WO2013067901A1 - 一个植物花粉发育晚期特异表达启动子及其应用 - Google Patents

一个植物花粉发育晚期特异表达启动子及其应用 Download PDF

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WO2013067901A1
WO2013067901A1 PCT/CN2012/083999 CN2012083999W WO2013067901A1 WO 2013067901 A1 WO2013067901 A1 WO 2013067901A1 CN 2012083999 W CN2012083999 W CN 2012083999W WO 2013067901 A1 WO2013067901 A1 WO 2013067901A1
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sequence
plant
gene
promoter
dna
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PCT/CN2012/083999
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English (en)
French (fr)
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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
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Priority to CN2012800037715A priority Critical patent/CN103261417A/zh
Publication of WO2013067901A1 publication Critical patent/WO2013067901A1/zh

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    • 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
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C12N15/8289—Male sterility

Definitions

  • the invention belongs to the technical field of plant bioengineering and plant improvement genetic engineering, and particularly relates to the isolation and identification of a late-specific expression promoter of plant pollen development and its application in regulating plant pollen fertility.
  • Heterosis is a common phenomenon in the biological world, and the use of heterosis can significantly increase crop yield, quality and resistance.
  • Crossbreeding has become the main route for many crops to breed new varieties. Effective control of crop self-pollination and fertilization is the key to obtaining high-purity hybrid Ft seeds and thus utilizing crop heterosis. Key questions that must be addressed in crossbreeding
  • the questions are: (1) Obtaining available male sterile lines: generally controlled by cytoplasmic infertility or recessive nuclear sterility genes; (2) Hybrid matching: Sterile lines can be combined with corresponding male plants to produce excellent traits Hybrid offspring; (3) Breeding of sterile lines: Sterile lines can restore fertility under certain conditions to maintain them. Therefore, the breeding of crop male sterile lines is a key link in the utilization of heterosis.
  • Wheat is a self-pollinating plant.
  • the core issue of wheat heterosis utilization is the technical system for efficient production of wheat hybrids.
  • the research on the utilization of wheat heterosis mainly focuses on: the use of nuclear infertility male sterility ("three-line method"), the use of chemical killing technology (“slaughter method”) and light temperature sensitivity Use of nuclear male sterility (“two-line method”:).
  • the three-line method has not been widely used in production because of the difficulty in breeding of sterile lines, narrow recovery sources, and cytoplasmic side effects.
  • the killing method avoids the relationship between recovery and maintenance.
  • the driving activity and specificity of plant pollen or anther promoters determine the success or failure of genetically engineered means to regulate pollen fertility, create plant sterile lines and restore lines.
  • genetically engineered means to regulate pollen fertility, 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 genetic engineering to regulate pollen fertility and creation of plant male sterile lines in wheat.
  • the promoter specifically expressed in the late stage of plant pollen development is a nucleoside cloned from wheat (Triticum aestivum L.) genomic DNA by a chromosome walking technique using a cDNA sequence specifically expressing a late stage of wheat pollen development.
  • the acid sequence, designated P TaPSG076 promoter, is 1957 bp in length and its nucleotide sequence is shown in SEQ ID NO: 1.
  • the late-specific expression promoter of plant pollen development comprises the nucleotide sequence shown by SEQ ID NO: 1 in the sequence listing, or comprises 90°/ of the nucleotide sequence listed in SEQ ID NO: 1. . a nucleotide sequence of the above similarity, or comprising 100 and more than 100 consecutive nucleotide fragments derived from the sequence of SEQ ID NO: 1, and which can drive a nucleotide sequence operably linked to the promoter in the plant Expression in pollen, especially in late-developed pollen.
  • 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: 1 promoter disclosed herein are also within the scope of the invention.
  • the promoter nucleotide sequence of this embodiment can be used to isolate corresponding sequences from plants other than wheat, especially Is homologous cloning 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: 1 promoter sequence (or a fragment thereof) set forth herein are also included in the embodiments.
  • the promoter region of this embodiment can be isolated from any plant, including but not limited to rice, Brassica, corn, wheat, sorghum, two genus, white mustard, castor, sesame, cottonseed, linseed, Soybeans, Arabidopsis, Beans, Peanuts, Alfalfa, Oats, Rapeseed, Barley, Oats, Rye, Millet, Coriander, Triticale, Spelt, Spelt, Double wheat, flax, gran
  • Gramma grass Gramma grass, rubbing grass, false scorpion, fescue, perennial wheat straw, sugar cane, cranberry moss, papaya,
  • Banana safflower, oil palm, cantaloupe, apple, cucumber, sarcophagus, gladiolus, chrysanthemum, lily family, cotton, dragonfly, sunflower, book
  • Brassica, beets, coffee, yam, ornamental plants and pines Brassica, beets, coffee, yam, ornamental plants and pines.
  • promoter refers to a DNA regulatory sequence which typically contains a TATA box which directs RNA polymerase I I to initiate transcription of a particular coding sequence at a suitable transcription initiation site. Promoters may also contain additional recognition sequences, which are usually located upstream or 5' of the TATA box and are referred to as upstream promoter elements, which can affect the rate of transcription. One of skill in the art will recognize that it is within the skill of the art to isolate and identify additional regulatory elements located upstream of a particular promoter region identified herein, after identifying the nucleotide sequences of the promoter regions disclosed herein. Thus, the promoter regions disclosed herein may additionally comprise upstream regulatory elements, such as elements responsible for tissue-specific and time-specific expression, elements that regulate constitutive expression, enhancers, and the like.
  • the activity and strength 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.
