WO2012065528A1 - 植物根特异表达启动子的鉴定和应用 - Google Patents

植物根特异表达启动子的鉴定和应用 Download PDF

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WO2012065528A1
WO2012065528A1 PCT/CN2011/082126 CN2011082126W WO2012065528A1 WO 2012065528 A1 WO2012065528 A1 WO 2012065528A1 CN 2011082126 W CN2011082126 W CN 2011082126W WO 2012065528 A1 WO2012065528 A1 WO 2012065528A1
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plant
dna
nucleic acid
sequence
expression
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PCT/CN2011/082126
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French (fr)
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李早霞
周君莉
吴洁芳
夏勉
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北京未名凯拓作物设计中心有限公司
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Publication of WO2012065528A1 publication Critical patent/WO2012065528A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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
    • C12N15/8223—Vegetative tissue-specific promoters
    • C12N15/8227—Root-specific

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  • the present invention is in the field of plant transgenic breeding, 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 at the root of a plant. Further, the present invention relates to an expression cassette, a plant or the like comprising the DNA, and to the use of the DNA.
  • 7,365,185 B2 discloses genomic promoter sequences from rice and other plants, but among which multiple cores Glycosidic acid sequence SEQ ID NO: 68961 is up to 2760 nucleotides, and this sequence is submerged in the numerous sequences disclosed in this document without specifying a specific function, and it is less clear whether it has the specificity of the plant expression site.
  • 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.
  • a specific promoter Currently widely used in the field of agricultural biotechnology are some constitutive strong promoters, such as CaMV.
  • the 35S promoter and the maize Ubiquitin-1 promoter when these promoters are used to induce the target gene to transform rice and other crops in order to improve the quality of the crop, it is often due to the time of expression of the target gene (developmental stage specificity) or space (tissue organs) Specificity) is not well controlled, resulting in insignificant improvement, or because these constitutive promoters induce high levels of gene expression and affect plant growth and development, these are currently using constitutive strong promoters to bind functional genes. Obstacles encountered in improving crop quality.
  • Root is an important organ for plant body to absorb water and nutrients.
  • the root-specific expression system can be used to study plant hyperosmotic stress tolerance, phytoremediation and rhizosphere secretion.
  • Chinese patent applications CN1196753A, CN1791677A, CN101389762A, and CN101589144A disclose a series of root-specific promoters, the structure of these promoter sequences is different, and a new root-specific promoter cannot be expected.
  • the present invention provides novel isolated DNA having the function of a root-specific promoter. Further, the present invention relates to an expression cassette, a transgenic plant or the like comprising the DNA, and to the application of the DNA and the like.
  • the invention provides an isolated DNA capable of directing specific transcription and/or expression of a nucleic acid operably linked downstream thereof at a plant root, the sequence of the isolated DNA being selected from the group consisting of the sequence of:
  • the DNA of the first aspect of the invention has a sequence length of less than 2000 nucleotides, preferably less than 1500 nucleotides, more preferably less than 1200 nucleotides.
  • the DNA of the first aspect of the invention is capable of directing the specific transcription and/or expression of a nucleic acid operably linked downstream thereof in the column sheath of the plant root.
  • the DNA sequence of the first aspect of the invention is set forth in SEQ ID NO: 1.
  • the invention provides an expression cassette comprising:
  • the expression cassette of the second aspect of the invention wherein the nucleic acid is capable of conferring a trait selected from the group consisting of:
  • the invention provides a transgenic plant, plant seed, plant tissue or plant cell transformed with the expression cassette of the second aspect of the invention, preferably wherein the DNA of the first aspect of the invention is capable of directing operably linked The nucleic acid downstream thereof is specifically transcribed and/or expressed at the root of the plant.
  • the plant, plant seed, plant tissue or plant cell of the third aspect of the invention wherein the plant is a monocot or a dicot, such as corn, rice, wheat, barley, sorghum, soybean, canola, cotton, tomato , potato, sugar cane, sugar beet, tobacco or Arabidopsis, preferably rice.
  • a monocot or a dicot such as corn, rice, wheat, barley, sorghum, soybean, canola, cotton, tomato , potato, sugar cane, sugar beet, tobacco or Arabidopsis, preferably rice.
  • the present invention provides a method of producing a plant of the third aspect of the present invention, comprising:
  • the plants obtained in the step (4) are propagated to obtain progeny.
  • the present invention also provides a method of imparting a plant trait comprising
  • the plant obtained in the step (5) is propagated to obtain progeny
  • Figure 1 is a T-DNA region map of the expression vector pHPG.
  • LB and RB are the left and right borders of T-DNA, respectively;
  • hpt indicates the hygromycin resistance gene;
  • Pnos indicates the promoter of the nos gene;
  • Tnos indicates the terminator of the nos gene;
  • GUS indicates the gus protein ( ⁇ -glucuronide) Acidase ( ⁇ -glucuronidase) gene;
  • T35s represents the terminator of the 35s gene;
  • HindIII and BamHI indicates the restriction sites of restriction endonucleases HindIII and BamHI, respectively;
  • the root-specific promoter is the isolated root-specific expression promoter of the present invention.
  • Figure 2 is a GUS staining of tissues and organs of KT630P transgenic rice.
  • A is the seedling stage of rice seedlings;
  • B is the transgenic resistant callus;
  • C is the seedling stage leaves;
  • D is the seedling stage stem;
  • E is the seedling stage root;
  • F is the rice flower organ.
  • Figure 3 is a result of RT-PCR detection of the expression of the GUS gene driven by the KT630P promoter of the present invention in various tissues and organs of transgenic rice.
  • Figure 4 is a GUS staining of KT630P transgenic Arabidopsis plants.
  • A is the seedling stage of Arabidopsis thaliana seedlings
  • B is the plant body of Arabidopsis thaliana
  • C is the planting stage of Arabidopsis flowering
  • D is the GUS staining of roots observed by stereogram.
  • the invention provides an isolated DNA capable of directing specific transcription and/or expression of a nucleic acid operably linked downstream thereof at the root of a plant, in particular capable of directing a nucleic acid operably linked downstream thereof Specific transcription and/or expression in the column sheath in plant roots.
  • such isolated DNA may also be referred to as a root-specific promoter and belongs to a cis-acting element.
  • a root-specific promoter the expression of genes downstream and operably linked thereto is often limited to the root or its tissue site and exhibits developmental regulation and other characteristics.
