WO2015161744A1 - 植物花药特异表达启动子pTaASG048的鉴定和应用 - Google Patents

植物花药特异表达启动子pTaASG048的鉴定和应用 Download PDF

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WO2015161744A1
WO2015161744A1 PCT/CN2015/076503 CN2015076503W WO2015161744A1 WO 2015161744 A1 WO2015161744 A1 WO 2015161744A1 CN 2015076503 W CN2015076503 W CN 2015076503W WO 2015161744 A1 WO2015161744 A1 WO 2015161744A1
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promoter
gene
plant
sequence
expression
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马力耕
李健
邓兴旺
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未名兴旺系统作物设计前沿实验室（北京）有限公司
河北博农农业技术开发有限公司
兴旺投资有限公司
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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
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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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • 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)

Definitions

  • the present invention is in the field of plant biotechnology, and in particular, the present invention relates to isolated DNA capable of directing the specific transcription and/or expression of a nucleic acid operably linked downstream thereof in a plant anther. Further, the present invention relates to an expression cassette, a plant or the like comprising the DNA, and to the use of the DNA.
  • Plant gene regulation is mainly carried out at the transcriptional level and is coordinated by a variety of cis-acting elements and trans-acting factors.
  • the promoter is an important cis-acting element, which is a DNA sequence that regulates gene transcription in the upstream region of the 5' end of the structural gene, which activates RNA polymerase to accurately bind to the template DNA, ensuring accurate and efficient transcription. It plays a key role in transcriptional regulation. Promoters are classified into constitutive promoters and specific promoters based on their different characteristics of driving gene expression.
  • the constitutive promoter can initiate transcription in all cells or tissues, regardless of time and space; specific promoters can be further divided into tissue-specific promoters and inducible promoters, in which inducible promoters usually do not initiate transcription or Transcriptional activity is low, but transcriptional activity can be significantly increased by stimulation of certain specific stress signals.
  • the exogenous DNA sequence initiates expression in a plant host by ligation to a specific promoter, and selection of the promoter type determines the time and location of expression of the gene.
  • constitutive strong promoters such as the CaMV 35S promoter and the maize Ubiquitin-1 promoter.
  • these promoters are used to induce the target gene to transform rice and other crops in order to improve the quality of crops.
  • the time of expression (developmental stage specificity) or space (tissue organ specificity) of the target gene is not well controlled, the improvement effect is not obvious, or the gene expression level is too high due to the constitutive promoter. It affects the growth and development of plants.
  • the driving activity and specificity of plant pollen or anther promoter determine the success or failure of genetically engineered means to regulate pollen fertility and to create plant sterile lines and restore lines.
  • genetically engineered means to regulate pollen fertility and to create plant sterile lines and restore lines.
  • wheat has more research on the molecular mechanism of pollen or anther development due to its large genome and complex structure. Therefore, the cloning and functional analysis of the promoters of wheat pollen-specific expression lay the foundation for the full utilization of wheat heterosis resources in wheat breeding by using genetic engineering to regulate pollen fertility and creating plant male sterile lines in wheat.
  • the results of high-throughput sequencing of wheat transcriptome were first sequenced by Trinity software, and the resulting splicing sequence further removed redundancy and similarity clustering.
  • the high-throughput sequenced sequences in each sample were first aligned with the results of transcript splicing by TopHat (http://tophat.cbcb.umd.edu/) software.
  • TopHat http://tophat.cbcb.umd.edu/
  • the Cufflink software is then able to calculate the homogenous expression of the transcript contigs on the alignment, using the "fragments per kilobase of exon model per million mapped fragments (FPKM)" .
  • comp180208_c2_seq1 sequence as shown in SEQ ID NO: 1 pollen is not expressed in the anthers of the meiosis phase but is expressed in the anthers of the pollen in the mononuclear, binuclear and trinuclear phases.
  • the gene corresponding to comp180208_c2_seq1 was named TaASG048 (Anther Specific Gene 048).
  • Wheat is a heterologous hexaploid composed of three sets of genomes A, B and D.
  • the average copy number of the gene is 2.8, of which nearly half of the genes (46%) have 3-4 copies, and 12% of the genes have 1 - 2 copies, 42% of gene copies ⁇ 5.
  • the cDNA sequences of the three TaASG048 genes are shown in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively, and the homology between the three is 95-97%.
  • Specific primers for TaASG048-1, TaASG048-2 and TaASG048-3 cDNA were designed, and the three genes were used in wheat roots, stems, leaves, anthers and anthers at different developmental stages by RT-PCR. Expression specificity analysis was performed in various tissue materials such as floral organs. As shown in Fig. 2, TaASG048-1, TaASG048-2 and TaASG048-3 genes were only in the pollen mononuclear, binuclear and trinuclear anthers.
  • TaASG048-1, TaASG048-2 and TaASG048-3 genes are anther-specific expression. And genes that are specifically expressed only in anthers of late pollen development.
  • the present invention also provides three anther-specific promoters obtained by the following methods: starting from the cDNA sequences of the TaASG048-1, TaASG048-2 and TaASG048-3 genes, using CerealsDB and IWGSC (International Wheat Genome Sequencing) Consortium) published information on common wheat sequencing, and electronic cloning of sequencing information from wheat ancestors Uraltu urartu (A genome donor) and Aegilops tauschii (D genome donor) published in Nature in 2013.
  • the promoters of the TaASG048-1, TaASG048-2 and TaASG048-3 genes were obtained, and were named TaASG048-1 promoter, TaASG048-2 promoter and TaASG048-3 promoter, respectively, and the promoter may also be used in the present invention.
  • Called pTaASG048-1, pTaASG048-2 and pTaASG048-3, which are 2085 bp, 2025 bp, and 2000 bp in length, respectively, are shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively.
  • the cis element analysis of the TaASG048-1 promoter, the TaASG048-2 promoter and the TaASG048-3 promoter was performed using the PlantCARE database and the PLACE database.
  • the translation initiation site ATG is indicated by a bold underline
  • a of the translation start site ATG is defined as "+1”
  • the AGAAA motif is indicated by a hatching
  • the GTGA motif is represented by a square
  • AGGTCA The motif is represented by a downward curve.
  • AGAAA motifs There are 14 AGAAA motifs and 6 GTGA motifs.
  • the AGAAA motif, GTGA motif and AGGTCA motif are cis-regulatory elements involved in pollen/anther specific expression, TaASG048-1 promoter, TaASG048-2 promoter and multiple AGAAA motifs in the TaASG048-3 promoter, GTGA
  • the presence of the motif and the AGGTCA motif indicates that the promoter may be a promoter associated with pollen/anther specific expression.
  • one of the promoters SEQ ID NO: 7, was ligated to the reporter gene GUS, transformed into plants, and the GUS gene was not detected in the vegetative organs such as roots, stems and leaves of transgenic rice. Expression, GUS gene expression was not detected in flower organs other than anthers in the mononuclear, binuclear and trinuclear stages of pollen meiosis, and the TaASG048-3 promoter could only be activated.