  • a GUS reporter gene There are three detection methods depending on the substrate used for GUS gene detection: histochemistry, spectrophotometry, and fluorescence (the highest sensitivity is spectrophotometric), the most common of which is histochemistry.
  • the histochemical method was carried out using 5-bromo-4-chloro-3-indol- ⁇ -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 a blue product under suitable conditions.
  • a DNA vector comprising a promoter operably linked to a heterologous nucleotide sequence comprising the DNA sequence SEQ ID NO: 1 of the present invention, or comprising a nucleotide sequence having a nucleotide sequence of 90% or more similar to the nucleotide sequence set forth in ID NO: 1, or comprising 100 and more than 100 consecutive nucleotide fragments derived from the sequence of SEQ ID NO: 1, and
  • the above heterologous nucleotide sequence is driven for expression in a plant cell.
  • Embodiments of the invention also provide expression vectors, and plants or plant cells comprising the above DNA vectors stably in the genome.
  • “Operatively linked” refers to the manner in which a heterologous nucleotide sequence is placed under the action of a promoter, and also refers to the joining of two nucleotide sequences such that the coding sequence of each MN fragment is maintained in the appropriate reading frame.
  • the SEQ ID NO: 1 promoter and variants and fragments thereof disclosed herein can be used in plant genetic engineering, for example, to prepare transformed or transgenic plants to produce a phenotype of interest.
  • Transformed plant or “transgenic plant” refers to a plant that contains a heterologous nucleotide sequence within the genome.
  • the transformed plant or transgenic plant genome stably contains these heterologous nucleotide sequences which are stably inherited to the next generation.
  • These heterologous nucleotide sequences may be present in the genome either alone or together with the recombinant Li A vector.
  • a “transgenic event” as used herein includes any cell, cell line, callus, tissue, plant part or whole plant as long as their genotype is altered by the presence of an exogenous nucleic acid, including by transgenic manipulation.
  • GMO events as used herein do not include alteration of the genome (chromosomal or extrachromosomal) by traditional planting methods or natural events (eg, random hybridization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition or spontaneous mutation). plant.
  • the SEQ ID NO: 1 promoter sequence disclosed herein can regulate the expression of any heterologous nucleotide sequence in a host plant.
  • the heterologous nucleotide sequence may be a structural gene operably linked to a promoter disclosed herein, a regulatory gene, an antisense gene of a structural gene, an antisense gene of a regulatory gene, or capable of interfering with an endogenous source. Gene expression of small RNA.
  • the construction of two promoter expression vectors of "promoter-GUS reporter gene” and "promoter-pollen lethal gene” is provided, and its expression and effect in transgenic plants are carried out. analysis.
  • any of the above functional genes of interest can be operably linked to the SEQ ID NO: 1 promoter sequence of the present invention and expressed in plants.
  • the provided pollen development-specific expression promoter of the present invention can be used for specific expression of a foreign gene in pollen, 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 plants.
  • Pollen It describes the functional analysis and identification of genes related to growth and development of the book; it 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 plant gene expression vector of the "promoter-GUS reporter gene” in the present invention has an expression cassette which is a pTaPSG076 promoter and a GUS ( ⁇ -glucuronidase gene) reporter gene and a nopaline synthase ( The transcription terminator of nos ), the selected marker gene is neomycin phosphotransferase II (NPTI I ), and the starting vector is pCAMBIA2300, GUS ( ⁇ -glucuronidase gene) reporter gene and nopaline purchased from CAMBIA company.
  • the transcription terminator of the synthase (nos) was from pBI121 of Clontech, and the new plant expression vector was named ⁇ 17.
  • the plant gene expression vector of the "promoter-pollen lethal gene” described in the present invention is a fusion gene of the pTaPSG076 promoter, the peptide of the Bt-1 gene of maize and the Amylase (AA) gene (SEQ ID N0: 2) and transcriptional terminator of nopaline synthase (nos);
  • the selected marker gene is neomycin phosphotransferase II (NPTII)
  • the starting vector is pCAMBIA2300 of CAMBIA
  • the new plant expression vector is named pTa68.
  • the rice callus was transformed by Agrobacterium-mediated transformation to obtain PCR-positive transgenic rice plants.
  • the promoter of the present invention is specifically expressed in the late stage of pollen development, and can be used for the specific expression of genes in the late stage of pollen development, and can also drive efficient or specific expression of homologous or heterologous genes in the late stage of plant pollen development. It avoids the adverse effects of sustained expression of the target gene in other parts, and is of great significance for the creation of plant male sterile lines and restorer lines.
  • Figure 1 is a T-DNA region map of the expression vector pTal7.
  • LB and RB are the left and right borders of T-DNA, respectively;
  • ⁇ indicates the neomycin pitytransferase gene;
  • P35S indicates the promoter of CaMV35S gene;
  • T35S indicates the terminator of CaMV35S gene;
  • GUS indicates ⁇ -glucuronide
  • the enzyme gene; Tnos represents the terminator of the nopaline synthase (nos) gene; HindIII, Pstl, Xbal, Sac I and EcoRI represent the restriction endonuclease sites, respectively;
  • the late specific promoter of pollen development is the invention The isolated and identified pollen development specifically expresses the promoter.
  • Figure 2 shows GUS staining of tissues and organs of pTal7 transgenic rice.
  • A is root; B stem; C leaf; D is flower with pollen at meiosis; E is flower with pollen in mononuclear stage; F is flower with pollen in dinuclear stage; G is flower with pollen in trinuclear stage; H is pollen in the mononuclear phase; I is pollen in the dinuclear phase; J is pollen in the trinuclear phase.
