，所述植物是与花粉发育相关的胼胝质合成酶的表达水平下降的植物。
作为本发明的一个优选方案，所述植物为根据本发明的第一个方面所述的方法培育的植物不育系。
作为本发明的一个优选方案，所述“降低花粉的发育速度”是通过降低植株生长的环境温度和/或减少植株的光照时间实现的。
作为本发明的一个优选方案，降低植株生长的环境温度包括将环境温度(平均温度)控制在18-23℃，如19℃、20℃或22℃等。
作为本发明的一个优选方案，降低植株生长的环境温度的时间包括花药形成阶段，花粉成熟阶段以及开花授粉阶段或其前后2周。
作为本发明的一个优选方案，在植株抽薹或抽穗时开始降低植株的生长温度，低温培育7-14天后，恢复正常温度培育。
本发明的第四个方面在于提供一种植物育种方法，包括如下步骤：
步骤1、维持植株不育；
步骤2、将植株由不育转为可育；
步骤3、维持植株可育并育种。
其中，所述维持植株不育为维持根据本发明提供的第一个方面所述的方法培育的植物不育系；
所述将植株由不育转为可育是根据本发明提供的第三个方面所述的方法将 植株由不育转为可育。
本发明的第五个方面，在于提供一种植物细胞，在由所述植物细胞发育成的植株中，与花粉发育相关的胼胝质合成酶的表达降低。
作为本发明的一个优选方案，所述降低满足以下条件：
A1/A0＝0-80％，优选为A1/A0＝0-60％，更优选为A1/A0＝0-40％，最优选为A1/A0＝0-30％；
其中，A1为所述植株中与花粉发育相关的胼胝质合成酶的酶活性；A0为野生型同种类型植物植株中相同胼胝质合成酶的酶活性。
作为本发明的一个优选方案，所述胼胝质合成酶为CalS5蛋白或其同源蛋白。
其中，“CalS5蛋白”、“CalS5多肽”指具有CalS5的氨基酸序列，如SEQ ID NO.1-6的蛋白或多肽，在未特别指出时，CalS5蛋白包括野生型CalS5蛋白和突变型CalS5蛋白。
本发明所述的胼胝质合成酶包含但并不仅限于SEQ ID NO.1-6所示氨基酸序列，根据植物种类或品种，所述蛋白的氨基酸序列可能有所不同。换言之，所述CalS5蛋白可以是突变型蛋白或其人工变体，只要有助于通过降低该蛋白的活性而培育植物不育系，所述突变型蛋白及其人工变体的氨基酸序列在SEQ ID NO.1-6所示氨基酸序列的一个或多个位置包含一个或多个氨基酸的取代、缺失、插入、添加或倒置。
本发明所述的“多个”，根据蛋白质中氨基酸残基三维结构的位置或类型而有所不同，优选为2–20个，更优选为2–10个，更优选为2–5个。此外，根据植物的个体或种类，氨基酸的取代、缺失、插入、添加或倒置包括人工变体或天然突变所致的氨基酸的取代、缺失、插入、添加或倒置。
本发明所述的“分离的”是指物质从其原始环境中分离出来，如果是天然的物质，则其原始环境即为自然环境，例如，活体细胞内天然状态下的多聚核苷酸和多肽是没有分离纯化的，而如果从天然状态中将同样的多聚核苷酸或多肽与其他物质分开，则为分离纯化的。
本发明所述的“分离的CalS5蛋白或多肽”是指基本上不含与CalS5蛋白相 关的其它蛋白、脂类、糖类或其它物质。本领域技术人员能够采用标准的蛋白质纯化技术纯化水稻等植物中的CalS5蛋白。基本上，纯的多肽在非还原聚丙烯酰胺凝胶上能产生单一的主带。
本发明所述的“多肽”可以是重组多肽、天然多肽或合成多肽等，优选为重组多肽。
本发明所述的多肽可以是天然纯化的产物，也可以是化学合成的产物，或使用重组技术从原核或真核宿主(例如，细菌、酵母、高等植物、昆虫和哺乳动物细胞)中产生；根据重组技术使用的宿主，所述的多肽可以是糖基化或非糖基化多肽；本发明所述多肽还可以包括起始的甲硫氨酸残基。
本发明所述的CalS5蛋白还包括CalS5蛋白的片段、衍生物及其类似物。
本发明中，术语“片段”、“衍生物”和“类似物”是指能保持与本发明所述CalS5蛋白相同的生物学功能或活性的多肽。
本发明所述的多肽片段、衍生物或类似物可以是：
(1)有一个或多个保守或非保守性氨基酸残基被取代的多肽，并优选为保守性氨基酸残基被取代的多肽，所述被取代的氨基酸残基可以由遗传密码编码；
(2)在一个或多个氨基酸残基中具有取代基团的多肽；
(3)成熟多肽与另一个化合物，如延长多肽半衰期的化合物，例如聚乙二醇，融合所形成的多肽；
