WO2009052687A1 - Gènes améliorant la tolérance au sel et à la sécheresse d'une plante et leurs utilisations - Google Patents

Gènes améliorant la tolérance au sel et à la sécheresse d'une plante et leurs utilisations Download PDF

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WO2009052687A1
WO2009052687A1 PCT/CN2007/071043 CN2007071043W WO2009052687A1 WO 2009052687 A1 WO2009052687 A1 WO 2009052687A1 CN 2007071043 W CN2007071043 W CN 2007071043W WO 2009052687 A1 WO2009052687 A1 WO 2009052687A1
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seq
plant
sequence
dna
drought
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PCT/CN2007/071043
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English (en)
French (fr)
Inventor
Min Lin
Ming Chen
Jin Wang
Jie Pan
Zhengfu Zhou
Wei Zhang
Wei Lu
Shuzhen Ping
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Biotechnology Research Institute, The Chinese Academy Of Agricultural Sciences
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Priority to US12/739,168 priority Critical patent/US8153861B2/en
Publication of WO2009052687A1 publication Critical patent/WO2009052687A1/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)
    • C12N15/8241—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
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits

Definitions

  • the present invention relates to a nucleotide coding sequence and another nucleotide coding sequence artificially synthesized by a plant preference codon, and the present invention also relates to the improvement of plant resistance to salt and drought stress by the nucleotide coding sequence.
  • Deinococcus radiodurans is one of the highest radiation-resistant organisms known to date.
  • the strain has extreme resistance to lethal doses of ionizing radiation, UV radiation, and DNA damage agents, and can completely restore the genome of hundreds of DNA double-strand breaks caused by ionizing radiation, without the ability to mutate.
  • Its extreme radiation tolerance and its DNA loss repair molecular mechanism have aroused great interest in the scientific community. It is not only important for exploring the basic disciplines of DNA repair molecular mechanisms, but also promoting the development of new DNA technologies and environmental protection. There are great potential applications for bioremediation, human health, biotechnology, and even the development and utilization of extraterrestrial space.
  • the 1999 Genetic Research Institute (TIGR) completed and published the complete sequence of the D. radiodurans genome (White 1999).
  • the object of the present invention is to discover and artificially synthesize a DNA sequence which enhances salt tolerance and drought resistance of a plant, and transfects the sequence into a plant to cultivate a salt-tolerant and drought-tolerant transgenic plant.
  • the present inventors have found that DNA sequences as shown in SEQ ID ⁇ :1 and SEQ ID NO: 2 have functions of improving plant salt tolerance and drought resistance.
  • the sequence SEQ ID NO: 1 is derived from a stretch of D. radiodurans;
  • SEQ ID NO: 2 is a sequence derived from SEQ ID ⁇ : 1, and artificially synthesized by plant preference codon.
  • the present invention also provides a recombinant vector comprising the DNA of SEQ ID NO: 1 or the DNA of SEQ ID NO: 2.
  • the present invention transforms host cells with the above recombinant vectors, including prokaryotic cells, including eukaryotic cells.
  • prokaryotic host cells include JM109
  • commonly used eukaryotic host cells include yeast cells and other plant cells.
  • the host cells are E. coli JM109 and tobacco.
  • a method of producing a polypeptide having SEQ ID NO: 1 or SEQ ID NO: 2 protein activity the steps of which are as follows:
  • step (1) (2) transferring the expression vector in step (1) into a host cell to form a recombinant cell;
  • step (3) cultivating the recombinant cell in step (2) under conditions suitable for expression of the protein polypeptide of SEQ ID NO: 1 or SEQ ID NO: 2;
  • a substantially pure polypeptide having the activity of SEQ ID NO: 1 or SEQ ID NO: 2 is isolated, the sequence of which is SEQ ID NO: 3.
  • the invention also provides a method for transforming SEQ ID ⁇ :1 or SEQ ID NO:2 into a plant by using transgenic technology.
  • the method of the object to increase the resistance of the plant to salt and drought stress, the steps are as follows:
  • step (1) (2) transferring the expression vector in step (1) to a plant cell;
  • Transformed cells are obtained by screening and the transgenic plants and their progeny are finally regenerated, including plant seeds and plant tissues.
  • operably linked means that certain portions of the linear DNA sequence are capable of affecting the activity of other portions of the same linear DNA sequence.
  • the signal peptide DNA is expressed as a precursor and is involved in the secretion of the polypeptide, then the signal peptide (secretion leader sequence) DNA is operably linked to the polypeptide DNA; if the promoter controls the transcription of the sequence, then it is operably linked to A coding sequence; if the ribosome binding site is placed at a position that enables translation, then it is operably linked to the coding sequence.
  • “operably linked to” means adjacent, and for secretory leader sequences means adjacent in the reading frame.
  • the expression vector in the step (1) is transferred to Agrobacterium, and the Agrobacterium containing the expression vector is co-cultured with the eukaryotic host cell, and dark culture is carried out at 22-28 ° C. After 2 days, transformed cells containing the SEQ ID NO: 1 or SEQ ID NO: 2 gene were obtained by screening, such as antibiotic screening, and finally the transgenic plants and their progeny, including plant seeds and plant tissues, were regenerated.
  • the above vectors may be selected from various carriers known in the art, such as commercially available vectors, including plasmids, cosmids and the like.
  • the present invention provides a nucleic acid molecule which can be used as a probe, which molecule usually has 8-100 contiguous nucleotides of the nucleotide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 2, preferably The ground has 15-50 contiguous nucleotides.
  • the probe can be used to detect the presence or absence of a nucleic acid molecule encoding SEQ ID NO: 1 or SEQ ID NO: 2 in the sample.