  • Figure 3 is a GUS staining of tissues and organs of ⁇ 17 transgenic wheat.
  • A is the root; B stem; C leaf; D is the flower in the early development of pollen; E is the pollen in the late developmental stage; F is the pollen in the late developmental stage; G is the anther in the late developmental stage; Pollen is in the late developmental pistil; I is in the late developmental pollen.
  • Figure 4 is a T-DNA region map of the expression vector pTa68.
  • LB and RB are the left and right borders of T-DNA, respectively;
  • ⁇ indicates the neomycin phosphotransferase ⁇ gene;
  • P35S indicates the promoter of CaMV35S gene;
  • T35S indicates the terminator of CaMV35S gene;
  • Tnos indicates nopaline synthase (nos) The terminator of the gene;
  • ZmTP-AA represents the fusion gene of the Bt-1 gene of the maize and the AA gene; HindI II, Pstl, Sai l , Xbal and Smal respectively indicate the restriction enzyme cleavage site;
  • pollen The late development-specific promoter is a promoter specifically expressed in the late stage of pollen identification and isolation identified in the present invention.
  • Figure 5 shows the PCR identification of pTa68 transgenic rice.
  • M is a DNA marker; 1 indicates a control without template; 2 indicates that the untransformed rice DNA is used as a template; and 3-12 indicates that the transgenic rice DNA is used as a template.
  • Figure 6 shows the RT-PCR identification of pTa68 transgenic rice.
  • Figure 7 is a ⁇ 2 - ⁇ staining observation of ⁇ 68 transgenic rice pollen.
  • ⁇ is a non-transgenic rice pollen;
  • B is a pollen of transgenic rice.
  • Figure 8 is the morphology of P Ta68 transgenic rice and non-transgenic control plants.
  • 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., during the construction of PCR kits and vectors.
  • the endonuclease was purchased from Bao Bioengineering Co., Ltd.
  • the pEASY-Tl ligation kit was purchased from Beijing Quanjin Biotechnology Co., Ltd.
  • the T4 DNA ligase was purchased from NEB.
  • the methods were all carried out according to the method provided by the kit.
  • the vector P2300 used in the experiment was modified from the laboratory, and the basic skeleton was obtained from CAMBIA's pCAMBIA2300.
  • the end sequence designated as the TaPSG076 promoter, has the nucleotide sequence shown as SEQ ID NO: 1.
  • Primer 1 5 ' - CTTCTCCAAGAACGGAGGCGAAT-3 '
  • the first genomic transfer resulted in a 5' 374 bp DNA sequence.
  • Primer 4 5 ' - AAGGGCTGGCGATTATGCAC -3,
  • the plant expression vector PBI121 was digested with restriction endonucleases Hindll and EcoRI, and the 35S:GUS fragment was ligated into the pCAMBIA2300 vector of CAMBIA, which was also digested with Hindi II and EcoRI, using T4 DNA l igase.
  • the vector was named P2300 35S: GUS.
  • Primer 7 5 ' - ctgcag GTGTTGCGGACCCAGGTT -3,
  • Primer 8 5, - tctaga AGGAAGGGAACCGTCGGC -3 '
  • the sequence ctgcag in primer 7 is the cleavage site of Pstl
  • the sequence tctaga in primer 8 is the cleavage site of Xbal.
  • 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 2 minutes 30 seconds; 32 cycles; extension at 72 °C for 10 minutes.
  • the PCR product was detected by 1% agarose gel electrophoresis, and the product was ligated into pEASY-Blunt vector, and the positive clone was screened and verified by sequencing.
  • the sequence is shown as SEQ ID NO: 1, and the plasmid is called pTal5.
  • pTal5 was digested with restriction endonucleases Pstl and Xbal, and the resulting TaPSG076 promoter was ligated into the p2300 35S:GUS vector, which was also digested with Pstl and Xbal, using T4 DNA ligase to obtain the plant expression vector pTal7. As shown in Figure 1.
  • the plant expression vectors P 17 and P Ta68 were transferred to the Agrobacterium AGL0 strain by heat shock.
  • Agrobacterium tumefaciens is used to infect rice embryogenic callus, which is cultured for 2-3 days in the dark. Then, after two steps of resistance screening, pre-differentiation, differentiation and rooting culture, the transgenic rice with kanamycin resistance is finally obtained.
  • the plant expression vector pTal7 was transferred into Agrobacterium AGL0 strain by heat shock method.
  • the callus was induced by secreting the mature embryo of wheat.
  • Wheat callus was infested with Agrobacterium and cultured for 3 days in the dark.
  • the callus co-cultured with Agrobacterium was placed in the induction medium supplemented with cefotaxime for 1 week in the dark, and then transferred to the screening medium for 4-6 weeks to transfer the resistant callus into differentiation.
  • the medium induces differentiation of the buds, and the differentiated buds are transferred to a rooting medium for rooting culture, and finally transgenic wheat (trans ⁇ 17 wheat) T having kanamycin resistance is obtained. Generation plants.
  • X-Glue base solution 50 mM PBS pH 7. 0, 10 mM EDTA-2Na, 0.1% Triton X-100, 5 mM iron potassium hydride, 0.5 mM potassium ferrous hydride.
  • X-Glue use solution 50 ⁇ ⁇ mother liquor + 950 ⁇ 1 base solution.
  • Transgenic seedlings or specific tissues with the appropriate size of GUS reporter gene 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 the tissues and organs of pTA17 transgenic rice and transgenic wheat are shown in Fig. 2 and Fig. 3.
