Title of Invention

GENE FOR IMPROVING SALT TOLERANCE AND DROUGHT TOLERANCE OF PLANT

Abstract

The present invention provides a nucleotide coding sequence and another nucleotide coding sequence artificially synthesized according to plant-biased codons. Recombinant vectors containing the above-synthesized genes art constructed and introduced into host cells including prokaryotic cells and eukaryotic cells. It is confirmed that the resulting transgenic plant has improved salt and drought tolerance after the said genes are expressed in the plant.

Full Text

FORM-2 THE PATENTS ACT, 1970 (39 of 1970) & THE PATENTS RULES, 2003 Provisional /Complete specification [See Section 10 and rule 13] 1 Title of the Invention. "GENES FOR IMPROVING SALT TOLERANCE AND DROUGHT TOLERANCE OF PLANT AND THE USES THEREOF" 2 Applicant (s) Applicant BIOTECHNOLOGY RESEARCH INSTITUTE, THE CHINESE ACADEMY OF AGRICULTURAL SCIENCES Nationality China Address No.12 Zhongguancun Nan Dajie, Haidian District, Beijing 100081, China. The following specification particularly describes the invention and the manner in which it is to be performed. TECHNICAL HELD The invention relates to a nucleotide coding sequence and another nucleotide coding sequence artificially synthesized according to plant-biased codons ° The invention also relates to the use of the said nucleotides in improving salt tolerance and drought tolerance of a plant. BACKGROUD ART Deinococcus radiodurans (D. radiodurans) is one of the most radiation-resistant organisms known so far. The bacterium has extremely high resistance to a lethal dose of ionizing radiation, UV radiation and a DNA damaging agent and is capable of repairing hundreds of breakages of Genomic DNA strands induced by ionizing radiation without mutagenesis. The bacterium has attracted great interests among the scientific community in its extraordinary radiation resistance and DNA damage repair mechanism, the study of which is very meaningful for the development of basic subjects exploring DNA repair mechanism and have potential application prospects in environment protection, bioremediation, human health, biotechnology, or even exploitation and development of the outer space. The Institute for Genomic Research ( TIGR) completed and brought out the whole genome sequence of D. radiodurans (White 1999) in 1999. There is no any report on Deinococcus radiodurans for improving salt tolerance and drought tolerance of plant in the art. DISCLOSURE OF THE INVENTION An object of the invention is to find and artificially synthesize the DNA sequences for improving salt tolerance and drought tolerance of a plant, introduce the sequences into the plant and grow the transgenic plant with salt and drought tolerance. The inventors found the DNA sequences set forth in SEQ ID NO:l and SEQ ID NO:2 both have improved salt and drought tolerance. The sequence of SEQ ID NO:l is derived from the DNA of D. radiodurans', SEQ ID NO:2 is derived from SEQ ID NO:l and artificially synthesized according to plant-biased codons. The invention also provides a recombinant vector, comprising the DNA fragment set forth in SEQ ID NO:l or the DNA fragment set forth in SEQ ID NO:2. The host cells are transformed with the said recombinant vector and the host cells include prokaryotic cells and eukaryotic cells. The commonly used prokaryotic host cells include JM109 and the commonly used eukaryotic host cells include yeast cells and other plant cells. In an embodiment of the invention, the host cells are E.coli JM109 and tobacco cells. In an aspect of the invention, it also provides a method for generating polypeptide with the protein activity of SEQ ID NO:l or SEQ ID NO:2, comprising the steps of: (1) SEQ ID NO:l or SEQ ID NO:2 is operatively linked to an expression regulation sequence to form protein expression vector of SEQ ID NO:l or SEQ ID NO:2; (2) The expression vector of the step (1) is introduced into host cells to form recombinant cells; (3)The recombinant cells obtained from the step (2) are cultured under the conditions suitable for the expression of the polypeptides of SEQ ID NO:l or SEQ ID NO:2; (4) The substantially pure polypeptides with the activity of the protein of SEQ ID NO:l or SEQ ID NO:2 are isolated, which sequence is shown in SEQ ID NO:3. The invention further provides a method of transferring