(4)附加的氨基酸序列融合到此多肽序列而形成的多肽，如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列或融合蛋白。
本发明所述的片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
在本发明的优选实施方式中，“CalS5蛋白””或“CalS5多肽”序列如SEQ ID NO.1-6所示。
在本发明的优选地实施方式中，“CalS5蛋白””或“CalS5多肽”还包括与CalS5蛋白功能相同的、SEQ ID NO.1-6序列的变异形式，这些变异形式包括但并不限于一个或多个，通常为1-50个，优选为1-30个，更优选为1-20个，更优选为1-10个，氨基酸的缺失、插入和/或取代，以及在碳末端和/或氮末端添加1个或数个氨基酸，通常为20个以内，优选为10个以内，更优选为5个以内。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不 会改变蛋白质的功能，在碳末端和/或氮末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。
在本发明的优选地实施方式中，“CalS5蛋白””或“CalS5多肽”还包括CalS5蛋白或多肽的活性片段和活性衍生物。
其中，所述CalS5多肽的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严谨度的条件下能与CalS5蛋白DNA杂交的DNA所编码的蛋白。
本发明还提供了其他多肽，如包含CalS5蛋白或其片段的融合蛋白。除了几乎全长的多肽外，本发明还包括了CalS5蛋白的可溶性片段。通常，所述片段具有CalS5蛋白序列的至少约10个连续氨基酸，通常具有至少约30个连续氨基酸，优选地，具有至少约50个连续氨基酸，更优选地，具有至少约80个连续氨基酸，最优选地，具有至少约100个连续氨基酸。
本发明中所述修饰通常不改变一级结构，所述修饰形式主要包括：体内或体外的多肽的化学衍生形式如乙酰化或羧基化；还包括糖基化。修饰形式还包括具有磷酸化氨基酸残基的序列，如磷酸酪氨酸、磷酸丝氨酸、磷酸苏氨酸；还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的多肽。
在本发明中，“CalS5保守性变异多肽”指与SEQ ID NO.：1-6所示的氨基酸序列相比，有至多10个，优选为至多8个，更优选为至多5个，最优选为至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表1进行氨基酸替换而产生。
表1
最初的残基 代表性取代 优选取代
Ala(A) Val，Leu，Ile Val
Arg(R) Lys，Gln，Asn Lys
Asn(N) Gln，His，Lys，Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro，Ala Ala
His(H) Asn，Gln，Lys，Arg Arg
Ile(I) Leu，Val，Met，Ala，Phe Leu
Leu(L) Ile，Val，Met，Ala，Phe Ile
Lys(K) Arg，Gln，Asn Arg
Met(M) Leu，Phe，Ile Leu
Phe(F) Leu，Val，Ile，Ala，Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr，Phe Tyr
Tyr(Y) Trp，Phe，Thr，Ser Phe
Val(V) Ile，Leu，Met，Phe，Ala Leu
其中，所述代表性取代是指本发明能够取代最初的残基的碱基，所述优选取代是本发明最优选的能够取代最初的残基的碱基。
本发明所述的多核苷酸可以是DNA形式或RNA形式。其中，DNA形式包括cDNA、基因组DNA或人工合成的DNA；DNA可以是单链的或是双链的；DNA可以是编码链的，也可以是非编码链的；编码成熟多肽的编码区序列可以与SEQ ID NO.7-12所示的编码区序列相同或者是简并的变异体。