  • the expression of GUS gene was not detected in the vegetative organs such as roots, stems and leaves of transgenic rice and wheat.
  • the expression of GUS gene was also not detected in flower organs other than the flower organ.
  • the TaPSG076 promoter only activated the GUS gene in pollen and only in the pollen of late development (dinuclear and trinuclear), indicating that the TaPSG076 promoter is a A promoter specifically expressed in the late stage of pollen development.
  • Primer 11 5 ' - ctgcag GTGTTGCGGACCCAGGTT -3 '
  • Primer 12 5 ' - gtcgac GCTCGCTCGCCGCTAGCT -3 '
  • the sequence tctaga in primer 9 is the restriction site of Xbal
  • the sequence cccggg in primer 10 is the restriction site of Smal
  • the sequence ctgcag in primer 11 is the cleavage site of Pstl
  • the sequence gtcgac in the primer 12 is Sail. Restriction sites.
  • Tnos and P TaPSG076 fragments were amplified by PCR using the specific primers described above, respectively, and passed through an agarose gel. Description
  • the ZmTP-Amylase (AA) gene sequence was obtained by artificial synthesis. During the synthesis, Xhol cleavage site (ctcgag) and Xbal cleavage site (tctaga) were added to the 5' and 3' of ZmTP-AA, respectively.
  • the -AA gene was ligated to the PEASY-T3 vector and verified by sequencing.
  • the pEASY-T3 plasmid harboring the ZmTP-AA gene was digested with the restriction sites Xhol and Xbal at both ends of the gene to obtain the ZmTP-AA gene with the correct sequence and corresponding restriction sites at both ends. Fragment.
  • the primers were designed to identify the transgenic rice plants by PCR.
  • 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 40 seconds; 30 cycles; extension at 72 ° C for 10 minutes.
  • the TaPSG076 promoter and a partial fragment of ZmTP-AA were amplified, and the length was 517b. The results of the identification are shown in Fig. 5.
  • the regenerated rice plants obtained by Agrobacterium-mediated rice transformation were positive plants transformed with ⁇ 68 gene.
  • Primer 15 ATGGCGGCGACAATGGCAGTG
  • Primer 15 and primer 16 are the detection primers of ZmTP-AA gene, and the amplified fragment size is 241 bp;
  • Material 17 and Primer 18 are the analysis primers of rice internal reference gene ⁇ / ⁇ , and the amplified fragment size is 554 bp.
  • the PCR detection system and procedures are: Instruction manual
  • PCR reaction conditions 94 ° C, pre-denaturation for 5 minutes; 94 ° C, denaturation for 30 seconds; 58 ° C, annealing for 30 seconds; 72 ° C, extension for 30 seconds; 28 cycles, 72 ° C, 10 minutes.
  • PCR results are shown in Fig. 6.
  • Fig. 6 In the roots, stems, leaves and ears of different developmental stages, the expression of the ⁇ / ⁇ / - ⁇ gene was detected only in the ears of pollen in the dinuclear and trinuclear stages.