SEQ ID NO:l or SEQ ID NO:2 into a plant by using transgenic technology to improve salt tolerance and drought tolerance of the plant, comprising the steps of: (1) A sequence set forth in SEQ ID NO:l or SEQ ID NO:2 is operatively linked to a regulation sequence of plant expression to form a plant expression vector; (2) The expression vector obtained in the step (1) is introduced into plant cells to obtain transformed cells; (3) The transformed cells are obtained via screening and ultimately regenerated into transgenic plants and progenies thereof, including the seeds and tissues of the plants. The term "operatively linked to" as described above means that such linkage allows that certain regions of a linear DNA sequence are capable of having an influence on the activity of the remaining regions of the same linear DNA sequence. For example, DNA of signal peptide (secretion leader sequence) is operatively linked to DNA of polypeptide if DNA of signal peptide is expressed as precursor and participates in the secretion of the polypeptide. The promoter control sequence is operatively linked to the coding sequence if it is transcribed; the ribosome bind site is operatively linked to the coding sequence if it is located in the position making it to be translated. In general, the term "operatively linked to" means to be adjacent and to be adjacent in reading frame for the secretion leader sequence. In one embodiment of the invention, the expression vector of step (Z) is transferred into Agrobacterium, and the transformed Agrobacterium containing the expression vector is co-cultured with eukaryotic host cells at 22-28 °C, the transformed cells containing SEQ ID NO:l or SEQ ID NO:2 gene are obtained via screening such as antibiotics screening after dark incubation for 1-2 days, and the cells are regenerated into transgenic plants and progenies thereof, including seeds and tissues of the plants. It is experimentally confirmed that the transgenic plants as described above have improved salt and drought tolerance. The vectors as described above may be selected from various vectors known in the art, such as commercially available vectors, including plasmid and cosmid and the like. In addition, the invention further provides a nucleic acid which can be used as a probe, generally comprising 8-100 consecutive nucleotides, preferably 15-50 consecutive nucleotides, derived from the nucleotide coding sequence of SEQ ID NO:l or SEQ ID NO:2. The probe may be used to detect whether there is nucleic acid molecules coding SEQ ID NO:l or SEQ ID NO:2 in samples. The invention still further provides a method for detecting whether there are nucleic acid molecules coding SEQ ID NO:l or SEQ ID NO:2 in samples, comprising that hybridizing the samples with the probe as described above and then determining whether the probe is combined. Preferably, the samples are produced by PCR amplification, wherein the primers used for the PCR amplification correspond to the nucleotide coding sequence of SEQ ID NO:l or SEQ ID NO:2 and may be located in flank and middle of the coding sequence. The primers are generally 15-50 nucleotides in length. According to the invention, "SEQ ID NO:l or SEQ ID NO:2" refers to the nucleotide sequence and the degenerate sequence thereof, coding the polypeptide with the protein activity of SEQ ID NO:l or SEQ ID NO:2. The degenerate sequence refers to the sequence where one or more codons are substituted with one or more degenerate codons coding the same amino acid. The degenerate sequence with as low as about 89% homology with SEQ ID NO:l or SEQ ID NO:2 also can code the sequence of SEQ ID NO:2. The term also includes the nucleotide sequence hybridized with the nucleotide sequence of SEQ ID NO:l under moderate stringent conditions, preferably high stringent conditions. The term further includes the nucleotide sequence with at least 89% homology with the nucleotide sequence of SEQ ID NO:l, preferably at least 80%, more preferably at least 90%, most preferably at least 95%. The term also includes variants of the open reading frame sequence of SEQ ID NO:l, which are capable of coding the proteins with the same function as natural SEQ ID NO:l or SEQ ID NO:2 does. These variants include, but are not limited to, the deletion, insertion and/or substitution of several nucleotides, generally 