  • the root-specific promoter can not only accumulate the expression product of the target gene mainly in the plant root or its tissue site, for example, the amount of downstream nucleic acid mRNA produced in the root or its tissue is 5 times and 10 times that of other plant parts. 100 times, 200 times, or even 1000 times, increasing the amount of expression in the area, and avoiding unnecessary waste of plant nutrition.
  • the root-specific promoter of the invention is directly discriminated by the macroscopically observable staining method to direct transcription and/or expression at the root of the plant, and the regulation continues at various stages of plant development, while in the plant In other parts, there is no transcription and/or expression that is clearly distinguishable by the naked eye.
  • operably linked refers to a mode of ligation that enables transcription and/or expression of a nucleic acid linked downstream to be initiated and regulated by a root-specific promoter of the invention.
  • the operably linked nucleic acids are contiguous.
  • the downstream nucleic acid can be ligated downstream of the root-specific promoter by a small amount of nucleotides while maintaining the reading frame, and is regulated.
  • isolated DNA refers to DNA that has been isolated from its natural environment (eg, the genome of a plant cell), or chemically synthesized or recombinantly expressed, and which is either free or substantially free of DNA. Other nucleic acids, cells and culture media from which the species are derived.
  • the isolated DNA of the present invention has a shorter nucleic acid sequence, preferably less than 2000 nucleotides in length, preferably less than 1500 nucleotides, more preferably less than 1200 nucleotides.
  • the isolated DNA of the invention is a SEQ of 1000 nucleotides ID NO: The sequence shown in 1.
  • the isolated DNA of the present invention further comprises a functional equivalent of the DNA having the sequence of SEQ ID NO: 1, ie having the SEQ ID DNA of a variant sequence of NO: 1, which is still capable of directing SEQ ID specific for transcription and/or expression of a nucleic acid operably linked downstream thereof at the plant root NO: sequence shown by 1.
  • Variant sequences include the ability to interact with SEQ ID under stringent conditions A DNA sequence of DNA hybridization of the sequence of NO:1.
  • stringent conditions are well known and include, for example, in the presence of 400 mM NaCl, 40 mM PIPES (pH 6.4), and 1 mM.
  • stringent conditions are well known and include, for example, in the presence of 400 mM NaCl, 40 mM PIPES (pH 6.4), and 1 mM.
  • the hybridization solution of EDTA was hybridized at 60 ° C for 12-16 hours, then washed with a wash solution containing 0.1 SDS, and 0.1% SSC at 65 °
  • the variant sequence also includes the SEQ ID A sequence of NO: 1 has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.
  • the percentage of sequence identity can be obtained by well-known bioinformatics algorithms, including Myers and Miller algorithm (Bioinformatics, 4(1): 11-17, 1988), and Needleman-Wunsch global alignment method (J. Mol. Biol., 48(3): 443-53, 1970), Smith-Waterman local alignment (J. Mol. Biol., 147: 195-197, 1981), Pearson and Lipman similarity search (PNAS, 85) (8): 2444-2448, 1988), Karlin and Altschul's algorithm (Altschul et al, J. Mol. Biol., 215(3): 403-410, 1990; PNAS, 90: 5873-5877, 1993). This is familiar to those skilled in the art.
  • the variant sequence also includes a fragment of the sequence set forth in SEQ ID NO: 1 capable of maintaining the function of the root-specific promoter, such as SEQ ID NO: A DNA sequence of at least 150, 200, 300, 500, 800, up to 999 contiguous nucleotides in 1.
  • the invention provides an expression cassette comprising:
  • the expression cassette of the present invention comprises the DNA of the first aspect of the invention, the nucleic acid operably linked downstream of the DNA of the first aspect of the invention, optionally the transcription and translation termination region, in the 5'-3' direction of transcription. (eg, transcription termination elements or polyadenylation signals).
  • the expression cassettes of the invention may also include an origin of replication required for replication in bacteria (eg, from pBR322 or P15A) The ORI region of ori), the element required for Agrobacterium T-DNA transfer (for example, the left and/or right border of T-DNA).
  • an enhancer examples include from CaMV 35S promoter, octopine synthase gene, rice actin I gene, corn alcohol dehydrogenase gene, maize dwarf I gene, TMV An enhancer element for the omega element and the promoter of the yeast.
  • Viral leader sequences can also be employed as a prodigy with enhancer utility, such as leader sequences from tobacco mosaic virus (TMV), maize chlorotic spot virus (MCMV), and alfalfa mosaic virus (AMV).
  • Exemplary plant introns include from Adh 1, bronze 1, actin 1, actin 2 intron, and sucrose synthase intron.
  • a nucleic acid encoding a nucleic acid downstream of the DNA of the first aspect of the invention encodes a heterologous protein, a polynucleotide encoding the fusion protein, an antisense sequence, a polynucleotide encoding a dsRNA sequence, and the like Wait.
  • a nucleic acid operably linked downstream of the DNA of the first aspect of the invention may encode a gene of a protein, such as an insect resistance gene, a bacterial disease resistance gene, a fungal disease resistance gene, a viral disease resistance gene, a nematode Disease resistance gene, herbicide resistance gene, gene affecting grain composition or quality, nutrient utilization gene, mycotoxin reduction gene, male sterility gene, selectable marker gene, selectable marker gene, negative selectable marker Gene, positive selectable marker gene, gene affecting plant agronomic characteristics (eg yield), environmental stress resistance gene (eg conferring resistance to drought, heat, cold, freezing, excessive humidity, salt stress or oxidative stress) Genes that are sexual or tolerant), genes that improve starch properties or quantity, oil quantity or quality, amino acid or protein composition, and the like.
  • a gene of a protein such as an insect resistance gene, a bacterial disease resistance gene, a fungal disease resistance gene, a viral disease resistance gene, a nematode Disease resistance gene, herb
  • nucleic acids operably linked downstream of the DNA of the first aspect of the invention may also encode dsRNA, antisense RNA, siRNA, miRNA, which are associated with unfavorable polynucleotides (eg, plant promoters that require downregulation, parasitic
  • unfavorable polynucleotides eg, plant promoters that require downregulation, parasitic
  • plant promoters that require downregulation, parasitic
  • the plant genes necessary for the survival, growth or reproduction of plant organisms are complementary and result in down-regulation of these polynucleotides.
  • the expression cassettes of the invention can be inserted into plasmids, cosmids, yeast artificial chromosomes, bacterial artificial chromosomes 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 provides a transgenic plant, plant seed, plant tissue or plant cell transformed with the expression cassette of the second aspect of the invention.