  • the GUS gene is expressed in the anthers of the pollen in the mononuclear, binuclear and trinuclear stages, indicating that the promoter provided by the present invention is a promoter for the specific expression of anthers in the late pollen development.
  • the plant anther-specific expression promoter provided by the present invention comprises the nucleotide sequence shown by SEQ ID NO: 5, 6 or 7 in the Sequence Listing or comprises the nucleotides listed in SEQ ID NO: 5, 6 or a nucleotide sequence having a sequence similarity of 90% or more, or comprising 100 and more than 100 consecutive nucleotide fragments derived from the sequence of SEQ ID NO: 5, 6 or 7, and which can be driven to operate with the promoter Expression of linked nucleotide 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: 5, 6 or 7 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 wheat, 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: 5, 6 or 7 promoter sequences (or fragments thereof) set forth herein are also included in the embodiments.
  • 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.
  • the promoter may also contain other recognition sequences, These recognition sequences are typically located upstream or at the 5' end of the TATA box and are commonly referred to as upstream promoter elements to 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 SEQ ID NO: 5, 6 or 7 promoter of the invention may be used in association with its own or a core promoter from another source.
  • 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 anther, or introduced into a callus for functional verification.
  • the activity and intensity of the promoter can be determined based on the amount of mRNA or protein expression of the reporter gene it drives.
  • a reporter gene is a gene encoding a protein or enzyme that can be detected, that is, a gene whose expression product is very easy to identify.
  • the coding sequence and the gene expression regulatory sequence are fused to form a chimeric gene, or fused with other gene of interest, and expressed under the control of a control sequence, thereby using its expression product to determine the expression regulation property of the target gene.
  • Commonly used reporter genes are the ⁇ -glucuronidase gene GUS and the green fluorescent protein gene GFP.
  • the present invention detects the activity and expression characteristics of a promoter by a GUS reporter gene.
  • the test material is soaked in a buffer containing a substrate, and if the tissue cells are transferred to the GUS gene and the GUS enzyme protein is expressed, the enzyme can hydrolyze X-Gluc to form a blue product under suitable conditions.
  • the promoter of the present invention may be ligated to a nucleotide sequence other than the TaASG048-1, TaASG048-2 or TaASG048-3 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.
  • the TaASG048-1 promoter, TaASG048-2 promoter and TaASG048-3 promoter disclosed in the present invention can be used to drive expression of the following heterologous nucleotide sequence to obtain a male sterile phenotype of the transformed plant.
  • the heterologous nucleotide sequence may encode an enzyme or a modified enzyme that promotes degradation of carbohydrates, an amylase, a debranching enzyme, and a pectinase, more specifically, an amylase gene, auxin, rot B, cells.
  • the toxin gene, diphtheria toxin, DAM methylase, avidin, or may be selected from a prokaryotic regulatory system, and may also be 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 promoter sequence provided by the present invention can be isolated from any plant, including but not limited to Brassica, corn, wheat, sorghum, two genus, white mustard, castor bean, sesame, cottonseed, linseed, soybean, and nan 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, ornamental plants and pines.
  • the plants include corn, soybean, safflower, mustard, wheat, barley, rye, rice
  • the present invention also encompasses constructs comprising a TaASG048-1 promoter, a TaASG048-2 promoter or a TaASG048-3 promoter, the construct comprising a so-called vector or expression cassette.
  • Other components may also be included in the above constructs, depending primarily on the purpose and use of the vector construction, for example, may further include a selectable marker gene, a targeting or regulatory sequence, a stable sequence or a leader sequence, an intron, and the like.
  • the expression cassette will also include a work in the plant at the 3' end of the target heterologous nucleotide sequence.
  • a transcriptional and translational terminator may be the terminator of the gene provided by the invention, or it may be a terminator from an exogenous source. More specifically, the above terminator may be a nopaline synthase or an octopine synthase termination region.
  • the expression cassette may also be included for A nucleotide sequence encoding a transit peptide.
  • transit peptides are well known in the art and include, but are not limited to, the small subunit of Rubisco, the plant EPSP synthase, the maize Brittle-1 chloroplast transit peptide, and the like.
  • a plurality of DNA fragments can be manipulated to provide a DNA sequence in the proper orientation or in the correct reading frame.
  • the DNA fragments can be ligated using adaptors or linkers, or further including other procedures to provide convenient restriction sites and the like.
  • constructs provided by the present invention may further comprise a selectable marker gene for selecting transformed cells or tissues.
  • the selectable marker gene includes a gene that confers antibiotic resistance or resistance to herbicides. Suitable selectable marker genes include, but are not limited to, chloramphenicol resistance gene, hygromycin resistance gene, streptomycin resistance gene, spectinomycin resistance gene, sulfonamide resistance gene, glyphosate resistance gene , grass cockroach resistance gene.
  • the selectable marker gene may also be a gene such as a red fluorescent gene, a cyan fluorescent protein gene, a yellow fluorescent protein gene, a luciferase gene, a green fluorescent protein gene, or an anthocyanin p1.
  • the expression cassette or vector provided by the present 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. Preferably, 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 invention may be present in the nucleus, chloroplast, mitochondria and/or plastid of a plant cell.
  • the expression cassette or vector of the invention is inserted into the chromosomal DNA of the plant cell nucleus.
  • the invention also encompasses the use of the disclosed TaASG048-1 promoter and/or TaASG048-2 promoter and/or TaASG048-3 promoter, in certain embodiments of the application, the TAASG048-1 provided by the present invention may be applied.
  • Promoter and/or TaASG048-2 and/or TaASG048-3 promoters for the propagation and maintenance of male sterile lines obtained by mutations of some fertility-related genes including but not limited to Ms26, Ms45, MSCA1 and so on.
  • 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 anthers.
  • 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, Plant male sterile line and restorer The creation of the complex is of great significance.
  • 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.
  • Figure 1 is a graph showing the expression level of comp180208_c2_seq1 in anthers of pollen in meiosis (WT-0), mononuclear (WT-1), binuclear (WT-2) and trinuclear (WT-3),
  • the abscissa is the different developmental stages of pollen, and the ordinate is FPKM, the expression level of the response gene.
  • Figure 2 is an RT-PCR analysis of three homologous genes of TaASG048 in different tissues and organs of wheat and anthers at different developmental stages. 1 indicates root, 2 indicates stem, 3 indicates leaf, 4 indicates pollen is in the meiosis stage, 5 indicates pollen in the mononuclear stage, 6 indicates pollen in the dinuclear stage, and 7 indicates pollen in the trinuclear stage. Anther, 8 means that the pollen is in the flower of the mononuclear stage except for the anthers, 9 means that the pollen is in the flower of the double-nuclear stage except for the anther, and 10 means that the pollen is in the flower of the trinuclear stage except the anther. Other flower organs.