  • the present invention also provides a method of detecting the presence of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 in a sample, which comprises hybridizing the sample with the probe described above, and then detecting whether the probe has bound.
  • the sample is a product after PCR amplification, wherein the PCR amplification primer corresponds to the nucleotide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 2 and may be located on either or both sides of the coding sequence.
  • Primers are typically 15-50 nucleotides in length.
  • SEQ ID NO: 1 or SEQ ID NO: 2 refers to a nucleotide sequence encoding a polypeptide having the activity of SEQ ID NO: 1 or SEQ ID NO: 2, and a degenerate sequence thereof.
  • the degenerate sequence refers to a sequence resulting from the substitution of one or more codons in the sequence by degenerate codons encoding the same amino acid. Due to the degeneracy of the codon, a degenerate sequence having a homology of less than about 89% with SEQ ID NO: 1 or SEQ ID NO: 2 can also encode the sequence set forth in SEQ ID NO: 2.
  • the term also encompasses nucleotide sequences which hybridize under moderately stringent conditions, preferably under high stringency conditions, to the nucleotide sequence of SEQ ID ⁇ :1.
  • the term also encompasses nucleotide sequences having at least 89%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% homology to the nucleotide sequence of SEQ ID ⁇ :1.
  • the term also encompasses variant forms of the open reading frame sequence of SEQ ID NO: 1 which encodes a protein having the same function as the native SEQ ID NO: 1 or SEQ ID NO: 2.
  • variants include, but are not limited to, a number of nucleotide deletions (usually 1-90, preferably 1-60, more preferably 1-20, optimally 1-10) , inserts and/or substitutions, and adding a few at the 5' and/or 3' end (usually within 60, preferably 30) Within one, more preferably within 10, optimally within 5 nucleotides.
  • a "substantially pure" protein or polypeptide means that it constitutes at least 20%, preferably at least 50%, more preferably at least 80%, and most preferably at least 90% (percent of the above) of the total material of the sample. Both refer to dry weight or wet weight). Purity can be measured by any suitable method, such as column chromatography, PAGE or HPLC to determine the purity of the polypeptide. A substantially pure polypeptide is substantially free of components that accompany it in its natural state.
  • the protein or polypeptide of SEQ ID NO: 3 refers to a protein-active polypeptide encoded by SEQ ID NO: 1.
  • the term also encompasses variant forms having the same function as day SEQ ID NO:3. These variants include, but are not limited to, a number (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) amino acid deletions, insertions and/or Substituting, and adding one or several (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminus and/or the N-terminus.
  • the function of the protein is usually not altered.
  • the addition of one or several amino acids at the C-terminus and/or N-terminus generally does not alter the function of the protein.
  • the term also encompasses active fragments and active derivatives of the protein of SEQ ID NO: 3.
  • Variant forms of the SEQ ID NO: 2 polypeptide of the invention include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, and SEQ ID NO: under high or low stringency conditions: Or a protein encoded by the DNA of the hybrid of SEQ ID NO: 2, and a polypeptide or protein obtained by using the antiserum of the polypeptide of SEQ ID NO: 3.
  • the invention also provides other polypeptides, such as fusion proteins comprising the polypeptide of SEQ ID NO: 3 or a fragment thereof. In addition to the nearly full length polypeptide, the invention also includes soluble fragments of the polypeptide of SEQ ID NO: 3.
  • the fragment has at least about 10 contiguous amino acids of the polypeptide sequence of SEQ ID NO: 3, typically at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, optimal. At least about 100 consecutive amino acids.
  • SEQ ID NO: 3 conservative variant polypeptide means having up to 10, preferably up to 8, more preferably up to 5 amino acids in nature compared to the amino acid sequence of SEQ ID NO:3. A similar or similar amino acid is replaced to form a polypeptide.
  • These conservative variant polypeptides are preferably produced by substitution according to Table 1.
  • the invention also includes analogs of the SEQ ID NO: 3 protein or polypeptide.
  • the difference between these analogs and the native SEQ ID NO: 3 polypeptide may be a difference in amino acid sequence, or may be a difference in the modified form that does not affect the sequence, or a combination thereof.
  • These polypeptides include natural or induced genetic variants. Induced variants can be obtained by a variety of techniques, such as random mutagenesis by irradiation or exposure to a mutagen, or by site-directed mutagenesis or other techniques known to molecular biology.
  • Analogs also include analogs having residues other than the native L-amino acid (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, ⁇ -amino acids). It is to be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.
  • Modifications include: chemically derivatized forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those produced by glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylation enzyme or a deglycosylation enzyme. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to increase their resistance to proteolytic properties or to optimize solubility properties.
  • chemically derivatized forms of the polypeptide such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those produced by glycosylation modifications in the synthesis and processing of the polypeptid
  • the expression of the SEQ ID NO: 1 or SEQ ID NO: 2 gene product can also be analyzed by Northern blotting, i.e., the presence or absence and amount of the RNA transcript of SEQ ID NO: 1 or SEQ ID NO: 2 in the cell.
  • SEQ ID NO: 1 or SEQ ID NO: 2 RNA and Western blot analysis of SEQ ID NO: 3 specific antibodies can be used in combination to confirm the presence of SEQ ID NO: 1 or SEQ ID NO: 2 in a biological sample. expression.
  • SEQ ID NO: 1 or SEQ ID NO: 2 homologous gene or homologous protein can be screened based on nucleic acid homology or homology of the expressed protein.
  • DNA probes can be used to screen D. radiodurans cDNA libraries under low stringency conditions with 32 P Radioactively labeled all or part of SEQ ID NO: 1 or SEQ ID NO: 2.