  • the root-specific promoter of the present invention It can be used to transform any plant species, including monocots and dicots, such as plants from the genus: genus, genus, genus Brassica, melon, Solanum, genus, cotton, apple, genus , snapdragon, populus, strawberry, Arabidopsis, spruce, capsicum, genus, chrysanthemum, petunia, pinus, pea, rice, maize, wheat, black Genus, rye, ryegrass, barley, soybean, cedar, galax, beet, sunflower, genus Nicotiana, genus Cucurbita, rose, strawberry, genus Genus, Trifolium, Hulu, Genus, Orange, Flax, Geranium, Cassava, Fenugreek
  • the present invention provides a method of producing a plant of the third aspect of the present invention, comprising:
  • the plants obtained in the step (4) are propagated to obtain progeny.
  • the transgenic plants of the invention are prepared using transformation methods known to those skilled in the art of plant biotechnology. Any method can be used to transform a recombinant expression vector into a plant cell to produce a transgenic plant of the invention. Transformation methods can include direct and indirect transformation methods. Suitable direct methods include polyethylene glycol-induced DNA uptake, liposome-mediated transformation, introduction using a gene gun, electroporation, and microinjection, and the like. In a particular embodiment of the invention, the invention uses 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, Volume 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, Volume 1, Engineering and Utilization, Academic Press, 1993, pp.
  • Agrobacterium strains eg, Agrobacterium tumefaciens or Agrobacterium rhizogenes
  • Agrobacterium tumefaciens or Agrobacterium rhizogenes comprise a plasmid (Ti or Ri plasmid) and a T-DNA element, which are transferred to plants after transfection with Agrobacterium, and the T-DNA is integrated Into the genome of plant cells.
  • the T-DNA can be located on the Ri-plasmid or Ti-plasmid, or independently in a so-called binary vector.
  • Agrobacterium-mediated transformation methods are described, for example.
  • Agrobacterium-mediated transformation is most suitable for dicotyledons, but is also suitable for monocots.
  • the transformation of Agrobacterium to plants is described, for example. Transformation can result in transient or stable transformation and expression.
  • the nucleotide sequences of the present invention can be inserted into any of the plants and plant cells that fall into these broad categories, they are particularly suitable for use in crop plant cells.
  • the present invention also provides a method of imparting a plant trait comprising
  • the plant obtained in the step (5) is propagated to obtain progeny
  • selection can be either field or greenhouse selection, or genetic selection using genetic markers.
  • proliferation can be sexually propagated or asexually propagated. In sexual reproduction, it is clear that additional hybridization steps can be performed and progeny that inherit the transgenic traits are selected.
  • 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 Huada Gene.
  • the PCR kit and the vector for the construction of the vector are purchased.
  • Zibao Bioengineering Co., Ltd. pEASY-T1 connection kit was purchased from Beijing Quanjin Biotechnology Co., Ltd.
  • T4 DNA ligase was purchased from Promega and the methods were performed according to the method recommended by the kit provider.
  • the vector pCAMBIA1303 used in the experiment was purchased from CAMBIA.
  • Primer 1 5'- CCCaagcttGCTATATGTGTACGTGATAGTATAT -3'
  • Primer 2 5'- CGggatccTTAATTTGCTCTTGTATTAGCTCTA-3'
  • sequence aagctt in primer 1 is the restriction site of HindIII
  • sequence ggatcc in primer 2 is the restriction site of BamHI.
  • the genomic DNA of rice (Zhonghua 11) extracted by the plant genomic DNA extraction kit (Tiangen Biotechnology (Beijing) Co., Ltd.) was used as The template is amplified and the reaction conditions are: Pre-denaturation at 95 ° C for 5 minutes; denaturation at 94 ° C for 30 seconds; Annealing at 55 ° C for 30 seconds; extending at 72 ° C for 1 minute; 30 cycles; extending at 72 ° C for 10 minutes.
  • the PCR product was detected by 1% agarose gel electrophoresis, and the product was ligated into the pEASY-T1 vector, and the positive clones were screened and verified by sequencing.
  • the results showed that the amplified sequence was our expected KT630P promoter sequence, and its sequence was SEQ. ID NO: 1 is shown.
  • the pEASY-T1 plasmid into which the KT630P promoter sequence has been inserted will be verified by sequencing using HindIII and BamHI Double digestion, connected to HindIII and BamHI
  • the colonies were picked for PCR detection, and the colonies with positive PCR results were selected for sequencing.
  • the corresponding positive clone plasmid was extracted and named as pHPG-KT630P.
  • the primers required for colony PCR detection are primers on the pHPG vector, which are located on both sides of the cloned promoter fragment, and the amplified fragment is about the length of the promoter.
  • the bacterial liquid is used as a template for amplification detection.
  • the PCR reaction conditions are: 95 Pre-denaturation for 5 minutes at °C; denaturation at 94 °C for 30 seconds; annealing at 55 °C for 30 seconds; extension at 72 °C for 1 minute; 34 cycles; extension at 72 °C for 10 minutes.
  • the map of the T-DNA region of the constructed expression vector is shown in Figure 1, wherein: LB and RB are the left and right borders of T-DNA, respectively; hpt indicates hygromycin resistance gene; Pnos indicates initiation of nos gene Tnos represents the terminator of the nos gene; GUS represents the gus protein ( ⁇ -glucuronidase) gene; T35S represents the terminator of the 35S gene; HindIII and BamHI indicates the restriction sites of HindIII and BamHI, respectively; the root-specific promoter is the KT630P promoter obtained in Example 1.
  • the plasmid pHPG-KT630P was transferred into Agrobacterium tumefaciens strain AGL0 by heat shock method, and the rice was co-transformed by Agrobacterium-mediated method, and the Arabidopsis thaliana plants were transformed by Agrobacterium inflorescence infection method.
  • the tissues and organs were isolated from the transgenic plants, and the GUS activity was detected.
  • the tissues and organs were placed in a centrifuge tube containing GUS staining buffer, incubated in a 37 ° C incubator overnight, and then destained in absolute ethanol at room temperature. save.
  • the rice callus with hygromycin resistance obtained by transforming the KT630P promoter was subjected to GUS staining experiment, and as shown in Fig. 2B, the visible blue color of the callus was not observed in the resistant callus, indicating that There is essentially no expression of the GUS gene in this tissue.
  • the tissues and organs of the transgenic rice transformed with the KT630P promoter namely, leaves, stems, roots and flowers, were subjected to GUS staining, respectively.
  • the results are shown in Fig. 2C, Fig. 2D, Fig. 2E and Fig. 2F, respectively.