1-90, preferably 1-60, more preferably 1-20, most preferably 1-10 nucleotides, as well as the addition of several nucleotides, generally less than 60, preferably less than 30, more preferably less than 10, most preferably less than 5 nucleotides, at 5' and/or 3' end thereof. According to the invention, the term "substantially pure" proteins or peptides refers to that they are present in an amount of at least 20%, preferably at least 50%, more preferably at least 80%, most preferably at least 90%, of the sample by dry or wet weight. The purity of peptides can be measured by any suitable method, such as column chromatography, PAGE or HPLC. The substantially pure peptides substantially contain no components naturally occurring together with it. According to the invention, the protein or peptide of SEQ ID NO:3 refers to the peptide with the activity of the protein coded by SEQ ID NO:l, and the variants with the same function as SEQ ID NO:3. The variants include, but are not limited to, the deletion, insertion and/or substitution of several nucleotides, generally 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10 nucleotides, as well as the addition of several nucleotides, generally less than 20, preferably less than 10, at 5' and/or 3' end thereof. For example, in the said protein, the function of the protein generally is not changed when substitution is carried out with the amino acids with the close or similar function. Another example is the function of the protein generally may not be changed by adding one or more amino acids at C-terminus and/or N-terminus. The term also includes active fragments or active derivatives of SEQ ID NO:3 protein. The variants of the polypeptide of SEQ ID NO:2 according to the invention include: homologous sequences, conservative variants, allelic variants, natural variants, induced variants, proteins coded by DNA hybridized with SEQ ID NO:l or SEQ ID NO:2 under high or low stringent conditions and polypeptides and proteins obtained by using antiserum of the polypeptides as showed in SEQ ID NO:3. The invention also provides other polypeptides, such as fusion protein containing the polypeptides of SEQ ID NO:3 and fragments thereof. The invention also includes soluble fragments of the polypeptides of SEQ ID NO:3 in addition to nearly full length polypeptides. The fragments generally have at least about 10 consecutive amino acids, generally at least about 30 consecutive amino acids, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, most preferably at least about 100 consecutive amino acids. According to the invention, "conservative variant polypeptides of SEQ ID NO:3" refers to the polypeptides having at most 10, preferably at most 8, more preferably at most 5 amino acids substituted with amino acids with close or similar property compared to the amino acid sequence of SEQ ID NO:3. The conservative variant polypeptides are prepared with the substitutions according to table 1 as below. The invention also embraces analogs of the proteins or polypeptides of SEQ ID NO:3. The differences between these analogs and the natural polypeptide of SEQ ID NO:3 may be the differences between the amino acid sequences, or the differences between the modified versions that are not influencing the sequences, or both. These polypeptides include natural or induced genetic variants. The induced variants can be obtained by various technologies, such as random mutagenesis via radiation or exposing to mutagenic agent, or via site-directed mutagenesis or any other molecular biologic technology known in the art. The analogs also include analogs which have residues differing from natural L-amino acids (such as D-amino acids) and analogs which have non-naturally occurring or synthetic amino acids (such as p, y-amino acids). It should be understood that the polypeptides of the invention aren't limited to the representative polypeptides as described above. The versions of modification (the primary structure is generally not modified) include: chemically derived polypeptides in vivo or in vitro, such as acetylation or acylation of polypeptides. The modifications also include glycosylation, such as the polypeptides produced by glycosylation during synthesis and processing or further processing steps of polypeptides. The modification can be accomplished by exposing