CN101260403B - Plant strong salt-resistant gene AtNHXS1 and its coding protein and application - Google Patents

Abstract

The invention provides a novel plant strong salt tolerant gene AtNHXS1 acquired through DNA reshuffling technology, and the ionic transport activity of Na+/H+ antiporter protein coded by the gene is stronger than that of wild Na+/H+ antiporter protein ATNHX1. A nucleotide sequence of the gene as shown in SEQ ID NO:1 also comprises genes of the nucleotide sequence of sequence 1 in a sequence tablewith the homology between 70 and 100 percent, or a nucleotide sequence of an amino acid sequence of sequence 2 in a coded sequence table. The new Na+/H+ antiporter has a protein of the amino acid sequence of sequence 2 in the sequence table, protein of the amino acid sequence of the sequence 2 with the homology between 70 and 100 percent, or protein which replaces, deletes or adds one or a plurality of amino acids in the amino acid sequence of the sequence 2 with the same homology. The invention also provides the construction of recombinant vector, the transgenic plant and other methods so asto apply the gene and the protein, thereby cultivating a novel variety of transgenic plant with strong capacity of salt tolerance or other improved biologic characters.

Description

The strong resistant gene of salt AtNHXS1 of a kind of new plant and proteins encoded and application

Technical field

The present invention relates to plant genetic engineering field, utilize the DNA shuffling technology to obtain the plant Na that function significantly improves specifically ⁺/ H ⁺The gene of reverse transport protein the invention still further relates to and utilizes this gene to cultivate the transgenic salt-tolerant wheat plant.

Background technology

The soil salinization is an important abiotic stress factor that influences farm crop production and ecotope.At present, nearly in the world 20% arable land and be subjected to serious harm (the Flowler T J of salt marsh near 50% irrigation land used, Yeo A R.Breeding for salinity resistance in crop plants.Where next? Aust.J.Plant Physiol.1995,22:875～884).The nearly 500,000,000 mu of saltingss of China, and its area has ever-increasing trend.Cultivate salt-resistant plant and be and promote the saltings development and use, improve the soil and the effective way of ecological management.Therefore; utilize the modern biology technology; by to plant salt tolerance physiology and molecular biological further investigation; clone and the important resistant gene of salt of plant modification; and then with these gene transformation in the plant to cultivate the transgenic salt-tolerant wheat new variety of plant, all have great importance for agriculture production, environment protection and ecological management etc.

The salinity of high density causes serious injury plant, show that mainly osmotic stress and ion poison two aspects (Blumwald E, Aharon G S, Apse M P.Sodium transport in plant cells.Biochem Biophy Acta.2000,1465:140～151).On the one hand, thus it is osmotic stress that the flow of water that the salinity of soil middle and high concentration makes soil water potential be lower than vegetable cell causes the vegetable cell water deficit; On the other hand, the various salt ions of excessive infiltration vegetable cell can cause ion to poison by pair cell again, mainly are Na ⁺Poison.Increased by the permeability of the plasma membrane of the vegetable cell that osmotic stress and ion poison, cause that peroxidation is coerced, the secondary injury effects such as disorder of nutritional deficiency and a series of physiological metabolism reaction, cause serious harm thereby plant grown normally.

The occurring in nature plant is in order to adapt to the salt of high density, formed a series of defense mechanism (Bohnert H J, Nelson D E, Jensen R G.Adaptation to environmental stress.PlantCell.1995,7:1099～1111), the balanced two kinds of strategies of ion that mainly comprise osmoregulation and reconstituted cell.The former refers to that vegetable cell utilizes some mineral ions or more synthetic small molecules organic compound as Osmolyte regulator, to reduce the cell flow of water, resists osmotic stress.The latter refers to that mainly vegetable cell passes through excessive N a in the tenuigenin ⁺Transport out cell or separating in vacuole, realize the ion equilibrium, eliminate Na ⁺The purpose of poisoning.A notable feature of vegetable cell is exactly to have the isolated vacuole of the very big film of volume, and under the condition of salt stress, vegetable cell keeps tenuigenin lower concentration Na ⁺Direct mode be exactly with Na ⁺Isolate in the vacuole.So both can make the kytoplasm enzyme avoid ion poisons, the osmotic pressure that can improve cell integral body again plays the effect that promotes the cell suction, be the main salt tolerant mechanism of occurring in nature salt-tolerant plant (Flower T J, Troke P F, Yeo A R.Themechanisms of salt tolerance in halophytes.Annu Rev PlantPhysiol.1977,28:89～121.).