在本发明中，“简并的变异体”在本发明中是指编码具有SEQ ID NO.1-6的蛋白质，但与SEQ ID NO.7-12所示的编码区序列有差别的核酸序列。
编码SEQ ID NO.1-6的成熟多肽的多核苷酸选自以下任意一种：
(1)只编码成熟多肽的编码序列；
(2)成熟多肽的编码序列和各种附加编码序列；
(3)成熟多肽的编码序列和任选的附加编码序列以及非编码序列。
作为本发明的一个优选实施例，所述的CalS5多肽的编码序列选自下组：
(1)编码如SEQ ID NO.1-6所述多肽的多核苷酸序列；
(2)如SEQ ID NO.7-12所示的多核苷酸序列；
(3)与(1)或(2)所述的多核苷酸序列互补的多核苷酸。
在本发明中，“编码多肽的多核苷酸”可以是包括编码该多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
本发明还涉及所述多核苷酸的变异体，所述编码与本发明有相同的氨基酸序列的多肽或多肽的片段、类似物和衍生物，所述多核苷酸的变异体可以是天然发生的等位变异体或非天然发生的变异体。这些核苷酸变异体包括取代变异体、缺失变异体和插入变异，如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可以是一个或多个核苷酸的取代、缺失或插入，但不会从实质上改变其编码的多肽的功能。
作为本发明的一个优选实施例，本发明还涉及与上述的序列杂交，且两个序列之间具有至少50％，优选为至少70％，更优选为至少80％相同性的多核苷酸。
本发明还涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。
本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC，0.1％SDS，60℃；或(2)杂交时加有变性剂，如50％(v/v)甲酰胺，0.1％小牛血清/0.1％菲可400，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。并且，可杂交的多核苷酸编码的多肽与SEQ ID NO.1-6所示的成熟多肽有相同的生物学功能和活性。
本发明还涉及与上述的序列杂交的核酸片段，所述“核酸片段”的长度至少含15个核苷酸，优选为至少含30个核苷酸，更优选为至少含50个核苷酸，最优选为至少含100个核苷酸。所述核酸片段可用于核酸的扩增技术，如PCR，以确定和/或分离编码CalS5蛋白的多聚核苷酸。
本发明所述的CalS5蛋白核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成法获得。PCR扩增法，可根据本发明所公开的有关核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增得到有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。重组法是将序列克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。人工合成法尤其适用于片段长度较短时，通常，先合成多个小片段，然后再进行连接可获得序列很长的片段。
本发明也涉及包含所述多核苷酸的载体以及用所述载体或CalS5蛋白编码 序列经基因工程产生的宿主细胞，以及经重组技术产生本发明所述多肽的方法。通过常规的重组DNA技术(Science，1984；224：1431)，利用本发明的多聚核苷酸序列表达或生产重组的水稻CalS5蛋白。一般来说包含以下步骤：
(1)用本发明编码CalS5蛋白的多核苷酸或变异体，或用含有所述CalS5蛋白的多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2)在合适的培养基中培养宿主细胞；
(3)从培养基或细胞中分离、纯化蛋白质。
在本发明中，编码所述CalS5蛋白的多核苷酸序列可以插入到重组表达载体中。
术语“重组表达载体”指本领域熟知的细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒或其他载体。总之，所述重组表达载体为能在宿主体内复制和稳定的任何质粒和载体。所述表达载体的一个重要特征是：通常含有复制起点、启动子、标记基因和翻译控制元件。
