WO2015154689A1 - Identification et utilisations du promoteur d'expression spécifique d'anthère de plante ptaasg027 - Google Patents

Identification et utilisations du promoteur d'expression spécifique d'anthère de plante ptaasg027 Download PDF

Info

Publication number
WO2015154689A1
WO2015154689A1 PCT/CN2015/076147 CN2015076147W WO2015154689A1 WO 2015154689 A1 WO2015154689 A1 WO 2015154689A1 CN 2015076147 W CN2015076147 W CN 2015076147W WO 2015154689 A1 WO2015154689 A1 WO 2015154689A1
Authority
WO
WIPO (PCT)
Prior art keywords
gene
promoter
plant
sequence
expression
Prior art date
Application number
PCT/CN2015/076147
Other languages
English (en)
Chinese (zh)
Inventor
马力耕
李健
邓兴旺
Original Assignee
未名兴旺系统作物设计前沿实验室（北京）有限公司
兴旺投资有限公司
河北博农农业技术开发有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 未名兴旺系统作物设计前沿实验室（北京）有限公司, 兴旺投资有限公司, 河北博农农业技术开发有限公司 filed Critical 未名兴旺系统作物设计前沿实验室（北京）有限公司
Publication of WO2015154689A1 publication Critical patent/WO2015154689A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C12N15/8289—Male sterility
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/823—Reproductive tissue-specific promoters
    • C12N15/8231—Male-specific, e.g. anther, tapetum, pollen
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/827—Flower development or morphology, e.g. flowering promoting factor [FPF]