the polypeptides to the enzymes (such as the glycosidase or the deglycosidase of mammals) for glycosylation. The versions of modification also include a sequence with phosphorylated amino acid residues (phosphotyrosine, phosphoserine and phosphothreonine). A polypeptide modified to improve the ability of resisting hydrolysis or optimizing the solubility property is also included. The product of gene expression of SEQ ID NO:l or SEQ ID NO:2 is analyzed by using the Northern blot method, i.e. to analyze whether the RNA transcripts of SEQ ID NO:l or SEQ ID NO:2 are present in the cells and the amount thereof. The Northern blot analysis of RNA of SEQ ID NO:l or SEQ ID NO:2 and the Western blot analysis of the specific antibody of SEQ ID NO:3 can be combined, to confirm the expression of SEQ ID NO.'l or SEQ ID NO:2 in a biological sample. In addition, genes or proteins which are homologous to SEQ ID NO:l or SEQ ID NO:2 may be screened according to the nucleotide sequences and the amino acid sequences of the invention, on the basis of homology of nucleic acids and proteins expressed. To obtain dot matrix of D. radiodurans cDNA that is related to the gene of SEQ ID NO:l or SEQ ID NO:2,D. radiodurans cDNAmay be screened using DNA probes, and these probes are produced by radioactively labeling the whole or partial SEQ ID NO:l or SEQ ID NO:2 with 32P under the low stringent condition. The most suitable cDNA library to be screened is from D. radiodurans library. The methods for constructing cDNA library from interested cells or tissues are well known in the field of molecular biology. In addition, many of such libraries may be commercially available, for example, from Clontech, Stratagene, Palo Alto, Cal.. The screening methods can identify a nucleotide sequence of gene family which is related to SEQ ID NO:l or SEQ ID NO:2. Once the related sequences are identified, the related sequences can be obtained in a large scale using a recombinant method. It is generally cloned into a vector, introduced into a cell and then the related sequences are isolated from the host cells after proliferation by conventional methods. In addition, the related sequences are also synthesized by artificial chemical synthesis method. A plurality of small fragments of polynucleotide are firstly synthesized and then they are linked to give the nucleotide sequences coding SEQ ID NO:2 protein of D. radiodurans of the invention according to the prior art prior to the invention. The nucleotide sequences can be subsequently introduced into various exiting DNA molecules (such as a vector) and cells in the art. Also, the mutation can be induced in the protein sequences of the invention by chemical synthesis. The fragments of the protein of the invention can be produced through solid phase method and direct peptide synthesis (Stewart et al. (1969), Solid-Phase Synthesis, WH Freeman Co., San Francisco; Merrifield J. (1963) J. Am. Chem. Soc 85:2149-2154) in addition to recombinant method. The synthesis of protein in vitro may be performed by hand or automatically. For example, the peptides can be synthesized using 431A Model Peptide Synthesizer from Applied Biosystems( Foster City, CA). The respective fragments of the protein of the invention can be chemically synthesized and then they are linked into full length molecular by chemical methods. BRIEF DESCRIPTION OF THE DRAWINGS Fig.l is the photo showing the growth state of E. coli containing the expression vector comprising SEQ ID NO:l in the medium containing 0.75M NaCl, to demonstrate that SEQ ID NO:l has the property to resist salt and drought. The contents in 5 tubes in the figure as follow: No.l isE. coli JM109 strain. No.2 is E. coli JM109 containing empty pMD18T vector. No.3, 4 and 5 are E. coli JM109 strains containing expression vectors comprising SEQ ID NO: 1 sequence. Fig.2, Fig. 3 and Fig. 4 are the photos showing that the expression vector containing the nucleotide sequence of SEQ ID NO:2 is expressed in tobacco cells. Fig.2 is the photo showing the good growth state of the transgenic tobacco in MS2 medium. Fig.3 is the photo showing the root growth state of the negative and positive seedlings of the transgenic tobacco in MS3 medium, wherein the roots of the transgenic tobacco grow well; and Fig. 4 is the photo showing the good growth state of the