  • the GUS gene is strongly expressed only in the roots of the transgenic rice seedlings, showing a strong blue color which can be clearly observed by the naked eye; No expression of the GUS gene was detected in all organs (leaves, stems, and flowers).
  • the whole transgenic rice seedlings were subjected to GUS staining. As a result, as shown in Fig.
  • the promoter of the present invention is capable of directing the expression of GUS protein downstream thereof in the root of the transgenic plant, and that the expression has root specificity; and, at the seedling stage or the flowering stage, the developmental stage exhibits such a plurality of development stages. Expression has root specificity.
  • KT630P transgenic rice plants were extracted, RNA was extracted, reverse transcribed into cDNA as a template, and the rice ACTIN gene was used as an internal reference to analyze the KT630P promoter-driven GUS reporter gene in transgenic rice. Express the situation.
  • the primers for RT-PCR detection are:
  • Primer 4 5'- CGGCGAAATTCCATACCTG -3'
  • primer 3 and primer 4 are detection primers of GUS gene, and the amplified fragment size is 321 bp; primer 5 and primer 6 are analytical primers for rice internal reference gene ACTIN, and the amplified fragment size is 252 bp.
  • the PCR detection system and procedures are:
  • PCR reaction conditions 95 ° C, pre-denaturation for 5 minutes; 94 ° C, denaturation for 30 seconds; 55 ° C, annealing for 30 seconds; 72 ° C, extension for 25 seconds; 28 cycles, 72 ° C, 10 minutes.
  • the KT630P promoter was transformed into Arabidopsis thaliana and the plant body at each development stage, and the whole tree was subjected to GUS staining. The results are shown in Figures 4A, 4B and 4C.
  • the reporter gene GUS showed strong expression only at the root of the plant, while the degree of staining in other tissues and organs was in the naked eye. Invisible level. This again shows that for a variety of plants, even heterologous plants, the promoters of the invention are capable of directing the expression of heterologous proteins downstream of the transgenic plant roots at various developmental stages, and that this expression has root specificity .
  • the root staining of KT630P transgenic Arabidopsis thaliana was observed by stereoscopic analysis. The results were shown in Figure 4D.
  • the GUS gene was only expressed in the central column sheath of the root system. This indicates that the promoter-directed expression of the present invention has root-stem sheath expression specificity.

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Abstract

一种分离的DNA，其具有SEQIDNO:1所示序列或类似序列，该DNA能够指导可操作地连接在其下游的核酸在植物根部特异转录和/或表达。还提供包含该DNA的表达盒和转基因植物。

Description

植物根特异表达启动子的鉴定和应用 发明领域
本发明属于植物转基因育种领域，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根部特异转录和/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
尽管许多植物（如，水稻，尤其是Oryza sativa.japonica等）的基因组序列已经被揭示（如可参见GenBank登录号AP003843.3、GenBank登录号AP004670.4等），但是大量基因组序列的功能仍旧未知。另外，美国专利申请US2006075523A1公开了来自水稻和其他植物的非生物性胁迫应答相关的多肽和多核苷酸，其中多核苷酸序列SEQ ID NO：15442长达2000个核苷酸，并且该序列淹没在该文献公开的众多序列中而未被指定特定功能；而美国专利US7365185B2公开了来自水稻和其他植物的基因组启动子序列，但是其中多核苷酸序列SEQ ID NO：68961长达2760个核苷酸，并且该序列淹没在该文献公开的众多序列中而未被指定特定功能，更不清楚其是否具有植物体表达部位的特异性。