  • FIG. 3 shows the TaASG048-1 promoter sequence (A), the TaASG048-2 promoter sequence (B) and the TaASG048-3 promoter sequence (C).
  • the translation initiation site ATG is indicated by a bold underline.
  • the A of the translation start site ATG is defined as "+1", the AGAAA motif is shaded, the GTGA motif is represented by a square, and the AGGTCA motif is represented by a downward curve.
  • AGAAA, GTGA and AGGTCA represent three conserved motifs associated with pollen/anther specific expression promoters.
  • Figure 4 is a T-DNA region map of the expression vector p249.
  • LB and RB are the left and right borders of T-DNA, respectively;
  • NPTII indicates the neomycin phosphotransferase II gene;
  • P35S indicates the promoter of CaMV35S gene;
  • T35S indicates the terminator of CaMV35S gene;
  • GUS indicates ⁇ -glucuronidase Gene;
  • Tnos represents the terminator of the nopaline synthase (nos) gene;
  • HindIII, PstI, XbaI, BamHI, SacI and EcoRI represent the restriction endonuclease sites, respectively;
  • the TaASG048-3 promoter is isolated by the present invention.
  • the promoter of wheat anther-specific expression is isolated by the present invention.
  • Figure 5 is a GUS staining of tissues and organs of p249 transgenic wheat.
  • A is root; B leaf; C stem; D is flower with pollen in meiosis; E is flower with pollen in mononuclear stage; F is flower with pollen in binuclear stage; G is flower with pollen in trinuclear stage; H is an anther of pollen in the binuclear phase; I is a pollen in the binuclear phase, DAPI-stained pollen in the upper right corner; J is a pollen in the trinuclear phase, and DAPI-stained pollen in the upper right corner.
  • the methods used in the following examples are conventional methods unless otherwise specified.
  • the primers used are synthesized by Shanghai Yingjun Biotechnology Co., Ltd., and the sequencing is completed by Beijing Sanbo Yuanzhi Biotechnology Co., Ltd., during the construction of PCR kits and vectors.
  • the endonuclease was purchased from Bao Bioengineering Co., Ltd., pEASY-T1-simple ligation kit and TransStart FastPfu DNAPolymerase were purchased from Beijing Quanjin Biotechnology Co., Ltd., and T4 DNA ligase was purchased from NEB. The method was based on the method provided by the kit. get on.
  • Example 1 Whole genome expression profiling of wheat anthers at different developmental stages and acquisition of anther expression contig in late pollen development
  • the results of high-throughput sequencing of wheat transcriptome were first sequenced by Trinity software, and the resulting splicing sequence further removed redundancy and similarity clustering.
  • the high-throughput sequenced sequences in each sample were first aligned with the results of transcript splicing by TopHat (http://tophat.cbcb.umd.edu/) software.
  • TopHat http://tophat.cbcb.umd.edu/
  • the Cufflink software is then able to calculate the homogenous expression of the transcript contigs on the alignment, using the "fragments per kilobase of exon model per million mapped fragments (FPKM)" .
  • comp180208_c2_seq1 sequence as shown in SEQ ID NO: 1 pollen is not expressed in the anthers of the meiosis phase but is expressed in the anthers of the pollen in the mononuclear, binuclear and trinuclear phases.
  • the gene corresponding to comp180208_c2_seq1 was named TaASG048 (Anther Specific Gene 048).
  • Wheat is a heterologous hexaploid composed of three sets of genomes A, B and D.
  • the average copy number of the gene is 2.8, of which nearly half of the genes (46%) have 3-4 copies, and 12% of the genes have 1 - 2 copies, 42% of gene copies ⁇ 5.
  • the cDNA sequences of the three TaASG048 genes are shown in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively, and the homology between the three is 95-97%.
  • Specific primers for TaASG048-1, TaASG048-2 and TaASG048-3 cDNA were designed, and the three genes were used in wheat roots, stems, leaves, anthers and anthers at different developmental stages by RT-PCR. Expression specificity analysis was performed in various tissue materials such as floral organs. As shown in Fig. 2, TaASG048-1, TaASG048-2 and TaASG048-3 genes were only in the pollen mononuclear, binuclear and trinuclear anthers.
  • TaASG048-1, TaASG048-2 and TaASG048-3 genes are anther-specific expression. And genes that are specifically expressed only in anthers of late pollen development.
  • the RT-PCR primers for the TaASG048-1 gene are:
  • Primer 1 5'-CGTGTGGTGGCTGGCTATAGG-3' (SEQ ID NO: 8)
  • Primer 2 5'-TCGTCGATGTCCTGCAGCTG-3' (SEQ ID NO: 9)
  • the RT-PCR primer for the TaASG048-2 gene is:
  • Primer 3 5'-GTATCTATCCATCCATCTCCTCTGACA-3' (SEQ ID NO: 10)
  • Primer 4 5'-CGTCGATGTCCTGCAGCCC-3' (SEQ ID NO: 11)
  • RT-PCR primers for the TaASG048-3 gene are:
  • Primer 6 5'-AGTCGTCGATGTTCTGCAGGTC-3' (SEQ ID NO: 13)
  • TaASG048 Based on the cDNA sequences of the TaASG048-1, TaASG048-2, and TaASG048-3 genes, sequencing information of common wheat published by CerealsDB and IWGSC (International Wheat Genome Sequencing Consortium), and wheat ancestral Uraltu wheat published in Nature 2013 (The sequencing information of Triticum urartu (A genome donor) and Aegilops tauschii (D genome donor) was electronically cloned, and the promoters of TaASG048-1, TaASG048-2 and TaASG048-3 genes were obtained, respectively named TaASG048.
  • the -1 promoter, the TaASG048-2 promoter and the TaASG048-3 promoter are 2085 bp, 2025 bp and 2000 bp in length, respectively, and the sequences are shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively.
  • the cis element analysis of the TaASG048-1 promoter, the TaASG048-2 promoter and the TaASG048-3 promoter was performed using the PlantCARE database and the PLACE database.
  • the translation initiation site ATG is indicated by a bold underline
  • a of the translation start site ATG is defined as "+1”
  • the AGAAA motif is indicated by a hatching
  • the GTGA motif is represented by a square
  • AGGTCA The motif is represented by a downward curve.
  • AGAAA motifs There are 14 AGAAA motifs and 6 GTGA motifs.
  • the AGAAA motif, GTGA motif and AGGTCA motif are cis-regulatory elements involved in pollen/anther specific expression, TaASG048-1 promoter, TaASG048-2 promoter and multiple AGAAA motifs in the TaASG048-3 promoter, GTGA
  • the presence of the motif and the AGGTCA motif indicates that the promoter may be a promoter associated with pollen/anther specific expression.
  • the plant expression vector pBI121 was digested with restriction endonucleases HindIII and EcoRI, and the 35S:GUS fragment was ligated into the pCAMBIA2300 vector of CAMBIA, which was also digested with HindIII and EcoRI, using T4 DNAligase.