transgenic sterile seedlings after they are transferred to perlite. Fig. 5 is the analytic result of Northern blot of some positive transgenic tobaccos via PCR detection, and the result of hybridization shows that the nucleotide sequence of SEQ ID NO:2 can be expressed in the transgenic tobaccos. Fig.6 and Fig.7 are the photos showing the comparison of the result of an identification for salt and drought tolerance of the transgenic plant strains containing the nucleotide sequence of SEQ ID NO:2. Fig.6 is the picture of the comparison between the transgenic tobaccos and the non-transgenic tobaccos in the medium with Ommol NaCl, and Fig. 7 is the picture of the comparison between the transgenic tobaccos and the non-transgenic tobaccos in the medium with 250mmol NaCl after 15-days incubation, and the transgenic tobaccos can grow in the medium with 250mmol NaCl, while the non-transgenic tobaccos cannot grow in the medium with 250mmol NaCl. THE EXAMPLES OF THE INVENTION The invention will be further described by the following examples. It should be understood that the examples are intended to illustrate the methods of the invention and are not intended to limit the scope of the invention. All experiment conditions not described herein are according to the conventional conditions well known in the art, for example the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturers. Example 1: Expression of the nucleotide sequence of SEQ ID NO:l in E. coli and analysis of the property of salt and drought tolerance 1. Cloning of the nucleotide sequence of SEQ ID NO:l The published genome sequences of Deinococcus radiodurans relate to a pair of PCR specific primers and the whole nucleotide sequences are amplified from the genomic DNA of Deinococcus radiodurans. 2. Construction of Is. coli expression vector and molecular verification The cloned fragments as described above are digested with two enzymes, Nde I and SacII, and linked to vector pTtSacB containing E. coli universal promoter groE to replace SacB gene, in order to produce E. coli expression vector for the genes. E. coli JM109 is transformed with the E. coli expression vector, and spread onto solid LB medium containing Amp. Then the white clones are selected to isolate the plasmids by using alkaline lysis to screen the different recombinants. These obtained recombinants are then digested with Bgl II and verified by sequencing, and one strain of E. coli JM109 containing the nucleotide sequence expression vector is obtained. The analysis of enzyme cutting shows that the gene fragment containing groE promoter is 1.2kb. 3. Verification of salt and drought tolerance of E. coli expression vector E. coli JM109 strain containing SEQ ID NO:l nucleotide sequence expression vector, pMD18T and host strain E. coli JM109 as controls, the OD value of which are all the same, are inoculated into MM medium comprising 0.75M NaCI in 1% inoculum size respectively, shaking cultured at 37°C for 15 hours, and OD value is detected at 550nm. From Fig.l, E. coli JM109 strain containing SEQ ID NO:l nucleotide sequence expression vector can tolerate 0.75M NaCI and it is well grown, whereas E. coli JM109 strain only containing empty expression vector and E. coli JM109 strain cannot grow in the medium with 0.75M NaCl. Example 2: The artificial synthesis of SEQ ID NO:2 nucleotide sequence According to the known SEQ ID NO:l nucleotide sequence, it is firstly divided into 7 regions, and then single strand oligonucleotide fragments in 150-200bp length and with cohesive terminus are synthesized according to the positive strand and the negative strand, respectively. The 7 complementary single strand oligonucleotide fragments corresponding to the positive strand and the negative strand are annealed to form 7 double strands oligonucleotide fragments with cohesive terminus. The double strands oligonucleotide fragments are combined, and assembled catalytically into a whole gene via T4 ligase. The two ends of the synthesized DNA fragments contain Xbal and Sad sites. SEQ ID NO:2 which is artificially synthesized as described above and 5' and 3' end enzyme cutting sites of which are Xbal and SacI is used to construct the plant expression vector with high salt and drought tolerance as described below. Example 3: The eukaryotic cell expression of the nucleotide sequence