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。
根是植物体吸收水分和营养物质的重要器官, 根特异表达系统可用于研究植物的高渗胁迫耐受、植物修复和根际分泌等。尽管中国专利申请CN1196753A、CN1791677A、CN101389762A和CN101589144A等文献公开了一系列根特异性启动子，但是这些启动子序列结构各异，无法预期得到新的根特异性启动子。
为此，本发明人经过长期研究，令人惊讶地发现了一种新的根特异性启动子，其不但相对于美国专利US7365185B2等文献公开的序列更短并且确认了具有根特异性启动子的功能，而且在双子叶和单子叶植物中的各个发育阶段能保持这一功能，极大地拓宽了其使用的范围。
发明概述
本发明提供了新的分离的DNA，其具有根特异性启动子的功能。另外，本发明还涉及包含该DNA的表达盒和转基因植物等，并涉及该DNA的应用等。
具体而言，在第一方面，本发明提供了分离的DNA，其能够指导可操作地连接在其下游的核酸在植物根部特异转录和/或表达，所述分离的DNA的序列选自下列组的序列：
（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）与（a）所述序列有至少90%（优选为至少95%）序列同一性的DNA序列；
（d）包含SEQ ID NO：1中至少150个（优选为至少200个）连续核苷酸的DNA序列；和
（e）与（a）-（d）之任一所述序列互补的DNA序列。
优选本发明第一方面的DNA，其序列长度小于2000个核苷酸，优选小于1500个核苷酸，更优选小于1200个核苷酸。
优选本发明第一方面的DNA，其能够指导可操作地连接在其下游的核酸在植物根部中柱鞘中特异转录和/或表达。
在本发明的一个具体实施方式中，本发明第一方面的DNA序列如SEQ ID NO：1所示。
在第二方面，本发明提供了表达盒，其包含：
（a）本发明第一方面的DNA；和
（b）核酸，其可操作地连接在本发明第一方面的DNA的下游。
优选本发明第二方面的表达盒，其中所述核酸能够赋予植物选自下列组的性状：
（a）所述核酸所编码的蛋白质产量的提高；
（b）所述核酸所编码的抑制性RNA含量的提高；
（c）抗逆性（包括抗旱性、耐寒性、耐高温性、和耐盐性）的提高；
（d）抗植物病虫害能力的提高，尤其是抗发生在植物根部病虫害能力的提高；
（e）营养品质的改善，包括植物中天然具有的营养成分含量的提高和植物非天然具有的营养成分的赋予；和
（f）植物产量的提高，尤其是植物经济性部位产量的提高。
在第三方面，本发明提供了用本发明第二方面的表达盒转化的转基因的植物、植物种子、植物组织或植物细胞，优选其中，本发明第一方面的DNA能够指导可操作地连接在其下游的核酸在植物根部特异转录和/或表达。
优选本发明第三方面的植物、植物种子、植物组织或植物细胞，其中所述植物是单子叶植物或双子叶植物，如，玉米、水稻、小麦、大麦、高粱、大豆、油菜、棉花、番茄、马铃薯、甘蔗、甜菜、烟草或拟南芥，优选是水稻。
在第四方面，本发明提供了本发明第三方面的植物的生产方法，其包括：
（1）构建本发明第二方面的表达盒；
（2）将步骤（1）获得的表达盒导入植物细胞；
（3）再生出转基因植物；和
（4）选择出转基因植物，其中本发明第一方面的DNA能够指导可操作地连接在其下游的核酸在植物根部特异转录和/或表达；并且
（5）任选地，增殖步骤（4）获得的植物以获得后代。
在第五方面，本发明还提供了赋予植物性状的方法，其包括
（1）选择能够赋予植物性状的核酸：
（2）使用步骤（1）获得的核酸构建本发明第二方面的表达盒；
（3）将步骤（2）获得的表达盒导入植物细胞；
（4）再生出转基因植物；和
（5）选择出赋予了植物性状的转基因植物；并且
（6）任选地，增殖步骤（5）获得的植物以获得后代，
其中，所述性状选自下列组：
（a）所述核酸所编码的蛋白质产量的提高；
（b）所述核酸所编码的抑制性RNA含量的提高；
（c）抗逆性（包括抗旱性、耐寒性、耐高温性、和耐盐性）的提高；
（d）抗植物病虫害能力的提高，尤其是抗发生在植物根部病虫害能力的提高；
（e）营养品质的改善，包括植物中天然具有的营养成分含量的提高和植物非天然具有的营养成分的赋予；和
（f）植物产量的提高，尤其是植物经济性部位产量的提高。
附图说明
图1是表达载体pHPG的T-DNA区图谱。LB和RB分别为T-DNA的左边界和右边界；hpt表示潮霉素抗性基因；Pnos表示nos基因的启动子；Tnos表示nos基因的终止子；GUS表示gus蛋白（β-葡糖醛酸酶（β-glucuronidase））基因；T35s表示35s基因的终止子；HindIII和 BamHI分别表示限制性内切酶HindIII和 BamHI的酶切位点；根特异启动子即为本发明的分离的根特异表达启动子。
图2是KT630P转基因水稻的组织、器官的GUS染色。其中，A为水稻幼苗期植株体；B为转基因抗性愈伤组织；C为苗期叶片；D为苗期的茎；E为苗期的根；F为水稻花器官。
图3是本发明KT630P启动子驱动的GUS基因在转基因水稻各组织器官中的表达的RT-PCR检测结果。
图4是KT630P转基因拟南芥植株的GUS染色。其中，A为拟南芥幼苗期植株体；B为拟南芥抽苔前植株体；C为拟南芥开花期植株体；D为体式镜观察的根部GUS染色情况。
发明详述
在第一方面，本发明提供了分离的DNA，其能够指导可操作地连接在其下游的核酸在植物根部特异转录和/或表达，尤其是其能够指导可操作地连接在其下游的核酸在植物根部中柱鞘中特异转录和/或表达。
在本文中，这种分离的DNA也可以被称为根特异性启动子，属于顺式作用元件。在根特异性启动子的驱动下，其下游并与其可操作地连接的基因的表达往往只限于根这一器官或其组织部位，并表现发育调节等特性。根特异性启动子不仅能使目的基因的表达产物主要在植物根部或其组织部位积累，例如在根部或其组织中产生的下游的核酸mRNA的量是其他植物体部位的5倍、10倍、100倍、200倍、甚至1000倍，增加区域表达量，同时也可以避免植物营养的不必要浪费。在本发明的具体实施方式中，通过肉眼可观测的染色法直接判别本发明的根特异性启动子在植物根部指导转录和/或表达，而且该调控持续在植物发育的各个阶段，而在植物的其他部位，没有肉眼可明显分辨的转录和/或表达。
在本文中，术语“可操作地连接”指的是一种连接方式，该方式使得连接在下游的核酸的转录和/或表达由本发明的根特异性启动子启始并调控。通常，可操作地连接的核酸是连续的。下游的核酸在保持读框的情况下可以间隔少量核苷酸而连接在根特异性启动子的下游，接受其调控。
在本文中，术语“分离的”DNA指的是DNA从其天然的环境（如，植物细胞的基因组）中被分离出来了，或者被化学合成或重组表达出来，并且DNA不含或者基本不含其来源物种的其他核酸、细胞和培养基。本发明的分离的DNA具有较短的核酸序列，优选序列长度小于2000个核苷酸，优选小于1500个核苷酸，更优选小于1200个核苷酸。在本发明的具体实施方式中，本发明的分离的DNA是具有1000个核苷酸的SEQ ID NO：1所示的序列。