  • the new vector was named p230035S. :GUS.
  • TaASG048 promoter sequence Since the TaASG048 promoter sequence has a segment with a high GC content, it is obtained in two segments.
  • the amplification primer for fragment 1 is:
  • Primer 7 5'-ctgcag TCTTAGCAAGTCACATCCAAAGAGAATAC-3' (SEQ ID NO: 14)
  • Primer 8 5'-GGTACCGGTTCTTTTGGTTTCTC-3' (SEQ ID NO: 15)
  • the fragment was 1325 bp in length, and the front end of the fragment was a PstI (ctgcag) restriction site, and the end of the fragment was a KpnI (GGTACC) restriction site.
  • primer 7 and primer 8 were used for amplification. The reaction conditions were: pre-denaturation at 94 ° C for 5 minutes; denaturation at 94 ° C for 30 seconds; annealing at 60 ° C for 30 seconds; extension at 72 ° C for 1 minute and 30 seconds; Cycle; 72°C extension Stretch for 10 minutes.
  • the plasmid was named p248.
  • the amplification primer for fragment 2 is:
  • Primer 9 5'-GGTACCAGAAAAATCACCTGCGCGCTG-3' (SEQ ID NO: 16)
  • Primer 10 5'-tctaga GCCGACGCCGACGCGATCCGATCG-3' (SEQ ID NO: 17)
  • the fragment length is 681 bp
  • the front end of the fragment is KpnI (GGTACC) cleavage site
  • the end of the fragment is XbaI (tctaga) cleavage site.
  • primer 9 and primer 10 were used for amplification under the conditions of pre-denaturation at 94 ° C for 5 minutes, denaturation at 94 ° C for 30 seconds, annealing at 60 ° C for 30 seconds, extension at 72 ° C for 2 minutes, and 35 cycles. ; extend at 72 ° C for 10 minutes.
  • the plasmid was named p244.
  • the pAS was digested with restriction endonucleases PstI and KpnI, and the fragment 1 of the TaASG048-3 promoter was obtained, and p244 was digested with restriction endonucleases KpnI and XbaI to obtain fragment 2 of the TaASG048-3 promoter. Fragment 1 and fragment 2 were simultaneously ligated into p230035S:GUS vector digested with PstI and XbaI using T4 DNAligase to obtain a plant expression vector p249, and the structure of the plasmid is shown in Fig. 4.
  • the plant expression vector p249 was transferred to the Agrobacterium AGL0 strain by heat shock.
  • the primers were designed to identify the transgenic rice plants by PCR.
  • Primer 11 5'-GGGAAAAGGGAAATAAGAAAACCAAG-3' (SEQ ID NO: 18)
  • Primer 12 5'-GCCGTCGAGTTTTTTGATTTCAC-3' (SEQ ID NO: 19)
  • the reaction conditions were: pre-denaturation at 94 ° C for 5 minutes; denaturation at 94 ° C for 30 seconds; annealing at 55 ° C for 30 seconds; extension at 72 ° C for 1 minute and 30 seconds; 30 cycles; and extension at 72 ° C for 10 minutes.
  • Amplified is a TaASG048-3 promoter and a partial fragment of GUS, which is 1450 bp in length.
  • X-Gluc mother liquor 100 mg X-Gluc was dissolved in 5 ml DMF.
  • X-Gluc base solution 50 mM PBS pH 7.0, 10 mM EDTA ⁇ 2Na, 0.1% Triton X-100, 5 mM iron hydride, 0.5 mM potassium ferrous hydride.
  • X-Gluc use solution 50 ⁇ l of mother liquor + 950 ⁇ l of base solution.
  • FIG. 5A-C The results of GUS staining on various tissues and organs of p249 transgenic rice plants are shown in Figure 5.
  • the expression of GUS gene was not detected in the vegetative organs such as roots, stems and leaves of transgenic rice (Fig. 5A-C), and the pollen was in meiosis.
  • the expression of GUS gene was not detected in flower organs and pollen in the mononuclear and mononuclear stages except for anthers in the dinuclear and trinuclear stages.
  • the TaASG048-3 promoter can only initiate the GUS gene in the pollen in the dual-nuclear phase. Expression in the anther of the trinuclear stage (Fig.
  • TaASG048-3 promoter is a promoter for the specific expression of anthers in the late pollen development.