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 wheat anthers in the meiotic, mononuclear, binucleic and trinuclear stages are taken, and the total RNA is extracted with Trizol (Invitrogen) and treated with DNaseI (Promega) to further purify the mRNA (Ambion).
  • the purified mRNA was subjected to reverse transcription (Invitrogen), ultrasonic disruption (Fisher), illumina preparation and amplification (illumina), and finally sequencing reaction was performed on an illumina machine.
  • 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)" .
  • comp188209_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 pollen in the mononuclear, binuclear and trinuclear phases.
  • the gene corresponding to comp188209_c2_seq1 was named TaASG027 (Anther Specific Gene 027).
  • the TaASG027 gene is specifically expressed only in the anthers of the pollen in the mononuclear, binuclear and trinuclear phases, and other organs and organs, roots, stems, leaves and meiotic stages of the organs in the same period. None of them were expressed, indicating that the TaASG027 gene is a gene specifically expressed in anthers and specifically expressed only in anthers of late pollen development.
  • the present invention also provides an anther-specific expression promoter obtained by the following method: starting from the CDS sequence of the TaASG027 gene, using the sequencing information of common wheat published by CerealsDB and IWGSC (International Wheat Genome Sequencing Consortium), and The sequencing information of the wheat ancestors Uraltu urartu (A genome donor) and Aegilops tauschii (D genome donor) published in Nature in 2013 was electronically cloned, and the promoter of TaASG027 gene was obtained, named The TaASG027 promoter, which may also be referred to as pTaASG027 in the present invention, is 1952 bp in length, and the nucleotide sequence of the promoter is shown in SEQ ID NO: 3.
  • the cis element analysis of the TaASG027 promoter was performed using the PlantCARE database and the PLACE database. As shown in Fig. 3, 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, and the GTGA motif is represented by a square, AGGTCA The motif is represented by a downward curve.
  • the TaASG027 gene promoter has three AGAAA motifs, seven GTGA motifs, and one AGGTCA motif.
  • the AGAAA motif, GTGA motif and AGGTCA motif are cis-regulatory elements involved in pollen/anther specific expression, and the presence of multiple AGAAA motifs, GTGA motifs and AGGTCA motifs in the promoter of the TaASG027 gene indicates the promoter It may be a promoter associated with pollen/anther specific expression.
  • SEQ ID NO: 3 was ligated to the reporter gene GUS, and the plants were transformed.
  • the expression of the GUS gene was not detected in the vegetative organs such as roots, stems and leaves of the transgenic rice, and the pollen was reduced.
  • the expression of GUS gene was also not detected in the flower organs of the mononuclear, dinuclear and trinuclear stages except for anthers in the mononuclear, dinuclear and trinuclear stages.
  • the TaASG027 promoter can only initiate the GUS gene in the pollen mononuclear stage. , dual-nuclear and trinuclear flowers
  • the expression in the drug indicates that the promoter provided by the present invention is a promoter specifically expressed in the late anther of pollen development.
  • the plant anther-specific expression promoter provided by the invention comprises the nucleotide sequence shown by SEQ ID NO: 3 in the sequence listing or comprises 90% or more similarity to the nucleotide sequence set forth in SEQ ID NO: 3. a nucleotide sequence, or comprising 100 and more than 100 contiguous nucleotide fragments derived from the sequence of SEQ ID NO: 3, and which can drive expression of a nucleotide sequence operably linked to the promoter in a plant anther .
  • 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: 3 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: 3 promoter sequence (or a fragment 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. Promoters may also contain additional recognition sequences, which are typically located upstream or 5' of the TATA box, commonly referred to as upstream promoter elements, which function to regulate transcription efficiency. It will be appreciated by those skilled in the art that while nucleotide sequences for the promoter regions disclosed herein have been identified, other regulatory elements for isolating and identifying upstream regions of the TATA box of a particular promoter region identified in the present invention are also Within the scope of the invention.
  • the promoter regions disclosed herein are generally further defined as comprising upstream regulatory elements, such as those elements, enhancers, and the like, for regulating tissue expression and temporal expression functions of the coding sequences.
  • upstream regulatory elements such as those elements, enhancers, and the like
  • promoter elements that enable expression in a target tissue can be identified and isolated for use with other core promoters to verify the preferential expression of male tissues.
  • the core promoter refers to the minimal sequence required for initiation of transcription, such as the sequence known as the TATA box, which is commonly found in the promoters of genes encoding proteins.
  • the SEQ ID NO: 3 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 histochemical method detected 5-bromo-4-chloro-3-indolyl- ⁇ -glucuronide (X-Gluc) as a reaction substrate.
  • 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 can be ligated to a nucleotide sequence other than the TaASG027 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 TaASG027 promoter disclosed herein can be used to drive expression of the following heterologous nucleotide sequences such that the transformed plants acquire a male sterile phenotype.
  • 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.
  • a 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.
  • Gene, regulatory gene, structure An antisense gene of a gene, an antisense gene that regulates a gene, or a small RNA capable of interfering with expression of an endogenous gene.
  • 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 the TaASG027 promoter, which include so-called vectors or expression cassettes.
  • 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 transcriptional and translational terminator that is functional in the plant at the 3' end of the heterologous nucleotide sequence of interest.
  • the 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 present invention also encompasses the use of the disclosed TaASG027 promoter, and in certain embodiments of the application, the TaASG027 promoter provided by the present invention can be used to achieve propagation of male sterile lines obtained by mutations of some fertility-related genes.
  • the fertility-related genes include, but are not limited to, Ms26, Ms45, MSCA1, and the like.
  • 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, The creation of plant male sterile lines and restorer lines 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 comp188209_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, vertical