  • the most suitable cDNA library for screening is the library from D. radiodurans. Methods for constructing cDNA libraries from cells or tissues of interest are well known in the art of molecular biology. In addition, many such cDNA libraries are also commercially available, for example, from Clontech, Stratagene, Palo Alto, Cal. This screening method can recognize the nucleotide sequence of the gene family associated with SEQ ID NO: 1 or SEQ ID NO: 2.
  • the recombination method can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • fragments of the proteins of the invention can also be produced by direct synthesis of peptides using solid phase techniques (Stewart et al., (1969) Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco; Merrifield J). (1963) J. Am Chem. Soc 85: 2149-2154).
  • the synthesis of proteins in vitro can be carried out manually or automatically.
  • peptides can be synthesized automatically using Applied Biosystems Model 431A Peptide Synthesizer (Foster City, CA).
  • Each fragment of the protein of the invention can be chemically synthesized separately and then chemically linked to produce a full length molecule.
  • Fig. 1 shows the growth state of Escherichia coli containing the expression vector of SEQ ID NO: 1 in a medium containing 0.75 M NaCl, demonstrating that SEQ ID NO: 1 has a salt-tolerant and drought-tolerant function.
  • the contents of the five tubes in the figure are as follows:
  • No. 1 is E. col i JM109 strain
  • No. 2 is an empty pMD18T carrier of E. col i JM109;
  • No. 3, No. 4 and No. 5 are E. col i JM109 strains containing the expression vector of SEQ ID ⁇ :1 sequence.
  • Figure 2 Figure 3 and Figure 4 are eukaryotic expression of expression vectors containing the nucleotide sequence of SEQ ID NO: 2 in tobacco cells.
  • Figure 2 shows the growth status of transgenic tobacco on MS 2 medium, and the growth state is good.
  • Figure 3 shows the root growth of transgenic tobacco negative and positive seedlings in MS 3 medium. The root growth of transgenic tobacco is good
  • Figure 4 is The growth of the transgenic sterile seedlings into the perlite was good and the growth was good.
  • FIG. 5 is a result of Northern blot analysis of a partially transgenic tobacco plant which was partially detected by PCR, and the hybridization result showed that the nucleotide sequence of SEQ ID NO: 2 can be expressed in transgenic tobacco.
  • Figure 6 and Figure 7 are comparisons of salt and drought resistance identification results of transgenic plants containing the nucleotide sequence of SEQ ID NO: 2.
  • Figure 6 is a comparison of the growth of transgenic tobacco and non-transgenic tobacco on a medium of 0 mmol NaCl.
  • Figure 7 is a comparison of the growth of transgenic tobacco and non-transgenic tobacco after 15 days of culture on a medium of 250 mmol NaCl.
  • the transgenic tobacco can contain 250 mmol. Normal growth was carried out on the medium of NaCl, and non-transgenic tobacco could not grow in the medium containing 250 mmol of NaCl.
  • the complete nucleotide sequence was amplified from D. radiodurans genomic DNA according to the published Deinococcus radiodurans genomic sequence involving a pair of PCR-specific primers.
  • the above cloned fragment was digested with Ndel and Sac II, and ligated with the vector pTtSacB containing the E. col i universal promoter groE, and the SacB gene was substituted to generate a gene E. coli expression vector.
  • Convert E. col i JM109, painted The cells were incubated on Amp-containing LB solid medium, and the white colony alkaline lysis method was used to extract different recombinants. Bgl ll digestion and sequencing confirmed that an E. coli JM109 containing the nucleotide sequence expression vector was obtained.
  • Strain. 2 ⁇ The restriction fragment analysis showed that the gene fragment containing the groE promoter was 1. 2kb.
  • E. coli JM109 strain containing the nucleotide expression vector of SEQ ID ⁇ :1 with the same 0D value, control
  • a single-stranded oligonucleotide fragment having a viscous end of 150 to 200 bp in length was synthesized from the positive and sub-chain sequences, respectively, in seven segments. Seven complementary single-stranded oligonucleotide fragments each corresponding to each other in the positive and the minor strands were annealed to form seven double-stranded oligonucleotide fragments with sticky ends. The double-stranded oligonucleotide fragments are mixed and catalyzed by T4 DNA ligase into a complete gene. The synthetic DNA fragment contains both Xbal and Sad sites at both ends.
  • the artificially synthesized 5' and 3' end cleavage sites were Xbal and Sacl sites SEQ ID NO: 2, and were used for the construction of the underlying high salt tolerance and drought resistance gene plant expression vector.
  • SEQ ID NO: 2 nucleotide sequence for eukaryotic expression in tobacco cells and identification of salt tolerance and drought resistance of transgenic plants
  • primers designed to amplify the entire coding reading frame are designed, and restriction endonuclease sites are introduced on the positive and negative primers respectively (this may depend on the selected vector) , in order to construct an expression vector.
  • an intermediate vector such as pBluescript
  • a binary expression vector such as pBI121 and PCAMBIA2200
  • the differentiated buds can be seen to grow. After the buds grow up, they are cut and placed in rooting medium (1/2MS+NAA). Rooting culture was carried out on 0.5 mg/L+Kan 25 mg/L), and rooting was carried out in about 2-7 days;
  • Figure 2 shows the growth status of transgenic tobacco on MS 2 medium, and the growth status is good.
  • Figure 3 shows the root growth of transgenic tobacco negative and positive seedlings in MS 3 medium. The root growth of transgenic tobacco is good, and Figure 4 is the transgenic. The growth of sterile seedlings into perlite is good.
  • RNA extraction Preparation reference "Molecular Cloning" (Sambrook et al., 1989)
  • RNA agarose gel electrophoresis separation 1) Take 6ml 25* running buffer, if 117ml sterile water, mix well.