  • the expression of GUS was not detected in the anther wall of the pollen in the binuclear phase (Fig. 5H), while the expression of GUS was detected in the pollen of the binuclear and trinuclear stages (Fig. 5I-J). Therefore, TaASG048-3 was activated. The child is a promoter specifically expressed in the late stage of wheat pollen development.

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Abstract

提供了一种在植物花药中特异表达的启动子及应用。

Description

植物花药特异表达启动子pTaASG048的鉴定和应用 技术领域
本发明属于植物生物技术领域，具体而言，本发明涉及分离的DNA，其能够指导可操作地连接在其下游的核酸在植物花药中特异转录和/或表达。另外，本发明还涉及包含该DNA的表达盒和植物等，并涉及该DNA的应用。
背景技术
植物基因调控主要是在转录水平上进行的，受多种顺式作用元件和反式作用因子的相互协调。启动子是重要的顺式作用元件，它是位于结构基因5′端上游区域调控基因转录的一段DNA序列，能活化RNA聚合酶,使之与模板DNA准确地结合，确保转录精确而有效地起始，在转录调控中起关键作用。根据其驱动基因表达的不同特点，启动子分为组成型启动子和特异性启动子。组成型启动子能在所有细胞或组织中，不分时间和空间地启动转录；特异性启动子又可分为组织特异性启动子和诱导型启动子，其中诱导型启动子平时不启动转录或转录活性很低，但在某些特定的逆境信号的刺激下，转录活性能够显著地提高。
外源DNA序列通过连接到特定的启动子从而启动在植物宿主中的表达，启动子类型的选择决定基因的表达时间和部位。目前在农业生物技术领域广泛应用的主要是一些组成型的强启动子，比如CaMV 35S启动子和玉米Ubiquitin-1启动子，然而在利用这些启动子诱导目的基因转化水稻等作物以期改良作物的品质时，往往会由于目的基因表达的时间(发育阶段特异性)或空间(组织器官特异性)不能很好地控制而导致改良效果不明显，或者由于这些组成型启动子诱导基因表达量太高而对植物的生长发育造成影响，这些都是目前利用组成型强启动子结合功能基因来改良作物品质时遇到的障碍。
此外，在研究某些代谢过程或调节途径时，常常需要将同一途径上的两个以上的基因转化到同一个株系中去，采用转化其中一个基因得到转基因植株后再转化另外一个基因，或者两个基因分别转化完成后再进行杂交，都需要等待较长的时间，为了提高效率缩短多个基因转化的时间，最近有报道可以利用新的载体同时进行多个基因的转化，但是在多基因转化时如果重复使用同一个启动子，也会由于启动子序列的高同源性可能导致基因沉默。
近年来，通过基因工程调控植物花粉育性、创造植物雄性不育系及其恢复系已在一些作物上获得成功，为作物杂种优势的利用开创了新的前景。目前利用基因工程创造雄性不育的策略主要是利用花粉发育的特异启动子与外源基因嵌合，构建表达载体，转化植物来阻断花 粉发育的过程从而达到雄性不育的目的。Mariani等利用烟草花药绒毡层特异启动子TA29与RNase T1或Barnase连接构建成嵌合基因，通过农杆菌介导转入烟草和油菜，获得了稳定的雄性不育的转化株(Mariani C等，Induction of male sterility in plants by a chimaeric ribonuclease gene，Nature，1990，347：737-741)。随后，他们又用TA29驱动Barstar基因，创建了上述雄性不育系的恢复系(Mariani C等，Achimaeric ribonuclease-inhibitor gene restores fertility to male sterile plants,Nature,1992,357:384-387)。此外，其他研究小组利用花药特异启动子启动反义苯基苯乙烯酮合成酶基因、反义肌动蛋白基因、β-1、3-葡聚糖酶基因等在花药中特异表达也分别成功地获得了雄性不育植物(Meer等，Promoter analysis of the chalcone synthase gene of petunia hybrid:a 67bp promoter region directs flower-specific expression,Plant Biol.,1990,15:95-109；李艳红等，将新的任红雄性不育基因导入小麦栽培品种的研究初报，农业生物技术学报，1999，7:255-258；Curtis等，Genomic male sterility in lettuce,a base line for the production of F1hybrid,Plant Sci.,1996,113:113-119)。
植物花粉或花药启动子的驱动活性和特异性决定了通过基因工程手段调控花粉育性、创造植物不育系及恢复系的成败。目前已知的驱动活性高且特异性良好的植物花粉或花药特异启动子还相对较少，而小麦因其基因组较大且结构复杂等原因，在花粉或花药发育分子机制方面的研究更加匮乏，因此，对小麦花粉特异表达启动子的克隆和功能分析对于在小麦中利用基因工程调控花粉育性、创造植物雄性不育系，从而为小麦杂种优势资源在小麦育种中的充分利用打下基础。
发明内容
本发明的目的是提供一种植物花粉发育晚期花药特异表达的启动子序列及克隆并应用该启动子的方法。
取花粉处于减数分裂期、单核期、双核期和三核期的小麦花药，用Trizol(Invitrogen)提取总RNA，并进行DNaseI(Promega)处理，进而纯化mRNA(Ambion)。将纯化的mRNA进行反转录(Invitrogen)、超声打断(Fisher)、制备文库(illumina)并扩增(illumina)，最后在illumina机器上进行测序反应。
小麦转录组高通量测序的结果首先通过Trinity软件进行序列拼接，得到的拼接序列进一步去除冗余以及相似性聚类。对于拼接得到的转录本contig的表达变化分析，各样品中高通量测序的序列首先通过TopHat(http://tophat.cbcb.umd.edu/)软件与转录本拼接的结果进行比对。 而后Cufflink软件能够计算比对上的转录本contigs的均一化表达量，用“外显子每百万比对片段的千碱基数(fragments per kilobase of exon model per million mapped fragments，FPKM)”表示。
通过对不同发育时期小麦花药的全基因组表达谱分析，找到花粉处于减数分离期的花药中不表达而在花粉处于单核、双核和三核期的花药中表达的转录本contig 7187个。如图1所示，comp180208_c2_seq1(序列如SEQ ID NO:1所示)花粉处于减数分离期的花药中不表达而在花粉处于单核、双核和三核期的花药中表达。将comp180208_c2_seq1所对应的基因命名为TaASG048(Anther Specific Gene 048)。
小麦是由A、B、D三套基因组组成的异源六倍体，基因的平均拷贝数为2.8个，其中接近一半的基因(46％)有3-4个拷贝，12％的基因有1-2个拷贝，42％的基因拷贝数≥5个。从comp180208_c2_seq1的序列(如SEQ ID NO:1所示)出发，利用CerealsDB和IWGSC(International Wheat Genome Sequencing Consortium)公布的普通小麦的测序信息，以及2013年Nature上发表的小麦祖先乌拉尔图小麦(Triticum urartu，A基因组供体)和粗山羊草(Aegilops tauschii，D基因组供体)的测序信息进行电子克隆，获得了3个TaASG048基因，分别命名为TaASG048-1，TaASG048-2和TaASG048-3，其中comp180208_c2_seq1对应于TaASG048-1。3个TaASG048基因的cDNA序列分别如SEQ ID NO:2，SEQ ID NO:3和SEQ ID NO:4所示，三者之间的同源性为95-97％。分别设计针对TaASG048-1，TaASG048-2和TaASG048-3cDNA的特异性引物，利用RT-PCR方法，对这三个基因在在小麦根、茎、叶、不同发育时期的花药及除花药以外的其他花器官等多种组织材料中进行表达特异性分析，结果如图2所示，TaASG048-1、TaASG048-2和TaASG048-3基因均只在花粉处于单核期、双核期和三核期的花药中特异性表达，在同时期的其他花器官和根、茎、叶及减数分裂期的穗子等组织器官中都不表达，说明TaASG048-1、TaASG048-2和TaASG048-3基因是花药特异表达、且只在花粉发育晚期的花药中特异表达的基因。