本领域技术人员熟知的方法能用于构建含CalS5蛋白编码DNA序列和合适的转录/翻译控制信号的表达载体。所述的本领域技术人员熟知的方法包括体外重组DNA技术、DNA合成技术以及体内重组DNA技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。
作为本发明的一个优选实施例，所述表达载体还包括翻译起始用的核糖体结合位点和转录终止子，优选地，包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP)，或用于大肠杆菌的四环素或氨苄青霉素抗性。
作为本发明的一个优选实施例，包含上述的适当DNA序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主细胞，以使其能够表达蛋白质。所述宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如植物细胞。代表性例子有：大肠杆菌，链霉菌属、农杆菌等。
本发明的多核苷酸在高等真核细胞中表达时，在载体中插入增强子序列时将会使转录得到增强。增强子是DNA的顺式作用因子，通常大约有10到300个碱基对，作用于启动子以增强基因的转录。
本领域技术人员清楚如何选择适当的载体、启动子、增强子和宿主细胞。重 组DNA转化宿主细胞可采用本领域技术人员熟知的常规技术。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl2法处理，采用本领域熟知的方法。另一种方法是使用MgCl2，转化也可用电穿孔的方法进行。当宿主细胞是真核生物时，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械法，如显微注射、电穿孔、脂质体包装等。
作为本发明的一个优选方案，所述转化植物可以使用农杆菌转化或基因枪转化等方法，例如叶盘法。对于转化的植物细胞、组织或器官可以用常规方法再生成植株，从而获得耐受性改变的植物。
获得的转化子可以采用常规方法培养，表达本发明的基因所编码的多肽。根据所使用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，采用合适的方法，如温度转换法或化学诱导法，诱导选择的启动子，将细胞再培养一段时间。
本发明的多核苷酸的一部分或全部可作为探针固定在微阵列(microarray)或DNA芯片，又称为“基因芯片”上，用于分析组织中基因的差异表达分析。用CalS5蛋白特异的引物进行RNA-聚合酶链反应(RT-PCR)体外扩增也可检测CalS5蛋白的转录产物。
本发明的主要优点在于：首次发现了对于某些特定的植物不育系，通过调控植物植株中与花粉发育相关的CalS5蛋白的表达或活性，来调控所述植株的育性，实现不育与可育之间的可控转换。此外，还开发了植物不育系在农业育种等方面的应用，大大简化了植物不育系育种方法。
附图说明
以下结合附图对本发明做进一步描述，其中：
图1为胼胝质壁缺陷导致花粉外壁模式异常
图1-1为CalS5突变体的T-DNA插入位置示意图
图1-2为CalS5突变体的RT-PCR分析
图1-3为CalS5三个等位突变体实时定量PCR表达
图1-4为CalS5等位突变体胼胝质壁缺失或减少、亚历山大染色及花粉外壁模式
图2为温度对雄性不育CalS5突变体育性的影响
图2-1为低温和短光照条件下CalS5突变体植株的育性
图2-2为CalS5突变植株恢复可育种子的数量
图3为恢复育性突变体的观察
图3-1为突变体四分体胼胝质壁的染色观察
图3-2为花粉发育的扫描电镜观察。
图3-3为CalS5突变体花药发育的半薄切片观察
图4为恢复的突变体花粉发育阶段的透射电镜分析
图5为环境温度对导拟南芥花粉发育速度的影响
图5-1为不同条件下花药完成发育过程的时间
图5-2为不同条件下产生的小孢子的直径分部
图5-3为不同条件下产生小孢子减数分裂进程统计
图6为CalS5不同突变体的育性恢复图
图7为高温条件下CalS5的育性
图7-1为高温条件下CalS5突变体植株的育性