  • RNA nylon membrane 1) Prior to transfer, soak the nylon membrane with 10*SSC. 2) Place the wet film on the film accurately, and moisten the two filter papers of the same size in the 2*SSC solution, cover the film, and remove the bubbles. 3) Place a stack of absorbent paper of the same size as the film on the filter paper, place a glass plate and a weight on the absorbent paper, place it horizontally, and transfer for 12-20 hours. 4) After transfer, bake the film at 80 ° C for 1-2 hours.
  • RNA on the membrane 1) Immerse the membrane in 4*SSC for 10 minutes, remove the membrane and remove excess liquid, and place the membrane in the pre-hybrid solution (50% formamide, 5*SSC, 50 mmol/L sodium phosphate (Ph6.4) 5*Dendart 0.1% SDS, O.lmg/ml salmon sperm DNA), prehybridized overnight at 42 °C. 2) To the pre-hybridization solution, exchange the same amount of the hybridization solution, denature the 32 P-labeled DNA probe in boiling water for 5 minutes, and add the hybridization solution (50% formamide, 5*SSC, 50 mmol/L sodium phosphate).
  • the transgenic plants are further characterized for salt tolerance and drought resistance and drought resistance.
  • the transgenic tobacco plants and non-transgenic tobacco plants were cultured in a medium containing 0 leg ol and 250 leg ol of NaCl, and the survival rate and development progress of the plants were studied; the observation was carried out on 5 d, 10 d and 15 d. It can be seen from Fig. 6, Fig. 7 that the transgenic tobacco can grow normally on 250 mmol of NaCl medium, and the non-transgenic tobacco can not grow on 250 mmol of NaCl medium. The results show that the sequence is resistant to salt stress.

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Description

提高植物耐盐及抗旱性的基因及其应用
技术领域
本发明涉及一种核苷酸编码序列和另一种按植物偏爱密码子人工合成的核苷酸编码 序列，本发明还涉及所述核苷酸编码序列在改良植物对盐、 干旱胁迫抗性上的应用。 背景技术
Deinococcus radiodurans (D. radiodurans ) 是至今已知生物中最高的辐射抗性的生 物之一。该菌具有对致死剂量的电离辐射、 UV辐射以及 DNA损伤试剂的极端抗性， 能将电 离辐射造成上百个 DNA双链断裂的基因组完整恢复如初， 且不发生突变的能力。 其极端的 耐辐射能力及其 DNA损失修复分子机制引起科学界的极大兴趣， 不仅对探索 DNA修复分子 机理的基础学科有着十分重要意义， 而且对促进新的 DNA技术的发展以及在环境保护和生 物修复、 人类健康、 生物技术， 乃至地外空间的开发和利用等方面的具有极大潜在应用前 景。 1999年基因研究所（TIGR)完成和公布了 D. radiodurans基因组全序列（White 1999)。
但现有技术中， 未见 D. radiodurans可提高植物对盐、 干旱胁迫的抗性的任何有关 报道。 发明内容
本发明的目的是发现并人工合成提高植物耐盐及抗旱性的 DNA序列， 并将该序列转 入植物中， 培育耐盐及抗旱性的转基因植物。 本发明发现如 SEQ ID ΝΟ:1和 SEQ ID NO:2所示的 DNA序列均具有提高植物耐 盐及抗旱性功能。其中序列 SEQ ID NO:l来源于 D. radiodurans其中的一段； SEQ ID NO:2 是源于 SEQ ID ΝΟ:1， 并按植物偏爱密码子人工合成的序列。 本发明还提供了一种重组载体， 它包含 SEQ ID ΝΟ:1所述的 DNA或包含 SEQ ID NO:2所述的 DNA。 本发明用上述重组载体转化宿主细胞， 这些宿主包括原核细胞， 也包括真核细胞。 常用的原核宿主细胞包括 JM109, 常用的真核宿主细胞包括酵母 细胞和其它植物细胞。在本发明举出的应用实例中， 宿主细胞是 E. coli JM109和烟 草。 在本发明的另一方面， 还提供了一种产生具有 SEQ ID NO:l或 SEQ ID NO:2蛋 白质活性的多肽的方法， 其步骤如下：