  • transgenic rice ⁇ 68 and the non-transgenic rice control were simultaneously planted in the field, and the development process and plant morphology were compared. As shown in Fig. 8, there was no significant difference between the transgenic plants and the non-transgenic plants.

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Abstract

提供了一种从小麦（Triticum aestivum L.）中分离的花粉发育晚期特异表达的启动子及其应用。该启动子具有时空表达特异性，能驱动目的基因在花粉发育晚期高效表达，而在根、茎、叶及除晚期花粉以外的花器官中不表达。

Description

一个植物花粉发育晚期特异表达启动子及其应用 技术领域
本发明属于植物生物工程和植物改良基因工程技术领域， 具体涉及一种植物花粉发育 晚期特异性表达启动子的分离鉴定及其在调控植物花粉育性方面的应用。
背景技术
杂种优势是生物界的一种普遍现象说， 利用杂种优势可以显著提高作物产量、 品质和抗 性。 杂交育种已经成为许多作物选育新品种的主要途径。 而有效控制作物自花授粉、 受精 是获得高纯度杂交 Ft种子、从而利用作物杂种优势的关键。杂交育种中必须要解决的关键问 书
题是： （1 ) 获得可用的雄性不育系： 一般由细胞质不育或隐性核不育基因控制； （2) 杂交 配组： 不育系可与相应的父本植物组合生产具有优良性状的杂交后代； （3 )不育系的繁殖: 不育系能在一定条件下恢复育性使其得到保持。 因此， 作物雄性不育系的选育是杂种优势 利用的关键环节。
小麦是自花授粉植物， 小麦杂种优势利用的核心问题是高效生产小麦杂交种的技术体 系。 综合近 50年来的研究进展， 小麦杂种优势利用研究主要集中于： 核质互作雄性不育的 利用 （"三系法")、 化学杀雄技术的利用 （"化杀法"） 和光温敏核雄性不育的利用 （"两系 法"：)。 三系法由于不育系难以繁殖、 恢复源较窄、 细胞质副效应等原因， 未能在生产上大 面积应用。 化杀法避开了恢复与保持间的相互关系， 曾被认为是一种很有希望的小麦杂交 制种新技术， 但由于其在制种过程中稳定性差、 制种成本高及环境污染严重等多方面原因， 在实际生产上也难以推广利用。 基于光温敏的两系法虽然具有制种成本低、 恢复源广泛， 较易获得优势组合等优点， 但也面临着两大关键问题一一环境因素的不稳定性对不育系育 性的影响和利用光温敏特性所选育的小麦不育系十分有限。
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 Tl 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 steril ity 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， A chimaeric ribonuc lease-inhibitor gene restores fertility to male sterile plants, Nature, 1992. 357: 384-387)。 此外， 其他硏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， 说
农业生物技术学报， 1999, 7: 255-258； Curtis等， Genomic male sterility in lettuce,
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a base line for the production of Fi hybrid, 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书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特异表达的启动子序列及克隆并应用该启 动子的方法。
本发明所提供的植物花粉发育晚期特异表达的启动子，是利用小麦花粉发育晚期特异表 达基因的 cDNA序列， 通过染色体步移技术， 从小麦 （Triticum aestivum L. )基因组 DNA 中克隆得到的核苷酸序列，称之为 PTaPSG076启动子，长度为 1957bp，其核苷酸序列如 SEQ ID N0:1所示。
本发明所提供的植物花粉发育晚期特异表达启动子， 含有序列表中 SEQ ID NO: 1所示的 核苷酸序列， 或包含与 SEQ ID NO: 1中所列核苷酸序列具有 90°/。以上相似性的核苷酸序列， 或包含来源于 SEQ ID NO: 1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粉中的表达， 尤其是在发育晚期的花粉中的 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 N0:1 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。
本实施方案中的启动子核苷酸序列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， 根据它们与本文所列的 SEQ ID N0 : 1 启动子序列 （或其片段） 间的序列相似性而分 离的相应片段， 也包括在实施方案中。 本实施方案的启动子区域可从任何植物中分离， 包 括 （但不限于） 水稻、 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 说
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、
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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、 书
芸苔、 甜菜、 咖啡、 薯蓣、 观赏植物和松类等。
本文中 "启动子"一词指 DNA调控序列， 其中通常含有一个 TATA盒， 该序列能够指导 RNA聚合酶 I I在合适的转录起始位点上起始特定编码序列的转录。 启动子也可另外含有其 他识别序列， 它们通常位于 TATA盒的上游或 5' 端， 被称作上游启动子元件， 这些元件能 够影响转录速率。 本领域技术人员应认识到当鉴别了本文所公开的启动子区域的核苷酸序 列后， 分离并鉴定位于本文所鉴别的特定启动子区域上游的其它调控元件就属于现有技术 范围了。 因此， 本文公开的启动子区域可另外包含上游调控元件， 如负责组织特异性和时 间特异性表达的元件、 调控组成型表达的元件和增强子等。