图7-2为高温条件下CalS5突变体植株产生的花粉
其中，a为野生型，b为CalS5-6，c为CalS5-2，d为CalS5-5
具体实施方式
下面结合具体实施例，进一步阐述本发明。应当理解的是，这些实施例仅用于解释本发明而不用于限定本发明的保护范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等，分子克隆：实验室手册(New York：Cold Spring Harbor Laboratory Press，1989)或植物分子生物学-实验手册(Plant Molecular Biology-A Laboratory Mannual，Melody S.Clark，Springer-verlag Berlin Heidelberg，1997)中所述的条件，或按照制造厂商所建议的条件。除非特别说明，涉及的百分比和份数均为质量百分比和质量份数。
材料与方法
植物材料与种植
本发明中拟南芥为Col生态型。4℃，种子预萌发于0.1％琼脂糖培养基上72h。然后将材料培养于蛭石中，培养条件为：室温24℃，光培养16h/暗培养8h(正常条件)，直至抽薹。之后，将抽薹株转移至不同条件下继续培养。低温培养(L)条件为：光照培养箱，18℃或21℃；普通光照(N)培养条件：24℃，光培养16h/暗培养8h；短光照培养(S)条件：24℃，光培养8h/ 暗培养16h。
细胞学分析
采用尼康数码相机(D-7000)拍摄植物材料。亚历山大染色与DAPI染色可参考Alex&Er，1969；Ross et al.，1996。对于半薄切片，选取花苞不同发育阶段进行固定并包埋于Spurr环氧树脂中。使用Powertome XL(RMC Products，Tucson，Arizona，USA)切片机进行1μm切片，并用甲苯胺蓝进行染色。使用Olympus DX51数码相机(Olympus，Japan)进行花药切片的拍摄。将8nm金颗粒包裹新鲜雄蕊和花粉粒材料进行扫描电镜实验，并利用JSM-840显微镜(JEOL，Japan)观察。对于透射电镜实验，将拟南芥花絮冰上固定于固定液中，固定液的配方为：含有2.5％戊二醛的0.1M磷酸缓冲液，pH值＝7.2。花苞材料进一步依次包埋至树脂(Hard Plus’Embedding Resin，Unite Kingdom)中。利用JEM-1230透射电镜(JEOL，Japan)进行观察超薄切片(50-70nm)。
RNA抽提和定量RT-PCR
总RNA可利用Trizol试剂(Invitrogen，USA)由成熟土培拟南芥植物花组织进行提取。poly-dT(12-18)作为引物；MMLV反转录酶和相应试剂将5μg的RNA反转第一条cDNA链(42℃转录60min)，合成好的cDNA链作为PCR模板。
定量RT-PCR是利用SYBR Green I master mix(Toyobo，Japan)通过ABI PRISM 7300系统(Applied Biosystems，USA)进行检测，以β-Tubulin作为对照。定量RT-PCR的程序参数是：95℃5min，94℃，10s变性40个循环，60℃退火延伸1min。
CalS5突变体的育性
本发明在拟南芥Col生态型的T-DNA突变体库中插入三个等位突变体，分别是CalS5-6，CalS5-2和CalS5-5(如图1-1)，考察拟南芥CalS5突变体的育性。
由图1-2～图1-4可知，CalS5-6和CalS5-2胼胝质壁缺失，CalS5-5胼胝质壁减少，图1-4中亚历山大染色显示，野生型和CalS5-5的花药中充满紫色有活力的花粉，而在CalS5-6和CalS5-2的花药中只有绿色的残余，说明CalS5-6和CalS5-2花粉败育；野生型花粉外壁模式呈现规则的网格状结 构，而CalS5-6和CalS5–2呈现不规则的球状沉积，CalS5–5也呈现出不同程度的外壁异常，均表明CalS5三个等位突变体花粉外壁模式异常。常温下，纯合的CalS5-6和CalS5-2突变体植株生长正常，但CalS5-6的育性完全丧失，而CalS5-2也只有个别正常花粉，即丧失绝大部分育性。
低温下CalS5突变体的育性恢复