(1)将 SEQ ID ΝΟ:1或 SEQ ID NO:2可操作地连于表达调控序列， 形成 SEQ ID ΝΟ:1或 SEQ ID NO:2蛋白表达载体；
(2)将步骤 (1)中的表达载体转入宿主细胞， 形成重组细胞；
(3)在适合表达 SEQ ID NO:l或 SEQ ID NO:2蛋白多肽的条件下， 培养步骤 (2)中 的重组细胞；
(4)分离出具有 SEQ ID NO:l或 SEQ ID NO:2蛋白活性的基本纯的多肽， 序列为 SEQ ID NO:3。 本发明还提供了一种利用转基因技术将 SEQ ID ΝΟ:1或 SEQ ID NO:2转化入植 物的方法， 以提高植物对盐、 干旱胁迫抗性， 其步骤如下：
(1) 将 SEQ ID ΝΟ:1或 SEQ ID NO:2所示序列可操作地连于植物表达调控序列， 形成植物表达载体；
(2) 将步骤 (1)中的表达载体转入植物细胞；
(3) 经筛选获得转化细胞并最终再生转基因植株及其后代， 包括植物种子及植物 组织。
上述 "可操作地连于"表示如下情况： 即线性 DNA序列的某些部分能够影响 同一线性 DNA序列其他部分的活性。例如， 如果信号肽 DNA作为前体表达并参与 多肽的分泌，那么信号肽 (分泌前导序列) DNA就是可操作地连于多肽 DNA;如果启 动子控制序列的转录， 那么它是可操作地连于编码序列； 如果核糖体结合位点被置 于能使其翻译的位置时， 那么它是可操作地连于编码序列。 一般， "可操作地连于" 意味着相邻， 而对于分泌前导序列则意味着在阅读框中相邻。 在本发明的一个实例中， 将步骤（1)中的表达载体转入农杆菌， 将含表达载体的农 杆菌同真核宿主细胞共培养，在 22-28°C条件下， 暗培养 1-2天后，通过筛选如抗生素筛 选， 获得含有 SEQIDNO:l或 SEQIDNO:2基因的转化细胞并最终再生转基因植株及 其后代， 包括植物种子及植物组织。
经实验证实， 上述转基因植株对植物盐、 干旱胁迫具有增强的抗性作用。
上述载体可选用本领域已知的各种载体， 如市售的载体， 包括质粒， 粘粒等。 此外，本发明还提供了一种可用作探针的核酸分子，该分子通常具有 SEQ ID NO： 1 或 SEQ ID N0:2 核苷酸编码序列的 8-100个连续核苷酸， 较佳地具有 15-50个连续核 苷酸。 该探针可用于检测样品中是否存在编码 SEQ ID N0:1或 SEQ ID N0:2 的核酸分 子。
本发明还提供了检测样品中是否存在 SEQ ID NO: 1或 SEQ ID N0:2核苷酸序列的 方法， 它包括用上述的探针与样品进行杂交， 然后检测探针是否发生了结合。 较佳 地， 该样品是 PCR扩增后的产物， 其中 PCR扩增引物对应于 SEQ ID NO: 1或 SEQ ID N0:2核苷酸编码序列， 并可位于该编码序列的两侧或中间。 引物长度一般为 15-50 个核苷酸。 在本发明中， "SEQ ID NO: 1或 SEQ ID NO: 2"指编码具有 SEQ ID NO: 1或 SEQ ID N0:2 蛋白活性的多肽的核苷酸序列及其简并序列。该简并序列是指所述序列中有一 个或多个密码子被编码相同氨基酸的简并密码子所取代后而产生的序列。 由于密码 子的简并性， 所以与 SEQIDNO:l或 SEQ ID N0:2同源性低至约 89%的简并序列也能 编码出 SEQ ID NO:2所述的序列。 该术语还包括能在中度严谨条件下， 更佳的在高 度严谨条件下与 SEQ ID ΝΟ:1核苷酸序列杂交的核苷酸序列。 该术语还包括与 SEQ ID ΝΟ:1中的核苷酸序列的同源性至少 89%， 较佳地至少 80%， 更佳地至少 90%， 最佳地至少 95%的核苷酸序列。
该术语还包括能编码具有与天然的 SEQ ID NO: 1或 SEQ ID N0:2相同功能的蛋白 的 SEQIDNO:l中开放阅读框序列的变异形式。这些变异形式包括 (但并不限于)： 若 干个 (通常为 1-90个， 较佳地 1-60个， 更佳地 1-20个， 最佳地 1-10个)核苷酸的缺 失、 插入和 /或取代， 以及在 5'和 /或 3'端添加数个 (通常为 60个以内， 较佳地为 30 个以内， 更佳地为 10个以内， 最佳地为 5个以内)核苷酸。
在本发明中， "基本纯的"蛋白质或多肽是指其至少占样品总物质的至少 20%, 较佳地至少 50%， 更佳地至少 80%， 最佳地至少 90% (以上各百分比均指按干重或 湿重计）。 纯度可以用任何合适的方法进行测量， 如用柱层析、 PAGE或 HPLC法测 量多肽的纯度。 基本纯的多肽基本上不含天然状态下的伴随其的组分。
在本发明中， SEQ ID NO:3蛋白或多肽指具有 SEQ ID ΝΟ:1编码的蛋白活性多 肽。 该术语还包括具有与天 SEQ ID NO:3的相同功能的变异形式。 这些变异形式包 括但并不限于若干个 (通常为 1-50个， 较佳地 1-30个， 更佳地 1-20个， 最佳地 1-10 个)氨基酸的缺失、 插入和 /或取代， 以及在 C末端和 /或 N末端添加一个或数个 (通 常为 20个以内， 较佳地为 10个以内， 更佳地为 5个以内)氨基酸。 例如， 在所述蛋 白中， 用性能相近或相似的氨基酸进行取代时， 通常不会改变蛋白质的功能。 又比 如， 在 C末端和 /或 N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。 该术语还包括 SEQ ID NO:3蛋白的活性片段和活性衍生物。