本发明还提供了三个花药特异表达的启动子，所述启动子通过以下方法获得：从TaASG048-1、TaASG048-2和TaASG048-3基因的cDNA序列出发，利用CerealsDB和IWGSC(International Wheat Genome Sequencing Consortium)公布的普通小麦的测序信息，以及2013年Nature上发表的小麦祖先乌拉尔图小麦(Triticum urartu，A基因组供体)和粗山羊草(Aegilops tauschii，D基因组供体)的测序信息进行电子克隆，获得了TaASG048-1、TaASG048-2和TaASG048-3基因的启动子，分别命名为TaASG048-1启动子、TaASG048-2启动子和TaASG048-3启动子，在本发明中所述启动子也可分别称为pTaASG048-1、 pTaASG048-2和pTaASG048-3，其长度分别为2085bp、2025bp和2000bp，序列分别如SEQ ID NO:5、SEQ ID NO:6和SEQ ID NO:7所示。
利用PlantCARE数据库和PLACE数据库，对TaASG048-1启动子、TaASG048-2启动子和TaASG048-3启动子进行顺式元件分析。如图3所示，翻译起始位点ATG用粗体下划线表示，以翻译起始位点ATG的A定义为“+1”，AGAAA基序用阴影表示，GTGA基序用方框表示，AGGTCA基序用下划曲线表示。TaASG048-1启动子中有15个AGAAA基序和7个GTGA基序，TaASG048-2启动子中有1个AGAAA基序、5个GTGA基序和1个AGGTCA基序，TaASG048-3启动子中有14个AGAAA基序和6个GTGA基序。AGAAA基序、GTGA基序和AGGTCA基序是与花粉/花药特异表达相关的顺式调控元件，TaASG048-1启动子、TaASG048-2启动子和TaASG048-3启动子中多个AGAAA基序、GTGA基序和AGGTCA基序的存在表明该启动子可能是与花粉/花药特异表达有关的启动子。
为了进一步验证这两个启动子的功能，将其中一个启动子SEQ ID NO:7与报告基因GUS相连，转化植物，在转基因水稻的根、茎和叶等营养器官中都检测不到GUS基因的表达，在花粉处于减数分裂期的穗子和花粉处于单核期、双核期和三核期的除花药以外的其它花器官中也检测不到GUS基因的表达，TaASG048-3启动子只能启动GUS基因在花粉处于单核期、双核期和三核期的花药中表达，说明本发明所提供的启动子是一个花粉发育晚期花药特异表达的启动子。
本发明所提供的植物花药特异表达启动子，含有序列表中SEQ ID NO:5、6或7所示的核苷酸序列，或包含与SEQ ID NO:5、6或7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:5、6或7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:5、6或7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小麦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，根据它们与本发明所列的SEQ ID NO:5、6或7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，本发明所述的SEQ ID NO:5、6或7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，例如阶段性花药，或引入愈伤组织，以进行功能验证。
启动子的活性和强度可以根据其驱动的报告基因的mRNA或蛋白质的表达量来测定。报告基因(reporter gene)是一种编码可被检测的蛋白质或酶的基因，也就是说，是一个其表达产物非常容易被鉴定的基因。把它的编码序列和基因表达调节序列相融合形成嵌合基因，或与其它目的基因相融合，在调控序列控制下进行表达，从而利用它的表达产物来确定目的基因的表达调控特性。常用的报告基因有β-葡萄糖苷酸酶基因GUS，和绿色荧光蛋白基因GFP。
本发明通过GUS报告基因来检测启动子的活性和表达特性。根据GUS基因检测所用的底物不同，有三种检测方法：组织化学法、分光光度法和荧光法(灵敏度为分光光度检测法最高)，其中最为常用的是组织化学法。组织化学法检测以5-溴-4-氯-3-吲哚-β-葡萄糖苷酸 (X-Gluc)作为反应底物。将被检材料用含有底物的缓冲液浸泡，若组织细胞转入了GUS基因，并表达出了GUS酶蛋白，在适宜的条件下，该酶就可将X-Gluc水解生成蓝色产物，这是由其初始产物经氧化二聚作用形成的靛蓝染料，它使各组织细胞中有GUS表达活性的部位或位点呈现蓝色，用肉眼或在显微镜下可看到，且在一定程度下根据染色深浅可反映出GUS活性的强弱。因此利用该方法可观察到外源基因在特定器官、组织，甚至单个细胞内的表达情况。
此外，本发明的启动子可与并非TaASG048-1、TaASG048-2或TaASG048-3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TaASG048-1启动子、TaASG048-2启动子和TaASG048-3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、生长素(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。
本发明所提供的启动子序列可分离自任何植物，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、观赏植物和松类等。优选地，植物包括玉米、大豆、红花、芥菜、小麦、大麦、黑麦、稻、棉花和高粱。
本发明还包括含有TaASG048-1启动子、TaASG048-2启动子或TaASG048-3启动子的构建体，所述构建体包括通常所说的载体或表达盒。上述构建体中还可包括其它组分，这主要取决于载体构建的目的和用途，例如可进一步包括选择标记基因、靶向或调控序列、稳定序列或引导序列、内含子等。表达盒还将在目标异源核苷酸序列的3’端包括在植物中具有功 能的转录和翻译终止子。终止子可以是本发明所提供基因的终止子，也可以是来自外源的终止子。更具体地，上述终止子可以是胭脂氨酸合酶或章鱼碱合酶终止区域。
在希望将异源核苷酸序列的表达产物引向特定细胞器，例如质体、造粉体，或者引向内质网，或在细胞表面或细胞外分泌的情况下，表达盒还可包含用于编码转运肽的核苷酸序列。此类转运肽是本领域所公知的，其包括但不限于Rubisco的小亚基、植物EPSP合酶、玉米Brittle-1叶绿体转运肽等。
在制备表达盒的过程中，可对多种DNA片段加以操作，以提供处于合适方向，或是处于正确读码框中的DNA序列。为达到此目的，可使用衔接子或接头，将DNA片段连起来，或者进一步包括其它操作，以提供方便的限制性酶切位点等。
进一步地，本发明所提供的构建体中还可包括选择标记基因，用于选择经转化的细胞或组织。所述选择标记基因包括赋予抗生素抗性或对除草剂抗性的基因。合适的选择标记基因包括但不限于:氯霉素抗性基因，潮霉素抗性基因，链霉素抗性基因，奇霉素抗性基因，磺胺类抗性基因，草甘磷抗性基因，草丁嶙抗性基因。所述选择标记基因还可以是红色荧光基因、青色荧光蛋白基因、黄色荧光蛋白基因、荧光素酶基因、绿色荧光蛋白基因、花青甙p1等基因。
本发明所提供的表达盒或载体可被插入质粒、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。