本发明的分离的DNA还包括具有SEQ ID NO：1所示的序列的DNA的功能等价体，即具有SEQ ID NO：1的变体序列的DNA，其仍旧能够指导可操作地连接在其下游的核酸在植物根部特异转录和/或表达的SEQ ID NO：1所示的序列。变体序列包括在严格条件下能够与具有SEQ ID NO：1所示的序列的DNA杂交的DNA序列。本文中使用的“严格条件”是公知的，包括诸如在含400mM NaCl、40mM PIPES（pH6.4）和1mM EDTA的杂交液中于60℃杂交12-16小时，然后在65℃下用含0.1SDS、和0.1%SSC的洗涤液洗涤15-60分钟。
变体序列还包括与SEQ ID NO：1所示序列有至少90%、95%、96%、97%、98%、或99%序列同一性的DNA序列。其中，序列同一性的百分比可以通过公知的生物信息学算法来获得，包括Myers和Miller算法(Bioinformatics，4(1)：11-17，1988)、Needleman-Wunsch全局比对法(J.Mol.Biol.，48(3)：443-53，1970)、Smith-Waterman局部比对法(J.Mol.Biol.，147：195-197，1981)、Pearson和Lipman相似性搜索法(PNAS，85(8)：2444-2448，1988)、Karlin和Altschul的算法(Altschul等，J.Mol.Biol.，215(3)：403-410，1990；PNAS，90：5873-5877，1993)。这对于本领域技术人员来说是熟悉的。
变体序列还包括能够保持根特异性启动子功能的SEQ ID NO：1所示的序列的片段，例如SEQ ID NO：1中至少150个、200个、300个、500个、800个、直至999个连续核苷酸的DNA序列。
在第二方面，本发明提供了表达盒，其包含：
（a）本发明第一方面的DNA；和
（b）核酸，其可操作地连接在本发明第一方面的DNA的下游。
本发明的表达盒在5’-3’的转录方向上包含本发明第一方面的DNA、可操作地连接在本发明第一方面的DNA的下游的核酸、任选的转录和翻译的终止区（如，转录终止元件或多聚腺苷酸化信号）。本发明的表达盒还可以包括，在细菌中复制所需的复制起点（例如，来自pBR322或P15A ori的ORI区），土壤杆菌T-DNA转移所需要的元件（例如，T-DNA的左边界和/或右边界）。本发明表达盒可以包含的其他成分包括，增强子、内含子、多克隆位点、操纵基因、阻遏物结合位点、转录因子结合位点等。示例性的增强子包括来自CaMV 35S启动子、章鱼碱合酶基因、稻肌动蛋白I基因、玉米醇脱氢酶基因、玉米矮缩I基因、TMV Ω元件和酵母的启动子的增强子元件。也可以采用病毒前导序列来作为具有增强子效用的远见，如来自烟草花叶病毒(TMV)、玉米褪绿斑点病毒(MCMV)和苜蓿花叶病毒(AMV)的前导序列等。示例性的植物内含子包括来自Adh 1、bronze 1、肌动蛋白1、肌动蛋白2的内含子、以及蔗糖合酶内含子。
在本文中，可操作地连接在本发明第一方面的DNA的下游的核酸编码异元蛋白的多核苷酸、编码融合蛋白的多核苷酸、反义序列、编码dsRNA序列的多核苷酸，等等。优选是能够赋予植物（尤其是植物根部）有益的性状的核酸，所述性状包括：
（a）所述核酸所编码的蛋白质产量的提高；
（b）所述核酸所编码的抑制性RNA含量的提高；
（c）抗逆性（包括抗旱性、耐寒性、耐高温性、和耐盐性）的提高；
（d）抗植物病虫害能力的提高，尤其是抗发生在植物根部病虫害能力的提高；
（e）营养品质的改善，包括植物中天然具有的营养成分含量的提高和植物非天然具有的营养成分的赋予；和
（f）植物产量的提高，尤其是植物经济性部位产量的提高。
相应地，可操作地连接在本发明第一方面的DNA的下游的核酸可以编码蛋白的基因，如昆虫抗性基因、细菌疾病抗性基因、真菌疾病抗性基因、病毒疾病抗性基因、线虫疾病抗性基因、除草剂抗性基因、影响谷粒组成或品质的基因、养分利用基因、真菌毒素减少基因、雄性不育基因、可选择标记物基因、可筛选标记物基因、阴性可选择标记物基因、阳性可选择标记物基因、影响植物农学特征(如产率)的基因、环境胁迫抗性基因(例如，赋予对干旱、热、寒冷、冷冻、过湿、盐胁迫或氧化胁迫的抗性或耐性的基因)、改善淀粉特性或数量、油品数量或品质、氨基酸或蛋白质组成的基因，等等。另外，可操作地连接在本发明第一方面的DNA的下游的核酸也可以编码dsRNA、反义RNA、siRNA、miRNA，其与不利的多核苷酸（如，需要下调表达的植物启动子，寄生植物体生物存活、生长或繁殖所必须的植物基因）互补并导致这些多核苷酸的下调。
本发明的表达盒可被插入质粒、粘粒、酵母人工染色体、细菌人工染色体或其他适合转化进宿主细胞中的任何载体中。优选的宿主细胞是细菌细胞，尤其是用于克隆或储存多核苷酸、或用于转化植物细胞的细菌细胞，例如大肠杆菌、根瘤土壤杆菌和毛根土壤杆菌。当宿主细胞是植物细胞时，表达盒或载体可被插入被转化的植物细胞的基因组中。插入可以是定位的或随机的插入。优选地，插入通过诸如同源重组来实现。另外，表达盒或载体可保持在染色体外。本发明的表达盒或载体可存在于植物细胞的核、叶绿体、线粒体和/或质体中。优选地，本发明的表达盒或载体被插入植物细胞核的染色体DNA中。
在第三方面，本发明提供了用本发明第二方面的表达盒转化的转基因的植物、植物种子、植物组织或植物细胞。
本发明的具体实施方式已经通过在两个有代表性的单子叶植物和双子叶植物中的有效应用证明了本发明的根特异性启动子的植物使用范围，因此本发明的根特异性启动子可用于转化任何植物物种，包括单子叶植物和双子叶植物，例如来自以下属的植物：苜蓿属、番茄属、芸苔属、香瓜属、茄属、核桃属、棉属、苹果属、葡萄属、金鱼草属、杨属、草莓属、拟南芥属、云杉属、辣椒属、藜属、菊属、牵牛属、松属、豌豆属、稻属、玉蜀黍属、小麦属、小黑麦属、黑麦属、黑麦草属、大麦属、大豆属、黄杉属、伽蓝菜属、甜菜属、向日葵属、烟草属、南瓜属、蔷薇属、草莓属、百脉根属、驴食草属、车轴草属、胡卢巴属、豇豆属、橘属、亚麻属、老鹳草属、木薯属、胡芦巴属、萝卜属、白芥属、颠茄属、曼陀罗属、天仙子属、烟草属、碧冬茄属、毛地黄属、菊苣属、莴苣属、雀麦属、天门冬属、金鱼草属、萱草属、水仙属、天竺葵属、稷属、狼尾草属、毛莨属、千里光属、喇叭舌属、蓝英花属、菜豆属、燕麦属和葱属，优选的植物包括玉米、水稻、小麦、大麦、高粱、大豆、油菜、棉花、番茄、马铃薯、甘蔗、甜菜、烟草或拟南芥，更优选是水稻。
在第四方面，本发明提供了本发明第三方面的植物的生产方法，其包括：
（1）构建本发明第二方面的表达盒；
（2）将步骤（1）获得的表达盒导入植物细胞；
（3）再生出转基因植物；和
（4）选择出转基因植物，其中本发明第一方面的DNA能够指导可操作地连接在其下游的核酸在植物根部特异转录和/或表达；并且
（5）任选地，增殖步骤（4）获得的植物以获得后代。