  • the coordinates are FPKM, the expression level of the reactive gene.
  • Figure 2 is an RT-PCR analysis of the TaASG027 gene in anthers of different tissues and organs of wheat 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
  • 7 indicates pollen in the trinuclear stage.
  • Anther 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
  • 10 means that the pollen is in the flower of the trinuclear stage except the anther.
  • 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
  • 7 indicates poll
  • Figure 3 shows the TaASG027 promoter sequence.
  • 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 p167.
  • 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, XbaI, SacI and EcoRI represent the restriction endonuclease sites, respectively;
  • the TaASG027 promoter is the specific expression of the wheat anther isolated by the present invention. Promoter.
  • Figure 5 is a GUS staining of tissues and organs of p167 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 all synthesized by Shanghai Yingjun Biotechnology Co., Ltd., and the sequencing is completed by Beijing Sanbo Yuanzhi Biotechnology Co., Ltd., PCR kit, pMD20-T vector,
  • the endonuclease in the construction process was purchased from Bao Bioengineering Co., Ltd.
  • TransStart FastPfu DNA Polymerase was purchased from Beijing Quanjin Biotechnology Co., Ltd.
  • T4 DNA ligase was purchased from NEB. The methods were all carried out according to the method provided by the kit.
  • Example 1 Whole genome expression profiling of wheat anthers at different developmental stages and acquisition of anther expression contig in late pollen development
  • the wheat anthers in the meiotic, mononuclear, binucleic and trinuclear stages are taken, and the total RNA is extracted with Trizol (Invitrogen) and treated with DNaseI (Promega) to further purify the mRNA (Ambion). Purified mRNA is reverse transcribed (Invitrogen), ultrasonically disrupted (Fisher), prepared (illumina) and amplified (illumina), most The sequencing reaction was then performed on an illumina machine.
  • 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)" .
  • comp188209_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 pollen in the mononuclear, binuclear and trinuclear phases.
  • the gene corresponding to comp188209_c2_seq1 was named TaASG027 (Anther Specific Gene 027).
  • the TaASG027 gene is specifically expressed only in the anthers of the pollen in the mononuclear, binuclear, and trinuclear stages, and other tissues of the flower organs and roots, stems, leaves, and meiotic stages of the ear. Not expressed in the organs, indicating that the TaASG027 gene is a gene specifically expressed in anthers and specifically expressed only in anthers of late pollen development.
  • the RT-PCR primers for the TaASG027 gene are:
  • Primer 1 5'-CGACGGCTACGACGACAGG-3' (SEQ ID NO: 4)
  • Primer 2 5'-TCGTCGTCATCGTTATCATCATTC-3' (SEQ ID NO: 5)
  • TaASG027 gene Based on the CDS sequence of the TaASG027 gene, sequencing information of common wheat published by CerealsDB and IWGSC (international wheat genome sequencing consortium), and wheat ancestral Uraltu wheat (Triticum urartu, A genome donor) published in Nature in 2013 and coarse
  • the sequencing information of Aegilops tauschii was electronically cloned, and the promoter of TaASG027 gene was obtained, which was named TaASG027 promoter, which was 1952 bp in length, and the promoter and partial coding region sequence was SEQ ID NO: 3. Shown.
  • the cis element analysis of the TaASG027 promoter was performed using the PlantCARE database and the PLACE database. As shown in Fig. 3, 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, and the GTGA motif is represented by a square, AGGTCA The motif is represented by a downward curve.
  • the TaASG027 gene promoter has three AGAAA motifs, seven GTGA motifs, and one AGGTCA motif.
  • the AGAAA motif, GTGA motif and AGGTCA motif are cis-regulatory elements involved in pollen/anther specific expression, and the presence of multiple AGAAA motifs, GTGA motifs and AGGTCA motifs in the promoter of the TaASG027 gene indicates the promoter It 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 as P2300 35S: GUS.
  • Primer 3 5'-aagctt ATATCCTTGCAAAACTGCCCC-3' (SEQ ID NO: 6)
  • Primer 4 5'-tctaga GCAGGAAGAGTAGTACCGCCAG-3' (SEQ ID NO: 7)
  • sequence aagctt in primer 3 is the restriction site of HindIII
  • sequence tctaga in primer 4 is the restriction site of XbaI.
  • primer 3 and primer 4 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 2 minutes and 30 seconds; One cycle; extending at 72 ° C for 10 minutes.
  • the PCR product was detected by 1% agarose gel electrophoresis, and the product was ligated into the pMD20-T vector, and the positive clone was screened and verified by sequencing.
  • the sequence was as shown in SEQ ID NO: 3, and the plasmid was named p165.
  • P165 was digested with restriction endonucleases HindIII and XbaI, and the resulting TaASG027 promoter was ligated into the p2300 35S:GUS vector, which was also digested with HindIII and XbaI, using T4 DNA ligase to obtain the plant expression vector p167.
  • the structure of the plasmid was obtained. As shown in Figure 4.
  • the plant expression vector p167 was transferred to the Agrobacterium AGL0 strain by heat shock.
  • the primers were designed to identify the transgenic rice plants by PCR.
  • Primer 5 5'-GTTATCTTGAGTAATGAAGAAGCAAGTACC-3' (SEQ ID NO: 8)
  • Primer 6 5'-GCCGTCGAGTTTTTTGATTTCAC-3' (SEQ ID NO: 9)
  • 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 40 seconds; 30 cycles; extension at 72 ° C for 10 minutes.
  • Amplified is a TaASG027 promoter and a partial fragment of GUS, which is 1569 bp in length.
  • the identification results indicated that the resistant regenerated plants obtained by Agrobacterium-mediated rice transformation were positive plants transfected with p167 gene.
  • 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.
  • the appropriately sized transgenic seedlings with GUS reporter gene or specific tissues were selected and immersed in GUS staining solution, stained overnight at 37 ° C, the reaction solution was aspirated, decolorized by ethanol gradient, and photographed by microscopy.
  • the results of GUS staining on various tissues and organs of p167 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 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 TaASG027 promoter can only initiate the GUS gene in the pollen in the dinuclear and trinuclear stages.
  • the expression in the anthers Fig.
  • the TaASG027 promoter is a promoter for the specific expression of anthers in the late pollen development. Further, the expression of GUS was not detected in the anther wall of pollen in the dinuclear phase (Fig. 5H), while the expression of GUS was detected in the pollen of the binuclear and trinuclear stages (Fig. 5I-J). Therefore, the TaASG027 promoter is A promoter specifically expressed in the late stage of wheat pollen development.