本发明还包括所公开的TaASG048-1启动子和/或TaASG048-2启动子和/或TaASG048-3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TaASG048-1启动子和/或TaASG048-2和/或TaASG048-3启动子来实现一些育性相关基因突变所获得的雄性不育系的繁殖和保持，所述育性相关基因包括但不限于Ms26、Ms45、MSCA1等。
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，还可以用于植物花药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。
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。
下面通过具体实施方式，结合附图对本发明做进一步详细描述，但不以任何方式限制本发明的范围。
附图说明
图1是comp180208_c2_seq1在花粉处于减数分裂期(WT-0)、单核期(WT-1)、双核期(WT-2)和三核期(WT-3)的花药中的表达水平分析，横坐标是花粉不同发育时期，纵坐标是FPKM，反应基因的表达水平。
图2是TaASG048的3个同源基因在小麦不同组织器官和不同发育时期的花药中的RT-PCR分析。1表示根，2表示茎，3表示叶片，4表示花粉处于减数分裂期的穗子，5表示花粉处于单核期的花药，6表示花粉处于双核期的花药，7表示花粉处于三核期的花药，8表示花粉处于单核期的花中除花药以外其它的花器官，9表示花粉处于双核期的花中除花药以外其它的花器官，10表示花粉处于三核期的花中除花药以外其它的花器官。
图3表示TaASG048-1启动子序列(A)、TaASG048-2启动子序列(B)和TaASG048-3启动子序列(C)。翻译起始位点ATG用粗体下划线表示，翻译起始位点ATG的A定义为“+1”，AGAAA基序用阴影表示，GTGA基序用方框表示，AGGTCA基序用下划曲线表示。 AGAAA、GTGA和AGGTCA表示三个与花粉/花药特异表达启动子相关的保守基序。
图4是表达载体p249的T-DNA区图谱。LB和RB分别为T-DNA的左边界和右边界；NPTII表示新霉素磷酸转移酶II基因；P35S表示CaMV35S基因的启动子；T35S表示CaMV35S基因的终止子；GUS表示β-葡萄糖苷酸酶基因；Tnos表示胭脂碱合成酶(nos)基因的终止子；HindIII、PstI、XbaI、BamHI、SacI和EcoRI分别表示限制性内切酶的酶切位点；TaASG048-3启动子就是本发明所分离的小麦花药特异表达的启动子。
图5是p249转基因小麦的组织器官GUS染色。A为根；B叶；C茎；D为花粉处于减数分裂期的花；E为花粉处于单核期的花；F为花粉处于双核期的花；G为花粉处于三核期的花；H为花粉处于双核期的花药；I为双核期的花粉，右上角是DAPI染色的花粉；J为三核期的花粉，右上角是DAPI染色的花粉。
具体实施方式
下述实施例中所用方法如无特别说明均为常规方法，所用引物均由上海英骏生物技术公司合成，测序由北京三博远志生物技术有限责任公司完成，PCR试剂盒、载体构建过程中的核酸内切酶购自宝生物工程有限公司，pEASY-T1-simple连接试剂盒和TransStart FastPfu DNAPolymerase购自北京全式金生物技术公司，T4DNA连接酶购自NEB公司，方法均参照试剂盒提供的方法进行。
实施例1.不同发育时期小麦花药的全基因组表达谱分析和花粉发育后期花药表达contig的获得
取花粉处于减数分裂期、单核期、双核期和三核期的小麦花药，用Trizol(Invitrogen)提取总RNA，并进行DNaseI(Promega)处理，进而纯化mRNA(Ambion)。将纯化的mRNA进行反转录(Invitrogen)、超声打断(Fisher)、制备文库(illumina)并扩增(illumina)，最后在illumina机器上进行测序反应。
小麦转录组高通量测序的结果首先通过Trinity软件进行序列拼接，得到的拼接序列进一步去除冗余以及相似性聚类。对于拼接得到的转录本contig的表达变化分析，各样品中高通量测序的序列首先通过TopHat(http://tophat.cbcb.umd.edu/)软件与转录本拼接的结果进行比对。而后Cufflink软件能够计算比对上的转录本contigs的均一化表达量，用“外显子每百万比对片段的千碱基数(fragments per kilobase of exon model per million mapped fragments，FPKM)”表示。
通过对不同发育时期小麦花药的全基因组表达谱分析，找到花粉处于减数分离期的花药中不表达而在花粉处于单核、双核和三核期的花药中表达的转录本contig 7187个。如图1所示，comp180208_c2_seq1(序列如SEQ ID NO:1所示)花粉处于减数分离期的花药中不表达而在花粉处于单核、双核和三核期的花药中表达。将comp180208_c2_seq1所对应的基因命名为TaASG048(Anther Specific Gene 048)。
实施例2.RT-PCR验证TaASG048基因的组织表达特异性
小麦是由A、B、D三套基因组组成的异源六倍体，基因的平均拷贝数为2.8个，其中接近一半的基因(46％)有3-4个拷贝，12％的基因有1-2个拷贝，42％的基因拷贝数≥5个。从comp180208_c2_seq1的序列(如SEQ ID NO:1所示)出发，利用CerealsDB和IWGSC(International Wheat Genome Sequencing Consortium)公布的普通小麦的测序信息，以及2013年Nature上发表的小麦祖先乌拉尔图小麦(Triticum urartu，A基因组供体)和粗山羊草(Aegilops tauschii，D基因组供体)的测序信息进行电子克隆，获得了3个TaASG048基因，分别命名为TaASG048-1，TaASG048-2和TaASG048-3，其中comp180208_c2_seq1对应于TaASG048-1。3个TaASG048基因的cDNA序列分别如SEQ ID NO:2，SEQ ID NO:3和SEQ IDNO:4所示，三者之间的同源性为95-97％。分别设计针对TaASG048-1，TaASG048-2和TaASG048-3cDNA的特异性引物，利用RT-PCR方法，对这三个基因在在小麦根、茎、叶、不同发育时期的花药及除花药以外的其他花器官等多种组织材料中进行表达特异性分析，结果如图2所示，TaASG048-1、TaASG048-2和TaASG048-3基因均只在花粉处于单核期、双核期和三核期的花药中特异性表达，在同时期的其他花器官和根、茎、叶及减数分裂期的穗子等组织器官中都不表达，说明TaASG048-1、TaASG048-2和TaASG048-3基因是花药特异表达、且只在花粉发育晚期的花药中特异表达的基因。
TaASG048-1基因的RT-PCR引物为：
引物1：5'-CGTGTGGTGGCTGGCTATAGG-3'(SEQ ID NO:8)
引物2：5'-TCGTCGATGTCCTGCAGCTG-3'(SEQ ID NO:9)
TaASG048-2基因的RT-PCR引物为：
引物3：5'-GTATCTATCCATCCATCTCCTCTGACA-3'(SEQ ID NO:10)
引物4：5'-CGTCGATGTCCTGCAGCCC-3'(SEQ ID NO:11)
TaASG048-3基因的RT-PCR引物为：
引物5：5'-GGATCCCATATATACATGCGCTCT-3'(SEQ ID NO:12)
引物6：5'-AGTCGTCGATGTTCTGCAGGTC-3'(SEQ ID NO:13)
实施例3.TaASG048-1、TaASG048-2和TaASG048-3基因启动子序列的获得和顺式元件分析
从TaASG048-1、TaASG048-2和TaASG048-3基因的cDNA序列出发，利用CerealsDB和IWGSC(International Wheat Genome Sequencing Consortium)公布的普通小麦的测序信息，以及2013年Nature上发表的小麦祖先乌拉尔图小麦(Triticum urartu，A基因组供体)和粗山羊草(Aegilops tauschii，D基因组供体)的测序信息进行电子克隆，获得了TaASG048-1、TaASG048-2和TaASG048-3基因的启动子，分别命名为TaASG048-1启动子、TaASG048-2启动子和TaASG048-3启动子，其长度分别为2085bp、2025bp和2000bp，序列分别如SEQ ID NO:5、SEQ ID NO:6和SEQ ID NO:7所示。