本发明的转基因植物使用植物生物技术领域技术人员已知的转化方法制备。任何方法可被用于将重组表达载体转化进植物细胞中，以产生本发明的转基因植物。转化方法可包括直接和间接的转化方法。合适的直接方法包括聚乙二醇诱导的DNA摄入、脂质体介导的转化、使用基因枪导入、电穿孔、以及显微注射，等。在本发明的具体实施方式中，本发明使用了基于土壤杆菌的转化技术（可参见Horsch RB等(1985)Science 225：1229；White FF，Vectors for Gene Transfer in Higher Plants，Transgenic Plants，第1卷，Engineering and Utilization， Academic Press，1993，pp.15-38；Jenes B等.Techniques for Gene Transfer，Transgenic Plants，第1卷，Engineering and Utilization， Academic Press，1993，pp.128-143，等）。土壤杆菌菌株(例如根瘤土壤杆菌或毛根土壤杆菌)包含质粒(Ti或Ri质粒)和T-DNA元件，所述质粒和元件在用土壤杆菌转染后被转移至植物，而T-DNA被整合进植物细胞的基因组中。T-DNA可位于Ri-质粒或Ti-质粒上，或独立地包含在所谓的双元载体中。土壤杆菌介导的转化方法描述于例如中。土壤杆菌介导的转化最适合双子叶植物，但是也适合单子叶植物。土壤杆菌对植物的转化描述于例如中。转化可导致瞬时或稳定的转化和表达。尽管本发明的核苷酸序列可被插入落入这些广泛种类中的任何植物和植物细胞中，但是其尤其适用于作物植物细胞。
在第五方面，本发明还提供了赋予植物性状的方法，其包括
（1）选择能够赋予植物性状的核酸：
（2）使用步骤（1）获得的核酸构建本发明第二方面的表达盒；
（3）将步骤（2）获得的表达盒导入植物细胞；
（4）再生出转基因植物；和
（5）选择出赋予了植物性状的转基因植物；并且
（6）任选地，增殖步骤（5）获得的植物以获得后代，
其中，所述性状选自下列组：
（a）所述核酸所编码的蛋白质产量的提高；
（b）所述核酸所编码的抑制性RNA含量的提高；
（c）抗逆性（包括抗旱性、耐寒性、耐高温性、和耐盐性）的提高；
（d）抗植物病虫害能力的提高，尤其是抗发生在植物根部病虫害能力的提高；
（e）营养品质的改善，包括植物中天然具有的营养成分含量的提高和植物非天然具有的营养成分的赋予；和
（f）植物产量的提高，尤其是植物经济性部位产量的提高。
在本文中，“选择”可以是田间或温室选择，也可以是使用遗传标记的遗传选择。在本文中，“增殖”可以有性繁殖，也可以是无性繁殖。在有性繁殖中，显然可以进行额外的杂交步骤，并选择出继承了转基因性状的后代。
本发明引用了公开文献，这些文献是为了更清楚地描述本发明，它们的全文内容均纳入本文进行参考，就好像它们的全文已经在本文中重复叙述过一样。
为了便于理解，以下将通过具体的实施例对本发明进行详细地描述。需要特别指出的是，这些描述仅仅是示例性的描述，并不构成对本发明范围的限制。依据本说明书的论述，本发明的许多变化、改变对所属领域技术人员来说都是显而易见的了。
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，测序由北京华大基因完成，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Promega公司，方法均参照试剂盒提供商推荐的方法进行。实验中所用的载体pCAMBIA1303可购自于CAMBIA公司。
实施例1．启动子KT630P的分离和鉴定
设计如下克隆启动子KT630P所需引物：
引物1：5'- CCCaagcttGCTATATGTGTACGTGATAGTATAT -3'
引物2：5'- CGggatccTTAATTTGCTCTTGTATTAGCTCTA-3'
引物1中序列aagctt为HindIII的酶切位点,引物2中序列ggatcc为BamHI的酶切位点。
利用启动子的正反向引物（其中带下划线部分的序列为启动子序列），以植物基因组DNA提取试剂盒（天根生化科技（北京）有限公司）提取的水稻(中花11)基因组DNA作为模板，进行扩增，反应条件是： 95℃预变性5分钟；94℃变性30秒； 55℃退火30秒；72℃延伸1分钟；30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载体，筛选阳性克隆并进行测序验证。结果表明，所扩增序列为我们预期的KT630P启动子序列，其序列如SEQ ID NO：1所示。
实施例2．表达载体的构建
将测序验证已经插入KT630P启动子序列的pEASY-T1质粒用HindIII和 BamHI 双酶切，连入同样用HindIII和 BamHI 双酶切的载体pHPG中，挑取菌落进行PCR检测，选取PCR结果为阳性的菌落进行测序，测序验证正确后，提取相应阳性克隆质粒，命名为pHPG-KT630P。其中，菌落PCR检测所需引物为pHPG载体上引物，位于所克隆的启动子片段两侧，扩增片段约为启动子的长度，以菌液为模板，扩增检测，PCR反应条件为：95℃预变性5分钟；94℃变性30秒；55℃退火30秒；72℃延伸1分钟；34个循环；72℃延伸10分钟。
所构建的表达载体的T-DNA区的图谱如图1所示，其中：LB和RB分别为T-DNA的左边界和右边界；hpt表示潮霉素抗性基因；Pnos表示nos基因的启动子；Tnos表示nos基因的终止子；GUS表示gus蛋白（β-葡糖醛酸酶（β-glucuronidase））基因；T35S表示35S基因的终止子；HindIII和 BamHI分别表示内切HindIII和 BamHI的酶切位点；根特异启动子即为实施例1获得的KT630P启动子。
实施例3．植物转化和功能鉴定
利用热激法将质粒pHPG-KT630P转入农杆菌AGL0菌株，利用农杆菌介导法对水稻进行共转化，同时利用农杆菌花序侵染法转化拟南芥植株。从转基因植株中分离各组织器官，进行GUS活性检测，将各组织器官置于含有GUS染色缓冲液的离心管中，放于37℃温箱温育过夜，然后室温条件下在无水乙醇中脱色保存。
3.1 转基因水稻苗的组织器官染色
将转化KT630P启动子得到的具有潮霉素抗性的水稻愈伤组织，进行GUS染色实验，结果如图2B所示，抗性愈伤组织中没有显示出肉眼可观测到的蓝色，表明在该组织中基本没有GUS基因的表达。
将转化KT630P启动子的转基因水稻的组织器官，即叶片、茎、根和花分别进行GUS染色。结果分别如图2C、图2D、图2E和图2F所示，GUS基因只在转基因水稻苗的根系中有很强的表达，显现出肉眼可明显观测到的很强的蓝色；而在其它各器官（叶片、茎、和花）中，基本没有检测到GUS基因的表达。另外，取完整的转基因水稻苗进行GUS染色，结果如图2A所示，通过明显的颜色对比可以发现，只有根部显示出肉眼可明显观测到的很强的蓝色，而其他器官部位都没有肉眼可识别的明显蓝色。这表明，本发明的启动子能够在转基因植株根中指导其下游的GUS蛋白表达，而且这种表达具有根部的特异性；而且，无论在苗期还是花期，多个发育阶段均表现出这种表达具有根部的特异性。
3.2 转基因水稻的RT-PCR表达分析
取KT630P转基因水稻植株纯系的根、茎、叶和花器官，提取RNA，反转录为cDNA作为模板，以水稻ACTIN基因作为内参照，分析KT630P启动子驱动的GUS报告基因在转基因水稻中的表达情况。
RT-PCR的检测引物是：
引物3：5'- TAATGTTCTGCGACGCTCAC -3'
引物4：5'- CGGCGAAATTCCATACCTG -3'
引物5：5'- TGTTCCTGCCATGTATGT -3'
引物6：5'- ATGTCCCTCACAATTTCC -3'
其中，引物3和引物4是GUS基因的检测引物，其扩增片段大小为321bp；引物5和引物6是水稻内参基因ACTIN的分析引物，其扩增片段大小为252bp。PCR检测体系和程序是：
10×buffer 2μL
10mM dNTP 0.4μL ?