of SEQ ID NO:2 in the tobacco cells and the identification of the salt and drought tolerance of the transgenic plant. (1) The construction of the expression vector containing the genes of interest Primers for amplifying the whole coding reading frame are designed according to the full length coding sequence (SEQ ID NO:2), and the cutting sites of restriction enzyme are introduced into the positive and negative primers (depending to the selected vectors), in order to construct the expression vector. The amplified product from example 1 is used as template, the sequence of cDNA is cloned into intermediate vector (such as pBluescript) after PCR amplification, and then it is further cloned into a binary expression vector (such as pBI121 and pCAMBIA2200) , the good expression vector is identified on the condition of ensuring the reading frame, it is transferred into Agrobacterium, and the model plant tobacco is transformed using leaf disc cocultivation. (2) Transformation of tobacco using leaf disc cocultivation 1.Picking the positive clones on the selective plate using sterile toothpicks, seed into 2ml YEB liquid (Sm+, Kan+), shaking culture for 24-36 hours at 28 °C, 200rpm; 2. Centrifuging at 4,000g at room temperature for lOmin; 3. Removing the supernatant, suspend the bacteria in 1/2 MS medium, and dilute 5-20 times as much as the initial volume to make the OD600 of liquid to be about 0.5; 4. Taking a sterile leaf of tobacco grown for about two weeks, remove the main vein, cut it into small pieces of about 1cm2; 5. Placing the leaf pieces into the liquid containing the bacteria prepared, immerse for 2-5 min, and suck off the liquid on the sterile filter paper; 6. Placing the infected leaf pieces onto the MS medium and culture for 48 hours at 28°C in the dark; 7. Transfering the leaf pieces onto the Callus medium (MS+6-BA l.Omg/L+NAA O.lmg/L+Kan 50mg/L+cb 250mg/L), culture at 25-28°C in the light and observe the formation of the callus tissues after 7-15 days; 8. Observing the differentiated buds coming out after about 20 days, cut the buds after it has grown up, put them into the root medium (1/2 MS+NAA 0.5mg/L+Kan 25mg/L) to perform rooting culture and observe the roots after about 2-7days; 9. Removing the plant after the root system is large, wash off the attached solid medium with sterile water, transfer it into the soil, cover it with a glass cover for several days at first, remove the cover after the plant is robust and plant it in the greenhouse. Fig.2 is the photo showing the growth state of the transgenic tobacco in MS2 medium, and the growth state is well; Fig.3 is the photo showing the root growth state of the transgenic tobacco negative and positive seedlings in MS3 medium, the growth state of the roots of the transgenic tobacco is well; and Fig. 4 is the photo showing the growth state of the transgenic sterile seedlings after they are transferred to perlite, the growth state is well. (3). Detection of the expression of SEQ ID NO:2 in the transgenic tobacco using Northern blot 1. Extracting RNA: refer to "molecular cloning" (Sambrook et al., 1989). 2. Quantifying RNA: refer to "molecular cloning*' (Sambrook et al., 1989), measure the OD260 using spectrophotometer; calculate the amount of RNA: 1 OD260=40ug/ml. 3. Isolating the total RNA through agarose gel electrophoresis: 1) taking 6ml 25x electrophoretic buffer, add 117ml sterile water and mix. 2) weighting 1.5g agarose into the solution as above, heating it to melt in the micro oven, and transfering it to the water bath at 55 °C. 3) taking 26.8ml formaldehyde in a fume cupboard, add it to the gel solution at 55 °C and mix. 4) pouring it rapidly into the plate for making gel, stand horizontally at the room temperature for 30mins to make the gel set. 5) allowing 30u.g extracted RNA to dissolve in 15ml RNA dilute solution, heat it at 55-65 °C for lOmin and then immediately put it onto the ice. 6) adding 2ul lOx loading buffer to the sample and mix. 7) dotting the sample under the condition that the electrophoresis buffer does not cover the gel, allowing it to electrophorse at 80V for lOmin, add the electrophoresis buffer to exceed the surface of the gel about half-centimeter after the sample comes into the gel totally. Allow it to elecrrophorese at 80-100V amino for 5 hours. 