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。
本发明的实施例中还包括 DNA载体,该载体含有操作性连接于异源核苷酸序列上的启动 子， 该启动子含有本发明所公开的 DNA序列 SEQ ID N0 : 1 , 或包含与 SEQ ID NO : 1中所列核 苷酸序列具有 90%以上相似性的核苷酸序列， 或包含来源于 SEQ ID NO : 1序列上的 100个及 100以上连续的核苷酸片段， 并可在植物细胞中驱动上述异源核苷酸序列进行表达。本发明 的实施方案还提供了表达载体， 以及在基因组中稳定包含上述 DNA载体的植物或植物细胞。
说
"操作性连接"指使异源核苷酸序列处于启动子作用下的连接方式， 也指将两个核苷酸序 列连接起来从而使每个丽 A片段的编码序列都保持在适当的阅读框内。 "异源核苷酸序列 " 书
指天然状态下没有与本文所述启动子序列 SEQ ID N0 : 1 操作性连接的序列， 对于植物宿主 来说可以是同源的， 或是异源的。
本文所公开的 SEQ ID NO : 1启动子及其变异体和片段可用于植物基因工程， 例如制备转 化或转基因植物， 以产生目的表型。 "转化植物"或 "转基因植物"指在基因组内含有异源 核苷酸序列的植物。 通常转化植物或转基因植物基因组稳定含有这些异源核苷酸序列， 可 将该异源核苷酸序列稳定地遗传给下一代。 这些异源核苷酸序列可单独或与重组丽 A载体 一起存在于基因组中。 这里所述的 "转基因事件"包括任何细胞、 细胞系、 愈伤组织、 组 织、 植物体部分或完整植物体， 只要它们的基因型被外源核酸的存在而改变， 包括通过转 基因操作而改变的起始宿主， 以及通过这些起始宿主进行有性或无性繁殖所得的后代。 这 里所用的 "转基因事件"并不包括通过传统植物种植方法或天然事件 （例如随机杂交、 非 重组病毒感染、 非重组细菌转化、 非重组转座或自发突变） 改变了基因组 （染色体或染色 体外） 的植物。
本文所公开的 SEQ ID NO : 1启动子序列可在宿主植物中调控任何异源核苷酸序列的表 达。 因此， 所述的异源核苷酸序列可以是操作性连接于本文所公开的启动子之上的结构基 因、 调节基因、 结构基因的反义基因、 调节基因的反义基因或者能够干扰内源基因表达的 小 RNA。 在本发明的实施中提供了 "启动子 -GUS报告基因"和 "启动子-花粉致死基因"两 种植物表达载体的构建， 并对其在转基因植物中的表达情况和所产生的效果进行了分析。
由上可见， 可将上述任何目的功能基因操作性连接到本发明中的 SEQ ID NO : 1启动子序 列上， 并在植物体中进行表达。
本发明的所提供的花粉发育晚期特异表达启动子可用于外源基因在花粉中特异性表达， 从而避免该外源基因在植物其他组织中持续表达所带来的不利影响， 还可以用于植物花粉 说 明 书 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。
具体地， 在本发明中所述 "启动子 -GUS报告基因"的植物基因表达载体， 其表达盒是 由 pTaPSG076启动子和 GUS ( β -葡萄糖苷酸酶基因）报告基因及胭脂碱合成酶（nos ) 的转 录终止子构成， 选择的标记基因为新霉素磷酸转移酶 II (NPTI I ), 出发载体为购自 CAMBIA 公司的 pCAMBIA2300， GUS ( β -葡萄糖苷酸酶基因）报告基因和胭脂碱合成酶 （nos ) 的转 录终止子来自 Clontech公司的 pBI 121， 新的植物表达载体命名为 ρ 17。 通过农杆菌介导 转化水稻和小麦， 取转基因植株的各个组织器官进行 GUS组织化学染色， 观察 GUS基因在 转基因植株的各个组织器官中的表达情况， 确定 PTaPSG076 启动子可以特异性地驱动 GUS 基因在花粉发育晚期表达。
具体地， 本发明中所述 "启动子-花粉致死基因" 的植物基因表达载体， 一个表达盒是 由 pTaPSG076启动子、 玉米 Bt-1基因的导肽与 Amylase (AA)基因的融合基因 （SEQ ID N0: 2) 以及胭脂碱合成酶 （nos ) 的转录终止子构成； 选择的标记基因为新霉素磷酸转移酶 II (NPTII ) , 出发载体为 CAMBIA公司的 pCAMBIA2300, 新的植物表达载体命名为 pTa68。 通过 农杆菌介导法转化水稻愈伤组织， 得到 PCR阳性的转基因水稻植株。通过对 T1代转基因水稻 植株花粉的 I2-KI染色分析， 50%花粉活力弱或无活力的结果进一步证实 pTaPSG076启动子是 一个植物花粉发育晚期特异表达的启动子， 同时说明该启动子在植物雄性不育的创造方面 具有重大的应用前景。
本发明的优点和积极效果： 本发明的启动子为花粉发育晚期特异表达， 可以用于基因 在花粉发育晚期特异表达的研究， 还可以驱动同源或异源基因在植物花粉发育晚期高效特 异表达， 避免了目的基因在其他部位持续表达所带的不利影响， 对植物雄性不育系和恢复 系的创造具有重要意义。
下面通过具体实施方式， 结合附图对本发明做进一歩详细描述， 但不以任何方式限制 本发明的范围。
附图说明
图 1是表达载体 pTal7的 T-DNA区图谱。 LB和 RB分别为 T-DNA的左边界和右边界; ΝΡΤΠ 表示新霉素憐酸转移酶 Π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、 Pstl、 Xbal、 Sac I 和 EcoRI 分别表示限制性内切酶的酶切位点； 花粉发育 晚期特异启动子即为本发明所分离鉴定的花粉发育晚期特异表达启动子。 说 明 书
图 2是 pTal7转基因水稻的组织器官 GUS染色。 A为根； B茎； C叶； D为花粉处于减 数分裂时期的花； 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为处于单核期的花粉； I为处于双核期的花粉； J为处于三核期的花粉。
图 3是 ρ 17转基因小麦的组织器官 GUS染色。 A为根； B茎； C叶； D为花粉处于发 育早期的花； E为花粉处于发育晚期的内桴； F为花粉处于发育晚期的外桴； G为花粉处于 发育晚期的花药； H为花粉处于发育晚期的雌蕊； I为处于发育晚期花粉。
图 4是表达载体 pTa68的 T-DNA区图谱。 LB和 RB分别为 T-DNA的左边界和右边界; ΝΡΤΠ 表示新霉素磷酸转移酶 Π基因； P35S表示 CaMV35S基因的启动子； T35S表示 CaMV35S基 因的终止子； Tnos表示胭脂碱合成酶 （nos )基因的终止子； ZmTP-AA表示玉米 Bt-1基因 的导肽与 AA基因的融合基因； HindI I I、 Pstl、 Sai l , Xbal和 Smal分别表示限制性内切酶 的酶切位点； 花粉发育晚期特异启动子即为本发明所分离鉴定的花粉发育晚期特异表达启 动子。