将CalS5突变体在常温下培养至抽薹，将其移入18℃连续培养，其后续的果荚皆恢复育性(如图2-1所示)，且种子数量较多(如图2-2所示)。在同样的低温条件下，野生型植株的育性并没有受到影响(如图2-1所示)。亚历山大染色显示，低温条件下突变体的花粉被染成紫红色(如图2-1所示)，与野生型的相同，而正常条件下的突变体花药内并无或只有较少可育花粉。这说明低温能够弥补CalS5突变体中雄配子体的发育缺陷。
本发明将抽薹的CalS5植株在18℃处理14天后，将CalS5突变体分别于18℃、20℃、23℃温度条件下进行培养，并观察育性恢复情况，结果表明：18℃下，CalS5-2突变体恢复全部育性，而在20℃和23℃的条件下育性分别降低至85％和48％，更高的温度则会导致完全不育(如图6、图7所示)，说明低温能够使CalS5突变体恢复育性，温度升高，育性降低，甚至完全丧失育性。
分别对野生型和低温恢复育性的CalS5-2突变体进行四分体胼胝质壁的染色观察，结果如图3所示，图3-1显示，与野生型植株相比，恢复育性的CalS5-2突变体植株的胼胝质壁仍然无法形成。图3-2的扫描电镜观察显示，野生型植株与恢复育性的CalS5-2植株的成熟花药中含有许多花粉，但外壁结构仍然异常。图3-3是分别对23℃和18℃培育的CalS5-2的花药发育第11期的半薄切片观察，结果显示，18℃培育的CalS5-2突变体中有明显的红色细胞内容物，而23℃培育的CalS5-2突变体的细胞内容物外漏到药室中。
图4为恢复育性的突变体发育阶段的透射电镜分析，结果显示，低温突变体与常温的一样无法形成花粉外壁。但野生型植株，在第6、7期，小孢子母细胞经历减数分裂形成四分体；随后，小孢子从四分体释放，并逐渐形成有正常花粉壁的三核花粉粒。常温CalS5突变体中，直到花药发育的第6期，均未观察到突变体和野生型的发育差异，这表明突变体雄配子体减数分裂不受影响。如图4所 示，至花药发育第7期早期，与野生型相比，突变体质膜波浪型起伏与野生型相比都较为正常，但缺少电子致密度较小的初生外壁，且电子致密度较大的孢粉素提前且不规则的沉积在小孢子外侧。花药发育第7期晚期，突变体前棒状体呈现球状沉积。至花药发育第8期，CalS5小孢子从四分体释放，与野生型相比，小孢子外壁呈现多处裂口的表型。花药发育第9期，大部分CalS5小孢子开始降解，随后，小孢子的细胞质收缩和瓦解。最终，药室内只有一些败育花粉的碎片，并没有正常的花粉形成。另一方面，在低温状态下(18℃)，CalS5小孢子在花药发育第8期的外壁裂口消失，在后续的发育阶段，大部分小孢子并没有破裂降解，而是逐渐恢复正常，低温下的药室中产生了正常的成熟花粉粒。
扫描电镜结果显示，CalS5-6和CalS5-2突变体在常温的药室中没有或只有较少花粉粒，但其在低温条件下的花粉粒数量和结构与野生型的基本一致(如图3所示)。Tinapol&DIOC2染色观察表明CalS5小孢子的细胞完整性在低温条件下得以恢复。在正常温度(23℃)下，CalS5小孢子的外壁结构仍然表现出不规则状态，其细胞质已明显泄漏，这导致了小孢子后期的破裂降解。该结果表明，尽管小孢子的外壁正常形成，但该基因的缺失导致了细胞壁完整性受损。在低温条件(18℃)下，CalS5小孢子花粉细胞质保持稳定(图3-3)，表明低温能够克服CalS5突变所带来的细胞壁完整性缺陷。
利用半定量的RT-PCR技术，检测CalS5突变体背景下，与花粉初生外壁合成相关基因的相对表达，结果显示初生外壁合成相关基因在CalS5背景下表达正常，初生外壁的合成不受影响(图4)。
为了阐明CalS5突变体温敏的机制，本发明对不同温度下的野生型和突变体花苞中的CalS5转录与蛋白水平进行了检测。定量PCR检测表明，常温(23℃)与低温(18℃)条件下突变体和野生型的CalS5在转录水平上没有显著差异。这些结果表明温度不会对CalS5产生诱导表达。
温度对花粉发育速度的影响
如图5所示，本发明考察了不同温度下，花粉发育的速度，并根据花苞的生长进程对花苞作小孢子的直径统计(图5-2)，同时根据花粉发育进程进行小孢子的减数分裂进程统计(图5-3)。