4. Transfer RNA onto the nylon membrane: 1) immersing the nylon membrane into 10x SSC before the transfer. 2) Putting the wetted membrane exactly onto the membrane, immerse two pieces of filter paper as big as the membrane into 2* SCC solution to wet, put them onto the membrane and exclude air bubbles. 3) Place a pile of cleaning paper as big as the membrane onto the filter paper, put a piece of glass and a heavy object onto-the cleaning papers, stand horizontally for the transfer for 12-20 hours. 4) Dry the membrane at 80 °C for 1-2 hours after the transfer. 5. Detect RNA on the membrane: l)immersing the membrane in 4x SSC for lOmin, removing the membrane to put it onto the filter paper to suck the excess liquid, put the membrane into prehybridization solution(50% formamide, 5x SSC, 50mmol/L sodium phosphate(Ph 6.4), 5x Dendart 0.1% SDS, 0.1ing/ml salmon sperm DNA), hybridize at 42 °C overnight. 2) pouring out the prehybridization solution, replace it with the same volume of hybridization solution, put DNA probe labeled with 32P into the boiling water to denature for 5min, add hybridization solution(50% formamide, 5x SSC, 50mmol/L sodium phosphate(Ph 6.4), 10% dextran sulfate, 5x Dendart 0.1% SDS, O.lmg/ml salmon sperm DNA), hybridize at 42 °C for 24-48 hours. 3) removing the membrane, put it into wash buffer I(1*SSC, 1% SDS), wash three times at 42°C, 5min every time. Transfer it into wash buffer II(0.1*SSC, 1% SDS), wash one to three times at 55-65 "C. Press the X-ray film onto the membrane for 1-7 days, develop the film and fix. Fig. 5 is the analytic result of Northern blot of some positive transgenic tobaccos via PCR detection. The result of hybridization of Fig.5 shows that the nucleotide sequence of SEQ ID NO:2 can be expressed in the transgenic tobaccos. (4) Identification of salt and drought tolerance of transgenic plant containing the nucleotide sequence of SEQ ID NO:2 The salt and drought tolerance of transgenic plant is further identified in view the fact that the sequence is confirmed to have salt tolerance in E.coli. The transgenic tobacco and non-transgenic tobacco are cultured in the medium with Ommol or 250mmol NaCl to investigate the viability and development of the plant under observation at 5d, lOd and 15d, respectively. From Fig.6 and Fig.7, the transgenic plant can grow normally in the medium with 250mmol NaCl, whereas non-transgenic tobacco cannot grow in the same medium. It is proved that the sequence has salt tolerance. We Claim: 1. A DNA sequence set forth in SEQ ID N0:1 for improving salt and drought tolerance of a plant. 2. A DNA sequence set forth in SEQ ID N0:2 for improving salt and drought tolerance of a plant. 3. An amino acid sequence encoded by the DNA sequence of claim 1, which is set forth in SEQ ID NO:3. 4. A recombinant vector comprising the DNA sequence of SEQ ID NO:l or SEQ ID NO:2. 5. A host cell transformed with the recombinant vector of claim 4, wherein, the host cell includes prokaryotic cell and eukaryotic cell. 6. The use of the DNA sequence of claim 1 or 2 in improving salt and drought tolerance of a plant. 7. A method of producing a transgenic plant with salt and drought tolerance by using the DNA sequence of claim 1 or 2, which comprises the steps: (1) The sequence set forth in SEQ ID NO:l or SEQ ID NO:2 is operatively linked to a regulation sequence of plant expression to form a plant expression vector; (2) The expression vector of the step (1) is introduced into plant cells to obtain the transformed cells; (3) The transformed cells are obtained via screening and they are ultimately regenerated into transgenic plants and progenies thereof, including seeds and tissues of the plants. 8. A method of detecting the presence of the DNA sequence of SEQ ID NO: 1 or SEQ ID NO:2 in samples, wherein the samples are hybridized with the antibody prepared with SEQ ID NO:l to detect whether the antibody is reacted with the sample probe; the said samples are produced by a PCR amplification, wherein the primers used in the PCR amplification correspond to the nucleotide encoding sequence of SEQ ID NO:l or SEQ ID NO:2 and may be located in flank or middle of the coding sequence, the primers are 15-50 nucleotides in length.

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787-MUMNP-2010-ABSTRACT(7-2-2013).pdf

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