利用PlantCARE数据库和PLACE数据库，对TaASG048-1启动子、TaASG048-2启动子和TaASG048-3启动子进行顺式元件分析。如图3所示，翻译起始位点ATG用粗体下划线表示，以翻译起始位点ATG的A定义为“+1”，AGAAA基序用阴影表示，GTGA基序用方框表示，AGGTCA基序用下划曲线表示。TaASG048-1启动子中有15个AGAAA基序和7个GTGA基序，TaASG048-2启动子中有1个AGAAA基序、5个GTGA基序和1个AGGTCA基序，TaASG048-3启动子中有14个AGAAA基序和6个GTGA基序。AGAAA基序、GTGA基序和AGGTCA基序是与花粉/花药特异表达相关的顺式调控元件，TaASG048-1启动子、TaASG048-2启动子和TaASG048-3启动子中多个AGAAA基序、GTGA基序和AGGTCA基序的存在表明该启动子可能是与花粉/花药特异表达有关的启动子。
实施例4.TaASG048-3启动子的克隆和植物表达载体的构建
将植物表达载体pBI121用限制性内切酶HindIII和EcoRI双酶切，得到的35S:GUS片段用T4DNAligase连入同样用HindIII和EcoRI双酶切的CAMBIA公司的pCAMBIA2300载体，新的载体被命名为p230035S:GUS。
由于TaASG048启动子序列中有GC含量较高的区段，故分两段获得。
片段1的扩增引物为：
引物7：5’-ctgcag TCTTAGCAAGTCACATCCAAAGAGAATAC-3’(SEQ ID NO:14)
引物8：5’-GGTACCGGTTCTTTTGGTTTCTC-3’(SEQ ID NO:15)
片段长度为1325bp，片段前端为PstI(ctgcag)酶切位点，片段末端为KpnI(GGTACC)酶切位点。以小麦的基因组DNA为模板，用引物7和引物8进行扩增，反应条件是：94℃预变性5分钟；94℃变性30秒；60℃退火30秒；72℃延伸1分30秒；35个循环；72℃延 伸10分钟。反应结束后，PCR产物经1％琼脂糖凝胶电泳检测回收，产物连入pEASY-T1-Simple载体中，筛选阳性克隆并进行测序验证，该质粒称为p248。
片段2的扩增引物为：
引物9：5’-GGTACCAGAAAAATCACCTGCGCGCTG-3’(SEQ ID NO:16)
引物10：5’-tctaga GCCGACGCCGACGCGATCCGATCG-3’(SEQ ID NO:17)
片段长度为681bp，片段前端为KpnI(GGTACC)酶切位点,片段末端为XbaI(tctaga)酶切位点。以小麦的基因组DNA为模板，用引物9和引物10进行扩增，反应条件是：94℃预变性5分钟；94℃变性30秒；60℃退火30秒；72℃延伸2分；35个循环；72℃延伸10分钟。反应结束后，PCR产物经1％琼脂糖凝胶电泳检测回收，产物连入pEASY-T1-Simple载体中，筛选阳性克隆并进行测序验证，该质粒称为p244。
用限制性内切酶PstI和KpnI双酶切p248，得到的TaASG048-3启动子的片段1，用限制性内切酶KpnI和XbaI双酶切p244，得到的TaASG048-3启动子的片段2，用T4DNAligase将片段1和片段2同时连入用PstI和XbaI双酶切的p230035S:GUS载体，得到植物表达载体p249，该质粒的结构如图4所示。
实施例5.农杆菌介导的水稻遗传转化及转基因植株的分子鉴定
利用热激法将植物表达载体p249转入农杆菌AGL0菌株。
用农杆菌侵染水稻胚性愈伤，暗中共培养2-3天，然后经过两步抗性筛选、预分化、分化和生根培养等步骤，最终获得具有卡那霉素抗性的、转p249水稻T0代植株。
设计引物对转基因水稻植株进行PCR鉴定。
引物11：5’-GGGAAAAGGGAAATAAGAAAACCAAG-3’(SEQ ID NO:18)
引物12：5’-GCCGTCGAGTTTTTTGATTTCAC-3’(SEQ ID NO:19)
反应条件为：94℃预变性5分钟；94℃变性30秒；55℃退火30秒；72℃延伸1分30秒；30个循环；72℃延伸10分钟。扩增的是TaASG048-3启动子和GUS的部分片段，长度为1450bp。鉴定结果表明，利用农杆菌介导的水稻转化获得的抗性再生植株是转p249基因的阳性植株。
实施例6.转基因水稻植株不同组织器官GUS基因表达的组织化学检测
X-Gluc母液：100mg X-Gluc溶于5ml DMF。
X-Gluc基液：50mM PBS pH7.0，10mM EDTA·2Na，0.1％Triton X-100，5mM铁氢化钾，0.5mM亚铁氢化钾。
X-Gluc使用液：50μl母液+950μl基液。
选择合适大小的带有GUS报告基因的转基因幼苗或特定组织浸入GUS染液中，37℃染 色过夜，吸去反应液，乙醇梯度脱色，显微镜观察照相。
对p249转基因水稻植株各组织器官的GUS染色结果如图5，在转基因水稻的根、茎和叶等营养器官中都检测不到GUS基因的表达(图5A-C)，在花粉处于减数分裂期、单核期的花及花粉处于双核期和三核期的除花药以外的其它花器官中也检测不到GUS基因的表达，TaASG048-3启动子只能启动GUS基因在花粉处于双核期和三核期的花药中表达(图5D-G)，说明TaASG048-3启动子是一个花粉发育晚期花药特异表达的启动子。进一步，花粉处于双核期的花药壁中检测不到GUS的表达(图5H)，而双核期和三核期的花粉中能够检测到GUS的表达(图5I-J)，因此，TaASG048-3启动子是一个小麦花粉发育晚期特异表达的启动子。

Claims (10)

  1. 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选自下列组的序列之一：
    (a)具有SEQ ID NO：5、6或7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)包含SEQ ID NO：5、6或7中至少100个连续核苷酸的DNA序列；和
    (d)与(a)-(c)之任一所述序列互补的DNA序列。
  2. 一种表达盒，具有在花药中特异表达的特性，其特征在于所述表达盒包含权利要求1所述的启动子序列。
  3. 一种表达载体，其特征在于所述表达载体包含权利要求2所述的表达盒。
  4. 一种工程菌，其特征在于所述工程菌含有权利要求3所述的表达载体。
  5. 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选自下列组的序列之一：
    (a)具有SEQ ID NO：5、6或7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)包含SEQ ID NO：5、6或7中至少100个连续核苷酸的DNA序列；和
    (d)与(a)-(c)之任一所述序列互补的DNA序列。
  6. 权利要求5所述的方法，其中所述的植物为单子叶植物，优选为禾本科植物，更优选的为水稻或小麦。
  7. 权利要求5所述的方法，其中所述的目的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。
  8. 权利要求5所述的方法，其中所述的目的核苷酸序列可以是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，更具体的如玉米a淀粉酶基因、生长素,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。
  9. 权利要求1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。
  10. 权利要求9所述的应用，其特征在于，所述的植物为单子叶植物，所述单子叶植物优选为禾本科植物，更具体的，所述禾本科植物优选为水稻或小麦。
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