将花粉发育过程分为3个时期：单核花粉期(uninucleate stage)、双核花 粉期(bicellular stage)以及三核花粉期(tricellular stage)。统计结果表明小孢子从四分体时期开始，常温下的直径大约为14μm，经过第一次有丝分裂后2天后，双核期的小孢子直径大约扩大了2倍。当形成成熟花粉后，花粉直径增大到了21μm。
本发明统计了不同温度下各时期的小孢子生长速度，结果表明，随着温度升高，小孢子直径增长速度加快。此外，DAPI染色结果表明，不同温度下减数分裂进程均正常，但随温度升高进程加快。该结果说明，低温延缓的生长发育时间是弥补CalS5小孢子缺陷的重要原因。
日照条件对CalS5突变体育性的影响
本发明在常温条件下(23℃)，正常光照(16h光照/8h黑暗)培养CalS5-2突变体至抽薹阶段，然后将其置入短日照(8h光照/16h黑暗)培养5天，以考察日照条件对CalS5小孢子发育缺陷的育性恢复。结果显示突变体的育性得到了恢复(如图2所示)。亚历山大染色实验表明在正常光照条件下，突变体花药内没有花粉，但在短日照条件下有一定量的花粉形成(如图2所示)。这些结果表明CalS5在短日照条件下的育性恢复机制类似于低温处理的机制。
图6、图7为高温条件下，CalS5突变体的种子量。在高温(28℃)下，正常光照培养的CalS5弱等位突变体的种子数量明显减少，且温度升高，突变体植株中的花粉数量减少，28℃时，突变体植株中几乎没有花粉，说明在该条件下，CalS5突变体的育性严重下降。
综上所述，对于Cals5突变体来说，相对于光照周期，环境温度对育性的恢复起到更重要的作用。
本发明提及的所有文献在本申请中引用只是作为参考，就如同每一篇文献被单独引用作为参考一样。此外还应理解的是，在阅读了本发明的上述内容之后，本领域技术人员可以对本发明做出各种改动或修改，这些等价形式同样属于本申请权利要求书所限定的范围。

Claims (10)

  1. 一种培育植物不育系的方法，其特征在于，包括步骤：降低所述植物植株中与花粉发育相关的胼胝质合成酶的表达或活性，所述胼胝质合成酶为CalS5蛋白或其同源蛋白。
  2. 根据权利要求1所述的培育植物不育系的方法，其特征在于，所述“降低所述植物植株中与花粉发育相关的胼胝质合成酶的表达或活性”满足：所述植株中与花粉发育相关的胼胝质合成酶的酶活性/野生型同种类型植物植株中相同胼胝质合成酶的酶活性＝0-80％。
  3. 根据权利要求1所述的培育植物不育系的方法，其特征在于，所述CalS5的野生型氨基酸序列选自SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6。
  4. 根据权利要求1所述的培育植物不育系的方法，其特征在于，所述胼胝质合成酶在植物花序或花药的细胞、组织或器官中特异性表达。
  5. 根据权利要求1所述的培育植物不育系的方法，其特征在于，通过以下任意一种方式降低所述胼胝质合成酶的表达或活性：
    1)编码所述胼胝质合成酶的多核苷酸部分或完全缺失；
    2)修饰表达调控序列以降低或抑制编码所述胼胝质合成酶的多核苷酸的表达；
    3)修饰染色体上的序列；或
    4)上述1)-3)中的任意组合。
  6. 一种编码如权利要求1所述胼胝质合成酶的基因在培育植物不育系或制备培育植物不育系的试剂或试剂盒中的用途。
  7. 一种将植物从不育转为可育的方法，其特征在于，包括步骤：降低花粉发育速度；其中：所述植物为根据权利要求1所述的方法培育的植物不育系。
  8. 根据权利要求7所述的将植物从不育转为可育的方法，其特征在于，所述降低花粉发育速度是通过降低植株生长的环境温度和/或减少植株的光照时间实现的。
  9. 一种植物育种方法，其特征在于，包括如下步骤：
    步骤1、维持植株不育；
    步骤2、将植株由不育转为可育；
    步骤3、维持植株可育并育种；
    其中，步骤1中所述“维持植株不育”为对根据权利要求1所述的方法培育的植物不育系进行维持；所述将植株由不育转为可育的方法为权利要求7所述的将植株由不育转为可育的方法。
  10. 一种植物细胞，其特征在于，在由所述植物细胞发育成的植株中，与花粉发育相关的胼胝质合成酶的表达降低。
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