本发明的 SEQ ID NO:2多肽的变异形式包括： 同源序列、 保守性变异体、 等位 变异体、天然突变体、诱导突变体、在高或低的严谨条件下能与 SEQ ID NO:l或 SEQ ID NO:2杂交的 DNA所编码的蛋白、 以及利用 SEQ ID NO:3多肽的抗血清获得的多肽 或蛋白。 本发明还提供了其他多肽， 如包含 SEQ ID NO:3多肽或其片段的融合蛋白。 除了几乎全长的多肽外， 本发明还包括 SEQ ID NO:3多肽的可溶性片段。 通常， 该 片段具有 SEQ ID NO:3多肽序列的至少约 10个连续氨基酸， 通常至少约 30个连续 氨基酸， 较佳地至少约 50个连续氨基酸， 更佳地至少约 80个连续氨基酸， 最佳地 至少约 100个连续氨基酸。
在本发明中， "SEQ ID NO:3保守性变异多肽"指与 SEQ ID NO:3的氨基酸序列 相比， 有至多 10个， 较佳地至多 8个， 更佳地至多 5个氨基酸被性质相似或相近的 氨基酸所替换而形成多肽。 这些保守性变异多肽最好根据表 1进行替换而产生。
Figure imgf000005_0001
Figure imgf000006_0001
发明还包括 SEQ ID N0:3蛋白或多肽的类似物。这些类似物与天然 SEQ ID N0:3 多肽的差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异， 或者兼而有之。 这些多肽包括天然或诱导的遗传变异体。 诱导变异体可以通过各种 技术得到， 如通过辐射或暴露于诱变剂而产生随机诱变， 还可通过定点诱变法或其 他已知分子生物学的技术。 类似物还包括具有不同于天然 L-氨基酸的残基 (如 D-氨 基酸)的类似物，以及具有非天然存在的或合成的氨基酸 (如 β、 Υ -氨基酸)的类似物。 应理解， 本发明的多肽并不限于上述例举的代表性的多肽。
修饰 (通常不改变一级结构)形式包括： 体内或体外的多肽的化学衍生形式如乙 酰化或羧基化。 修饰还包括糖基化， 如那些在多肽的合成和加工中或进一步加工步 骤中进行糖基化修饰而产生的多肽。 这种修饰可以通过将多肽暴露于进行糖基化的 酶 (如哺乳动物的糖基化酶或去糖基化酶)而完成。 修饰形式还包括具有磷酸化氨基 酸残基 (如磷酸酪氨酸， 磷酸丝氨酸， 磷酸苏氨酸)的序列。 还包括被修饰从而提高 了其抗蛋白水解性能或优化了溶解性能的多肽。
还可用 Northern印迹法技术分析 SEQ ID NO : 1或 SEQ ID NO : 2基因产物的表达， 即分析 SEQ ID N0 : 1或 SEQ ID NO : 2 的 RNA转录物在细胞中的存在与否和数量。
SEQ ID NO : 1或 SEQ ID NO : 2 RNA的 Northern印迹分析和 SEQ ID NO:3特异抗 体的 Western印迹分析可以联合使用， 以证实 SEQ ID NO : 1或 SEQ ID NO : 2在生物样 本中的表达。 此外， 根据本发明的核苷酸序列和氨基酸序列， 可以在核酸同源性或表达蛋白 质的同源性基础上， 筛选 SEQ ID N0 : 1或 SEQ ID N0 : 2 同源基因或同源蛋白。
为了得到与 SEQ ID N0 : 1或 SEQ ID NO : 2基因相关的 D. radiodurans cDNAs的 点阵， 可以用 DNA探针筛选 D. radiodurans cDNA文库， 这些探针是在低严紧条 件下， 用 32P对 SEQ ID NO : 1或 SEQ ID N0 : 2 的全部或部分做放射活性标记而得的。 最适合于筛选的 cDNA文库是来自 D. radiodurans的文库。 构建来自感兴趣的细胞 或者组织的 cDNA 文库的方法是分子生物学领域众所周知的。 另外， 许多这样的 cDNA文库也可以购买到， 例如购自 Clontech, Stratagene, Palo Alto, Cal.。 这种筛选 方法可以识别与 SEQ ID N0 : 1或 SEQ ID N0 : 2相关的基因家族的核苷酸序列。
一旦获得了有关的序列， 就可以用重组法来大批量地获得有关序列。 这通 常是将其克隆入载体， 再转入细胞， 然后通过常规方法从增殖后的宿主细胞中 分离得到有关序列。
此外， 还可用人工化学合成的方法来合成有关序列。 在本申请之前， 现有技术 已完全可以通过先合成多个多核苷酸小片段， 然后再进行连接而获得编码本发明 D. radiodurans的 SEQ ID NO:2蛋白的核酸序列。 然后， 可将该核酸序列引入本领域中 各种现有的 DNA分子 (如载体)和细胞中。此外， 还可通过化学合成将突变引入本发 明蛋白序列中。 除了用重组法产生之外， 本发明蛋白的片段还可用固相技术， 通过直接合成肽 而加以生产 (Stewart等人，（1969) Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco; Merrifield J. (1963) J. Am Chem. Soc 85: 2149-2154)。在体外合成蛋白质可 以用手工或自动进行。 例如， 可以用 Applied Biosystems的 431A型肽合成仪 (Foster City, CA)来自动合成肽。 可以分别化学合成本发明蛋白的各片段， 然后用化学方法 加以连接以产生全长的分子。 附图说明
图 1是 含有 SEQ ID NO : 1表达载体的大肠杆菌在含 0. 75 M NaCl的培养基中生长 状况， 证明 SEQ ID NO : 1具有耐盐及抗旱性功能。 图中 5个试管中内容物如下：
1号是 E. col i JM109菌株；
2号是空 pMD18T载体的 E. col i JM109;