图 5是 pTa68转基因水稻的 PCR鉴定。 M为 DNA marker; 1表示未加模板的对照； 2表 示以未转基因水稻 DNA为模板； 3-12表示以转基因水稻 DNA为模板。
图 6是 pTa68转基因水稻的 RT-PCR鉴定。
图 7是 ρ 68转基因水稻花粉的 Ι2-ΚΙ染色观察。 Α为未转基因的水稻花粉； B为转基因 水稻的花粉。
图 8是 PTa68转基因水稻与非转基因对照植株的形态。
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-Tl连接试剂盒购自北京全式金生物技术公 司， T4 DNA连接酶购自 NEB公司， 方法均参照试剂盒提供的方法进行。 实验中所用的载体 P2300由本实验室改造所得， 基本骨架来自于 CAMBIA公司的 pCAMBIA2300。
1. 启动子 TaPSG076的克隆
根据已知的小麦花粉发育晚期特异表达基因 TaPSG076的全长 cDNA序列，设计特异性引 物， 利用宝生物工程有限公司的 Genome Walking Kit , 经过两次步移从小麦的基因组 DNA 中共获得 1957bp的 5 ' 端序列， 命名为 TaPSG076启动子， 其核苷酸序列如 SEQ ID NO : 1所 示。
根据已知的小麦花粉发育晚期特异表达基因 TaPSG076的全长 cDNA序列， 设计三条第 一次基因组步移所需的特异性引物：
引物 1 : 5 ' - CTTCTCCAAGAACGGAGGCGAAT-3 '
引物 2: 5， - TGAGATATGCTAGGCCGATGGA -3 '
引物 3: 5， - CTCCGTTTCGGGTTAATGGTGT -3 '
第一次基因组歩移获得了 5' 端 374bp的 DNA序列。
根据第一次基因组步移获得的 DNA序列， 设计三条第二次基因组步移所需的特异性引 物：
说
引物 4: 5 ' - AAGGGCTGGCGATTATGCAC -3，
引物 5: 5， - TTTGGCCCGATCAACGTTTT -3，
引物 6: 5， - TGAGTTGTTTTGGGCTCCGTTT -3 '书
具体操作过程参照试剂盒中的说明书进行。
2. 植物表达载体 PTal7的构建
将植物表达载体 PBI 121用限制性内切酶 Hindll l和 EcoRI双酶切， 得到的 35S : GUS片 段用 T4 DNA l igase连入同样用 Hindi II和 EcoRI双酶切的 CAMBIA公司的 pCAMBIA2300载 体， 新的载体被命名为 P2300 35S : GUS。
从 TaPSG076启动子的 5 ' 端和 ATG上游设计引物：
引物 7: 5 ' - ctgcag GTGTTGCGGACCCAGGTT -3，
引物 8: 5， - tctaga AGGAAGGGAACCGTCGGC -3 '
引物 7中序列 ctgcag是 Pstl的酶切位点， 引物 8中序列 tctaga是 Xbal的酶切位点。 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°C预变性 5 分钟； 94 °C变性 30秒； 60 °C退火 30秒； 72 °C延伸 2分 30秒； 32个循环； 72 °C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-Blunt载体中， 筛选阳性克隆并进行测序验证， 序列如 SEQ ID N0 : 1所示， 该质粒称为 pTal5。
用限制性内切酶 Pstl和 Xbal双酶切 pTal5， 得到的 TaPSG076启动子用 T4 DNA ligase 连入同样用 Pstl和 Xbal双酶切的 p2300 35S : GUS载体， 得到植物表达载体 pTal7， 该质粒 的结构如图 1所示。
3. 农杆菌介导的水稻的遗传转化
利用热激法将植物表达载体 P 17和 PTa68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基因水稻 （转 pTal7水稻、 转 pTa68 说 明 书
水稻） T。代植株。
4. 农杆菌介导的小麦的遗传转化
利用热激法将植物表达载体 pTal7转入农杆菌 AGL0菌株。
以小麦成熟胚为材料暗中诱导愈伤。用农杆菌侵染小麦愈伤组织， 暗中共培养 3天。将 与农杆菌共培养后的愈伤置于加有头孢噻肟的诱导培养基上暗中恢复培养 1 周， 然后转到 筛选培养基上筛选 4-6周， 将抗性的愈伤转入分化培养基诱导芽的分化， 再将分化的芽转 入生根培养基进行生根培养， 最终获得具有卡那霉素抗性的转基因小麦 （转 ρ 17小麦） T。 代植株。
5. 转基因水稻和小麦植株不同组织器官 GUS基因表达的组织化学检测
X- Glue母液： lOOmg X- Glue溶于 5ml 丽？。
X- Glue基液： 50 mM PBS pH7. 0, 10 mM EDTA- 2Na, 0. 1 % Triton X- 100, 5 mM铁 氢化钾， 0. 5 mM亚铁氢化钾。
X- Glue使用液： 50 μ ΐ母液 +950 μ 1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°C染 色过夜， 吸去反应液， 乙醇梯度脱色， 显微镜观察照相。
对 pTA17转基因水稻和转基因小麦各组织器官的 GUS染色结果如图 2和图 3所示， 在 转基因水稻和小麦的根、 茎和叶等营养器官中都检测不到 GUS基因的表达， 在除花粉以外 的花器官中也检测不到 GUS基因的表达， TaPSG076启动子只能启动 GUS基因在花粉、 并且 只在发育晚期 （二核期和三核期） 的花粉中表达， 说明 TaPSG076启动子是一个花粉发育晚 期特异表达的启动子。
6. 植物表达载体 PTa68的构建
用于扩增 Tnos的特异性引物：
引物 9: 5， - tctaga GATCGTTCAAACATTTGGCAATAAAG -3，
引物 10: 5， - cccggg GATCTAGTAACATAGATGACACCGCG -3 '
用于扩增花粉发育晚期特异启动子 pTaPSG076的特异性引物：
引物 11 : 5 ' - ctgcag GTGTTGCGGACCCAGGTT -3 '
引物 12: 5 ' - gtcgac GCTCGCTCGCCGCTAGCT -3 '
引物 9中序列 tctaga是 Xbal的酶切位点，引物 10中序列 cccggg是 Smal的酶切位点，， 引物 11中序列 ctgcag是 Pstl的酶切位点， 弓 I物 12中序列 gtcgac是 Sail的酶切位点。