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Physiology (AREA)
  • Pregnancy & Childbirth (AREA)
  • Reproductive Health (AREA)
  • Virology (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un promoteur d'expression spécifique d'anthère de plante et ses utilisations.
PCT/CN2015/076147 2014-04-11 2015-04-09 Identification et utilisations du promoteur d'expression spécifique d'anthère de plante ptaasg027 WO2015154689A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201410144225 2014-04-11
CN201410144225.4 2014-04-11

Publications (1)

Publication Number Publication Date
WO2015154689A1 true WO2015154689A1 (fr) 2015-10-15

Family

ID=54287329

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2015/076147 WO2015154689A1 (fr) 2014-04-11 2015-04-09 Identification et utilisations du promoteur d'expression spécifique d'anthère de plante ptaasg027

Country Status (1)

Country Link
WO (1) WO2015154689A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109762815A (zh) * 2018-12-13 2019-05-17 海南波莲水稻基因科技有限公司 一种在水稻花药与花粉中特异表达的启动子pchf17及其应用
CN112251435A (zh) * 2020-08-27 2021-01-22 云南大学 植物花粉特异表达启动子POsPTD1及应用
CN113416735A (zh) * 2021-03-17 2021-09-21 云南中烟工业有限责任公司 一种烟草生殖细胞特异高表达基因及应用
CN113832151A (zh) * 2021-07-23 2021-12-24 电子科技大学 黄瓜内源启动子及其应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1309704A (zh) * 1999-03-26 2001-08-22 农业水产省农业生物资源研究所所长代表的日本国 在花药和花粉中表达的启动子序列
CN1334876A (zh) * 1998-11-03 2002-02-06 辛根塔参与股份公司 包含稻花药特异基因的dna及其转化的转基因植物
CN1912126A (zh) * 2006-09-07 2007-02-14 中国农业大学 一种植物花药特异性启动子及其应用
CN101182523A (zh) * 2007-11-22 2008-05-21 中国农业大学 植物花药特异性启动子及其应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1334876A (zh) * 1998-11-03 2002-02-06 辛根塔参与股份公司 包含稻花药特异基因的dna及其转化的转基因植物
CN1309704A (zh) * 1999-03-26 2001-08-22 农业水产省农业生物资源研究所所长代表的日本国 在花药和花粉中表达的启动子序列
CN1912126A (zh) * 2006-09-07 2007-02-14 中国农业大学 一种植物花药特异性启动子及其应用
CN101182523A (zh) * 2007-11-22 2008-05-21 中国农业大学 植物花药特异性启动子及其应用