3号、 4号禾卩 5号是含有 SEQ ID ΝΟ:1序列表达载体的 E. col i JM109菌株。 图 2， 图 3和图 4是含 SEQ ID NO:2核苷酸序列的表达载体在烟草细胞中进行真核 细胞表达。 其中图 2是转基因烟草在 MS2培养基上生长状况， 生长状态良好； 图 3是在 MS3培养基中转基因烟草阴性苗与阳性苗的根系生长状况， 转基因烟草的根系生长良好， 图 4是转基因无菌苗移入珍珠岩中的生长情况， 生长状态良好。 图 5是部分经 PCR检测的阳性转基因烟草植株的 Northern blot分析结果，杂交结果表 明 SEQ ID NO:2核苷酸序列能在转基因烟草中表达。 图 6、图 7是含 SEQ ID NO:2核苷酸序列的转基因植株的耐盐及抗旱性鉴定结果对比。 其中图 6是在 0 mmol NaCl 的培养基上转基因烟草和非转基因烟草生长对比， 图 7是在 250mmol NaCl的培养基上培养 15天后转基因烟草和非转基因烟草生长对比， 转基因烟草 能在含 250mmol的 NaCl的培养基上正常生长， 非转基因烟草在含 250mmol的 NaCl的培 养基不能生长。 具体实施方式
下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于举例说 明本发明的方法， 而不用于限制本发明的范围。 凡未注明具体实验条件的， 均为按 照本领域技术人员熟知的常规条件， 例如 Sambrook等分子克隆： 实验室手册 (New York: Cold Spring Harbor Laboratory Press, 1989)中所述的条件，或按照制造厂商所建 议的条件。 实施例 1 SEQ ID ΝΟ:1核苷酸序列在大肠杆菌中的表达和耐盐及抗旱性性能分析 1 . 核苷酸序列 SEQ ID NO:l的克隆
根据已公布的 Deinococcus radiodurans基因组序列涉及一对 PCR特异性引物， 从 D. radiodurans基因组 DNA中扩增完整的核苷酸序列。
2. 构建大肠杆菌表达载体及分子验证
用将上述克隆片段用 Ndel和 Sac II双酶切消化，与含 E. col i通用启动子 groE的载体 pTtSacB连接， 取代 SacB基因， 以产生基因的大肠杆菌表达载体。 转化 E. col i JM109,涂 布于含 Amp的 LB固体培养基上， 挑取白色菌落碱裂解法提质粒筛选不同的重组子， Bgl ll 酶切及测序验证， 得到一株含该核苷酸序列表达载体的 E. coli JM109菌株。 酶切分析表 明， 含 groE启动子的基因片段为 1. 2kb。
3. 大肠杆菌表达载体的耐盐及抗旱性性功能验证
将 0D值相同的含有 SEQ ID ΝΟ:1核苷酸序列表达载体的 E. coli JM109菌株， 对照
PMD18T和对照宿主菌株 E. coli JM109以 1 %的接菌量分别接种于含有 0. 75 M NaCl的 MM 培养基中， 37°C振荡培养 15个小时， 于 550nm波长测定 0D值。 从图 1中可看出含有 SEQ ID ΝΟ:1核苷酸序列表达载体的 E. coli JM109菌株能耐受 0. 75M NaCl , 生长状况良好， 而只含空载体的 E. coli JM109菌株和 E. coli JM109菌株在 0. 75M NaCl的培养基中不能生 长。 实施例 2 SEQ ID NO:2核苷酸序列的人工合成
根据已 SEQ ID ΝΟ:1核苷酸序列， 首先分 7个区段分别根据正链和副链序列， 分别 合成出长度 150_200bp、具有粘性末端的单链寡核苷酸片段。将正链和副链各一一对应的 7 个互补的单链寡核苷酸片段分别退火， 形成 7个带有粘性末端的双链寡核苷酸片段。 混合 双链寡核苷酸片段， 经 T4 DNA连接酶催化组装成一个完整的基因。该合成的 DNA片段的两 端含 Xbal和 Sad位点。
将上述人工合成的 5 ' 和 3 ' 端酶切位点为 Xbal和 Sacl位点 SEQ ID NO:2， 用于下 面高耐盐及抗旱性基因植物表达载体的构建。 实施例 3 SEQ ID NO:2核苷酸序列在烟草细胞中进行真核细胞表达及转基因植株 的耐盐、 抗旱性鉴定
(一） 含目的基因表达载体的构建
根据全长编码序列 (SEQ ID NO:. 2)， 设计扩增出完整编码阅读框的引物， 并在 正反引物上分别引入限制性内切酶位点 (这可视选用的载体而定)， 以便构建表达载 体。 以实施例 1 中获得的扩增产物为模板， 经 PCR扩增后， 将序列 cDNA克隆至 中间载体 （如 pBluescript )， 进一步克隆到双元表达载体 （如 pBI121 和 PCAMBIA2200) , 在保证阅读框架的前提下鉴定好的表达载体， 再将其转入农杆菌 中， 利用叶盘法技术转化模式植物烟草。
(二） 利用叶盘法转化烟草
1. 用无菌牙签挑取 YEB 选择平板上的阳性菌落,接种于 2 ml YEB 液体 (Sm+, Kan+),28度 ，200rpm振荡培养 24-36小时；
2. 室温下 4,000g离心 lOmin;
3. 弃上清， 菌体用 1/2MS 液体培养基悬浮， 稀释到原体积的 5-20倍， 使菌液的 OD600=0.5左右；
4. 取生长两周左右的烟草的无菌叶片， 去掉其主叶脉， 将其剪成约 1 平方厘米见 方的小叶片；
5. 将叶片放入制备好的菌液中， 浸泡 2-5min， 在无菌滤纸上吸干菌液；
6. 把经侵染的叶片放于 MS培养基上， 28°C暗培养 48小时；
7. 将叶片转到愈伤培养基 （MS + 6 -BA 1.0mg/L+NAA 0.1mg/L+Kan 50mg/L+cb 250mg/L) 上， 25-28°C光照下培养， 7-15天可见愈伤组织的形成；
8. 约 20天后可见分化芽长出， 待芽长大后， 切下， 置于生根培养基 (1/2MS+NAA 0.5mg/L+Kan 25mg/L)上进行生根培养， 2-7天左右生根；