先用上述特异性引物分别通过 PCR扩增得到 Tnos和 PTaPSG076片段， 经过琼脂糖凝胶 说 明 书
电泳和回收后连入 pEASY-Blunt和 pEASY-T3中。 阳性克隆经测序验证后， 分别根据引物两 侧所带的酶切位点进行双酶切， 得到序列正确、 两端带有相应酶切位点的上述片段。
ZmTP-Amylase (AA)基因序列由人工合成获得， 合成过程中在 ZmTP-AA的 5 ' 端和 3 ' 分 别加入 Xhol酶切位点 （ctcgag ) 和 Xbal酶切位点 （tctaga ) , 合成的 ZmTP-AA基因连在 PEASY-T3载体上并经测序验证正确。 用基因两端所带有的酶切位点 Xhol 和 Xbal 对连有 ZmTP-AA 基因的 pEASY-T3 质粒进行双酶切， 得到序列正确、 两端带有相应酶切位点的 ZmTP-AA基因片段。
在 pCAMBIA2300载体中依次连入 Tnos、 pTaPSG076和 ZmTP-AA， 最终得到植物表达载体 pTa68 (图 4)。
7. 转基因水稻的分子鉴定
设计引物对转基因水稻植株进行 PCR鉴定。
引物 13 : 5 ' -GACCCACCACACCATTAACC-3 '
引物 14 : 5， -ACCCCTGGAAGAGGACTTGT-3 '
反应条件为： 94°C预变性 5分钟； 94 °C变性 30秒； 55 °C退火 30秒； 72 °C延伸 40秒； 30个循环； 72 °C延伸 10分钟。 扩增的是 TaPSG076启动子和 ZmTP-AA的部分片段， 长度为 517b 鉴定结果如图 5所示， 利用农杆菌介导的水稻转化获得的再生水稻植株是转 ρ 68 基因的阳性植株。
8. RT-PCR表达分析
取 PTa68转基因水稻的根、 茎、 叶、 花粉处于减数分裂时期的穗、 花粉处于单核期的 穗和花粉处于双核及三核时期的穗， 提取 RNA, 用 ol i go-dT进行反转录。
以 cDNA作为模板， 以水稻 ACTIN基因作为内参， 分析 TaPSG076启动子驱动的 ZmTP-M 基因在转基因水稻中的表达情况。
RT-PCR鉴定用引物为：
引物 15 : ATGGCGGCGACAATGGCAGTG
引物 16 : GGACCAGGCCGCAGGCCGC
引物 17 : ACCTTCAACACCCCTGCTATG
引物 18： GCAATGCCAGGGAACATAGTG
其中引物 15和引物 16是 ZmTP-AA基因的检测引物， 其扩增片段大小为 241bp; 弓 |物 17和引物 18是水稻内参基因 ^ίΤ/Λ的分析引物， 其扩增片段大小为 554bp。 PCR检测体系 和程序是： 说 明 书
10 X buffer 2
10mM dNTP 0. 4
10 mM引物 F 1
10 mM引物 R 1
Taq polymerase 0. 2
cDNA 1
ddH20 14. 4
PCR反应条件： 94°C， 预变性 5分钟； 94°C， 变性 30秒； 58°C， 退火 30秒； 72°C， 延 伸 30秒； 28个循环, 72 °C , 10分钟。
反应结束后， 对 PCR产物进行 2%的琼脂糖凝胶电泳检测。 PCR检测结果如图 6所示， 在根、 茎、 叶和不同发育时期的穗等器官中， 只在花粉处于双核及三核期的穗中检测到了 Ζ/ζζΓ/ -^基因的表达。
9. 转基因水稻花粉的 Ι2-ΚΙ染色分析
Ι2-ΚΙ的配制： 称取 2 g KI溶于 10 ml蒸馏水中， 然后加入 1 g 12, 待全部溶解后， 加 蒸熘水定容至 300 ml o
染色： 取少量花粉置于载玻片上， 滴上 1-2滴 I2-KI染色液， 5分钟后在显微镜下观察。 结果判断： 凡是染成蓝黑色的为含有淀粉的活力的花粉粒， 成黄褐色的为发育不良的 花粉粒。 如图 7所示， 由于 Τ。代转基因植株均为杂合， 并且 TaPSG076启动子是一个花粉发 育晚期特异表达的启动子， 正如所预期的那样， 在转 pTa68的 T。代水稻植株中， 有 50%的花 粉由于 TaPSG076启动子驱动 Z ^-y^基因的表达而不育。
10. 转基因水稻 pTa68的形态分析
将转基因水稻 Ρ 68与非转基因水稻对照同时种植于田间， 对其发育过程和植株形态 进行比较观察， 如图 8所示， 转基因植株与非转基因植株相比没有明显差别。

Claims

权 利 要 求 书
1. 一种分离的 DNA序列， 其特征在于所述分离的 DNA的序列选自下列组的序列之一：
(a) 具有 SEQ ID N0: 1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: 1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. —种表达盒， 其特征在于所述表达盒包含权利要求 1所述的 DNA序列。
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异源核苷酸序列的方法，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异源核苷酸序列，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: 1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: 1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。
7. 权利要求 6所述的方法， 其中所述的单子叶植物为禾本科植物。
8. 权利要求 7所述的方法， 其中所述的禾本科植物为水稻或小麦。
9. 权利要求 5所述的方法， 其中所述的异源核苷酸序列是指天然状态下没有与所述启动子 序列操作性连接的序列， 对于植物宿主来说可以是同源或是异源的。
10.权利要求 5所述的方法， 其中所述的异源核苷酸序列可以是结构基因、调节基因、 结构 基因的反义基因、 调节基因的反义基因或者能够干扰内源基因表达的小 RNA， 其在花粉发 育晚期的特异性表达可以调节花粉的育性及花粉萌发。。
11.权利要求 1 所述的 DNA序列在以下 （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； 权 利 要 求 书
(d) 培育雄性不育植物品种或品系。
2.权利要求 11所述的应用， 其特征在于， 所述的植物为单子叶植物。3.权利要求 12所述的应用， 其中所述的单子叶植物为禾本科植物。4.权利要求 13所述的方法， 其中所述的禾本科植物为水稻或小麦。
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