10 mM引物3（对照分析使用10 mM引物5） 0.4μL ?
10 mM引物4（对照分析使用10 mM引物6） 0.4μL ?
Taq polymerase 0.4μL ?
cDNA 1μL ?
ddH2O 15.4μL ?
PCR反应条件：95℃，预变性5分钟；94℃，变性30秒；55℃，退火30秒；72℃，延伸25秒；28个循环，72℃，10分钟。
反应结束后，对PCR产物进行1.5%的琼脂糖凝胶电泳检测。PCR检测结果如图3所示，对照分析中所有ACTIN内参照的表达在根、茎、叶和花等器官中都被检测出来了，没有明显差异；而对于GUS基因的表达，在根、茎、叶和花等器官中，只在根组织中检测到了GUS基因的表达。这再一次表明，本发明的启动子能够在转基因植株根中指导其下游的异源蛋白表达，而且这种表达具有根部的特异性；而且，无论在苗期还是花期，多个发育阶段均表现出这种表达具有根部的特异性。
3.3 转基因拟南芥的组织器官染色
将KT630P启动子转化拟南芥所得的阳性苗及其各发育阶段的植株体，整棵进行GUS染色。结果如图4A、图4B和图4C所示，在转基因拟南芥的各个发育阶段，报告基因GUS都只在植物根部表现出很强的表达，而在其它组织器官中的染色程度均处于肉眼不可见水平。这再一次表明，对于多种植物，即使是异源植物，本发明的启动子也能够在各个发育阶段的转基因植株根中指导其下游的异源蛋白表达，而且这种表达具有根部的特异性。
用体式镜观察KT630P转基因拟南芥的根系染色情况，发现结果如图4D所示，GUS基因只在根系的中柱鞘中表达。这表明，本发明的启动子指导的表达具有根中柱鞘表达特异性。

Claims (1)

1.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根部特异转录和/或表达，所述分离的DNA的序列选自下列组的序列：
（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）与（a）所述序列有至少90%（优选为至少95%）序列同一性的DNA序列；
（d）包含SEQ ID NO：1中至少150个（优选为至少200个）连续核苷酸的DNA序列；和
（e）与（a）-（d）之任一所述序列互补的DNA序列。
2.权利要求1所述的DNA，其序列长度小于2000个核苷酸，优选小于1500个核苷酸，更优选小于1200个核苷酸。
3. 权利要求1或2所述的DNA，其能够指导可操作地连接在其下游的核酸在植物根部中柱鞘中特异转录和/或表达。
4. 权利要求1-3之任一所述的DNA，其序列如SEQ ID NO：1所示。
5.表达盒，其包含：
（a）权利要求1-4之任一所述的DNA；和
（b）核酸，其可操作地连接在权利要求1-4之任一所述的DNA的下游。
6.权利要求5所述的表达盒，其中所述核酸能够赋予植物选自下列组的性状：
（a）所述核酸所编码的蛋白质产量的提高；
（b）所述核酸所编码的抑制性RNA含量的提高；
（c）抗逆性（包括抗旱性、耐寒性、耐高温性、和耐盐性）的提高；
（d）抗植物病虫害能力的提高，尤其是抗发生在植物根部病虫害能力的提高；
（e）营养品质的改善，包括植物中天然具有的营养成分含量的提高和植物非天然具有的营养成分的赋予；和
（f）植物产量的提高，尤其是植物经济性部位产量的提高。
7.用权利要求5或6所述的表达盒转化的转基因的植物、植物种子、植物组织或植物细胞，其中，权利要求1-4之任一所述的DNA能够指导可操作地连接在其下游的核酸在植物根部特异转录和/或表达。
8.权利要求7所述的植物、植物种子、植物组织或植物细胞，其中所述植物是单子叶植物或双子叶植物，如，玉米、水稻、小麦、大麦、高粱、大豆、油菜、棉花、番茄、马铃薯、甘蔗、甜菜、烟草或拟南芥，优选是水稻。
9. 权利要求7或8所述的植物的生产方法，其包括：
（1）构建权利要求5或6所述的表达盒；
（2）将步骤（1）获得的表达盒导入植物细胞；
（3）再生出转基因植物；和
（4）选择出转基因植物，其中权利要求1-4之任一所述的DNA能够指导可操作地连接在其下游的核酸在植物根部特异转录和/或表达；并且
（5）任选地，增殖步骤（4）获得的植物以获得后代。
10. 赋予植物性状的方法，其包括
（1）选择能够赋予植物性状的核酸：
（2）使用步骤（1）获得的核酸构建权利要求5或6所述的表达盒；
（3）将步骤（2）获得的表达盒导入植物细胞；
（4）再生出转基因植物；和
（5）选择出赋予了植物性状的转基因植物；并且
（6）任选地，增殖步骤（5）获得的植物以获得后代，
其中，所述性状选自下列组：
（a）所述核酸所编码的蛋白质产量的提高；
（b）所述核酸所编码的抑制性RNA含量的提高；
（c）抗逆性（包括抗旱性、耐寒性、耐高温性、和耐盐性）的提高；
（d）抗植物病虫害能力的提高，尤其是抗发生在植物根部病虫害能力的提高；
（e）营养品质的改善，包括植物中天然具有的营养成分含量的提高和植物非天然具有的营养成分的赋予；和
（f）植物产量的提高，尤其是植物经济性部位产量的提高。
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