9. 等根系发达后， 将植株取出， 用无菌水洗净附着的固体培养基， 移入土壤中， 刚开始用玻璃罩罩几天， 待植株健壮后再取下玻璃罩， 温室中培养。 图 2是转基 因烟草在 MS2培养基上生长状况， 生长状态良好， 图 3是在 MS3培养基中转基因烟草 阴性苗与阳性苗的根系生长状况， 转基因烟草的根系生长良好， 图 4是转基因无菌苗 移入珍珠岩中的生长情况， 生长状态良好。
(三） 利用 Northern blot检测 SEQ ID NO:2在转基因烟草植株中的表达
1. RNA的提取： 制备参考 《分子克隆》 （Sambrook等， 1989)
2. RNA的定量: 参考 《分子克隆》 （Sambrook等， 1989)， 分光光度计测 0D26。； RNA含 量计算： 1 0D26。 = 40μ§/πι1 ο
3 总 RNA琼脂糖凝胶电泳分离： 1 )取 6ml 25*电泳缓冲液， 假如 117ml无菌水， 混匀。
2) 称取 1. 5g琼脂糖， 加入到上述溶液张， 于微波炉里加热融化， 转入 55°C水浴 中。 3 ) 于通风橱中取 26. 8ml甲醛， 加入到 55°C的凝胶溶液中， 混匀。 4 ) 迅速 倒入制胶板中， 室温水平放置 30分钟， 待胶凝固。 5 ) 将提取的 RNA30ug溶解于 15mlRNA稀释溶液中， 在 55°C-65°C下加热 10分钟， 然后立即放在冰上。 6 ) 在样 品中加入 2ull0*上样缓冲液， 混匀。 9) 在电泳液未盖过胶的条件下点样， 80V 电压电泳 10分钟， 待样品全部进入胶后， 加电泳液盖过胶面约半厘米。 80-100V 电泳 5小时。
4. RNA尼龙膜上转移： 1 ) 转移之前， 将尼龙膜用 10*SSC浸泡。 2) 将湿润的膜准确 地盖在膜上， 将两张与膜大小相同的滤纸置 2*SSC溶液中湿润， 盖在膜上， 排除 气泡。 3) 滤纸上放一叠与膜大小相同的吸水纸， 在吸水纸上放一玻璃板和一重 物， 水平放置， 转移 12-20小时。 4 ) 转移后， 将膜于 80°C烘烤 1-2小时。
5. 膜上 RNA的检测： 1 ) 将膜浸在 4*SSC中 10分钟， 取出膜置滤纸上吸去多余的 液体， 将膜放入预杂交液中 （50%甲酰胺， 5*SSC， 50mmol/L磷酸钠 （Ph6.4) 5*Dendart 0.1%SDS, O.lmg/ml鲑鱼精 DNA) ,42°C下预杂交过夜。 2) 到出预 杂交液， 换入等量的杂交液， 将用 32P标记的 DNA探针在沸水中变性 5分钟， 加入杂交液 （50%甲酰胺， 5*SSC， 50mmol/L磷酸钠 （Ph6.4) 10%葡聚糖硫酸 月旨， l*Dendart ， 0.1%SDS , 0.1mg/ml鲑鱼精 DNA) 中， 于 42。C杂交 24-48 小时。 3 ) 取出膜 ， 置洗膜液 I ( 1*SSC， 1%SDS ) 中， 于 42°C漂洗 3次， 每 次 5分钟。 转入洗膜液 II (0.1*SSC， 1%SDS ) 中， 于 55°C-65°C漂洗 1-3次。 用 X光片压片 1-7天， 然后显影、 定影。 图 5是部分经 PCR检测的阳性转基因烟 草植株的 Northern blot分析结果。图 5的杂交结果表明 SEQ ID NO:2核苷酸序列能在 转基因烟草中表达。
(四） 含 SEQ ID NO:2核苷酸序列的转基因植株的耐盐及抗旱性鉴定
鉴于该核苷酸序列已被证明在大肠杆菌中对盐胁迫具有抗性，进一步对转基因 植株进行耐盐及抗旱性和抗旱性鉴定。
用含有 0腿 ol、 250腿 ol的 NaCl的培养基培养转基因烟草植株和非转基因烟草植 株， 研究其对植株的存活率和发育进度； 通过 5 d、 10 d和 15 d 的观察。 从图 6， 图 7 上可知转基因烟草可在 250mmol的 NaCl培养基上正常生长，非转基因烟草在 250mmol的 NaCl培养基上不能生长， 结果证明， 该序列对盐的胁迫确有抗性。

Claims

权 利 要 求
1. 一种提高植物耐盐及抗旱性的 DNA序列， 如 SEQ ID N0:1所示。
2. 一种提高植物耐盐及抗旱性的 DNA序列， 如 SEQ ID N0:2所示。
3. 权利要求 1所述 DNA序列编码的氨基酸序列， 如 SEQ ID NO: 3所示。
4. 包含 SEQ ID NO: 1所述的 DNA或包含 SEQ ID NO: 2所述的 DNA的重组载体。
5. 用权利要求 4所述的重组载体转化的宿主细胞， 包括原核细胞和真核细胞。
6. 权利要求 1或 2所述的 DNA序列用于提高植物耐盐及抗旱性的用途。
7. 一种用权利要求 1或 2所述的 DNA耐盐及抗旱性转基因植物的方法， 步骤如下：
(1) 将 SEQ ID NO: 1或 SEQ ID NO: 2所示序列可操作地连于植物表达调控序列， 形成植物表达载体；
(2) 将步骤（1)中的表达载体转入植物细胞；
(3) 经筛选获得转化细胞并最终再生转基因植株及其后代， 包括植物种子及植物 组织。
8. 一种检测样品中是否含有权利要求 1或 2所述的 DNA序列的方法， 其特征在于 用 SEQ ID NO: 1制备的抗体与样品进行杂交， 检测抗体与样品探针是否发生结合； 所 述样品是 PCR扩增后的产物，其中 PCR扩增引物对应于 SEQIDN0:1或 SEQIDN0:2 核 苷酸编码序列， 并可位于该编码序列的两侧或中间， 引物长度为 15〜50个核苷酸。
PCT/CN2007/071043 2007-10-22 2007-11-09 Gènes améliorant la tolérance au sel et à la sécheresse d'une plante et leurs utilisations WO2009052687A1 (fr)

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