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109762815A (zh) * 2018-12-13 2019-05-17 海南波莲水稻基因科技有限公司 一种在水稻花药与花粉中特异表达的启动子pchf17及其应用
CN109762815B (zh) * 2018-12-13 2023-03-14 海南波莲水稻基因科技有限公司 一种在水稻花药与花粉中特异表达的启动子pchf17及其应用
CN112251435A (zh) * 2020-08-27 2021-01-22 云南大学 植物花粉特异表达启动子POsPTD1及应用
CN112251435B (zh) * 2020-08-27 2022-04-29 云南大学 植物花粉特异表达启动子POsPTD1及应用
CN113416735A (zh) * 2021-03-17 2021-09-21 云南中烟工业有限责任公司 一种烟草生殖细胞特异高表达基因及应用
CN113832151A (zh) * 2021-07-23 2021-12-24 电子科技大学 黄瓜内源启动子及其应用

Similar Documents

Publication Publication Date Title
US6407315B1 (en) Seed-preferred promoter from barley
CN111154756B (zh) 植物花药花粉发育后期特异性表达启动子及其应用
WO2015154689A1 (fr) Identification et utilisations du promoteur d'expression spécifique d'anthère de plante ptaasg027
US6921815B2 (en) Cytokinin Oxidase Promoter from Maize
WO2013067901A1 (fr) Promoteur d'expression spécifique d'un stade de développement tardif de pollens de plante et son utilisation
WO2015161744A1 (fr) Identification et utilisation du promoteur ptaasg048 spécifiquement exprimé par l'anthère d'une plante
WO2005113771A1 (fr) Promoteur d'expression preferentielle dans l'embryon et sa methode d'utilisation
CN106148344B (zh) 一种具有增强植物基因表达活性的5′utr序列及其应用
CN112175955B (zh) 一种在植物花粉中特异表达的强启动子cp09及其应用
US8044263B2 (en) Cytokinin oxidase promoter from maize
CN105316333B (zh) 植物花药特异表达启动子pTaASG005的鉴定和应用
CN109355291B (zh) 一种植物胚乳特异性表达启动子pOsEnS93的鉴定和应用
CN105316334B (zh) 植物花药特异表达启动子pTaASG019的鉴定和应用
CN104975024B (zh) 植物花药特异表达启动子pTaASG042的鉴定和应用
CN105039338B (zh) 植物花药特异表达启动子pTaASG004的鉴定和应用
JP4505626B2 (ja) 花粉特異的発現活性を有するプロモーター
JP4452823B2 (ja) カルス及び種子胚特異的発現活性を有するプロモーター
CN109097364B (zh) 一种植物胚乳特异性表达启动子pOsEnS100的鉴定和应用
CN104975025B (zh) 植物花药特异表达启动子pTaASG033的鉴定和应用
CN105274105B (zh) 植物花药特异表达启动子pTaASG050的鉴定和应用
CN104975021B (zh) 植物花药特异表达启动子的鉴定和应用
CN104975022B (zh) 植物花药特异表达启动子pTaASG036的鉴定和应用
JP4505627B2 (ja) 緑色組織特異的発現活性を有するプロモーター
CN115927324A (zh) 一种在植物各时期花药中特异性表达的强启动子cp17及其应用
KR20100043820A (ko) 단자엽 식물 형질 전환을 위한 종자 배유 특이 발현 프로모터

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15777070

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 06.03.2017)

122 Ep: pct application non-entry in european phase

Ref document number: 15777070

Country of ref document: EP

Kind code of ref document: A1