Plant Na ⁺/ H ⁺Reverse transport protein is a kind of Na ⁺The antiport body, distribution is all arranged, wherein vacuole skin Na on plasmalemma of plant and vacuole skin ⁺/ H ⁺Reverse transport protein is to be responsible for Na ⁺Separating is to the major protein of vacuole.Plant Na ⁺/ H ⁺Translocator is found on the barley plasma membrane at first, generally detects Na afterwards on various plants plasma membrane and vacuole skin ⁺/ H ⁺The activity of transhipment.From first plant Na in the world ⁺/ H ⁺Reverse transport protein gene A tNHX1 is cloned back (Gaxiola R A, Rao R, Sherman A, Grisafi P, Alper S, and FinkG R.The Arabidopsis thaliana proton transporters, AtNhx1 and Avp1, the vacuole skin Na of high plant such as canfunction in cation detoxify cation in yeast.Proc Natl Acad Sci USA.1999,96:1480～1485), paddy rice, Bei Binli, alkali be fluffy ⁺/ H ⁺The reverse transport protein gene obtains the clone in succession, be respectively OsNHX1 (Fukuda A, Nakamura A, TanakaY.Molecular cloning and expression of the Na+/H+ exchanger gene in Oryzasativa.Biochim Biophys Acta.1999,1446:149～155), AgNHX1 (HamadaA, Shono M, Xia T, Ohta M, Hayashi Y, Tanaka A, Hayakawa T.Isolation andcharacterization of a Na+/H+ antiporter gene from the halophyte Atriplexgmelini.Plant Molecular Biology.2001,46:35～42), SsNHX1 (Ma X L, ZhangQ, Shi H Z, Zhu J K, Zhao Y X, Ma C L, Zhang H.Molecular cloning anddifferent expression of a vacuolar Na+/H+ antiporter gene in Suaeda salsaunder salt stress.Biological Plantarum.2004,48 (2): 219～225) etc.Under the energy that the vacuole skin proton pump provides, be positioned at the Na of vacuole skin ⁺/ H ⁺Reverse transport protein is with Na ⁺Thereby separating enters and reduces Na in the tenuigenin in the vacuole ⁺Concentration, make too much Na ⁺Leave the metabolism site, alleviate its injury, also can reduce the cell flow of water, the osmotic stress that the opposing salinity causes enzyme and film system.So, Na ⁺/ H ⁺Reverse transport protein plays an important role to the salt resistance of plant.

With Na ⁺/ H ⁺Reverse transport protein (Na ⁺/ H ⁺Antiporter) gene transformation is carried out the effective means that overexpression is a cultivation transgenic salt-tolerant wheat plant in target plant.Apse etc. analyze overexpression Na to the Arabidopis thaliana plant that changes the AtNHX1 gene ⁺/ H ⁺The transgenic arabidopsis plant of reverse transport protein can grow by normal growth in 200mmol/L NaCl.Immunoblotting shows, contains in transformed plant blade purification vacuole than more AtNHX1 product from wild-type blade purification vacuole, Na on the vacuole ⁺/ H ⁺The reverse transport protein vigor significantly strengthens (Apse M P, Aharon G S, Snedden W A, Blumward E.Salt toleranceconferred by overexpression of a vacuolar Na+/H+ antiporter inArabidopsis.Science.1999,285:1256～1258).Equally, Arabidopis thaliana AtNHX1 gene is changed in the tomato, the transgenosis tomato can normal growth under 200mmol/L NaCl coerces, bloom and solid.Although Na in the blade ⁺Concentration height, but Na in the tomato fruit ⁺Concentration very low (Zhang H X, Blumward E.Transgenic salt-tolerant tomato plants accumulate salt infoliage but not in fruit.Nature Biotechnology.2001,19:765～768).These researchs have all shown Na ⁺/ H ⁺Reverse transport protein gene and albumen thereof, are considered to be hopeful to utilize its characteristic to cultivate the gene of transgenic salt-tolerant wheat kind at present most for the importance in the high reactant salt plant.

Yet, on production application, the Na that in plant, has cloned at present ⁺/ H ⁺The reverse transport protein gene still exists the problem that activity is not high, salt tolerance is not strong, has limited the production application and the popularization of this gene to a great extent.How to utilize specific modern biology technology; clone new strong resistant gene of salt or this gene is carried out molecular evolution and transformation; and then with these new gene transformation in the plant to cultivate the transgenic salt-tolerant wheat new variety of plant, all have great importance for agriculture production, environment protection and ecological management etc.

DNA reorganization (DNA Shuffling) technology is the method rapidly and efficiently of lactam enzyme by directional anagenesis in vitro such as present protein, enzyme and monoclonal antibody, is with a wide range of applications at the aspects such as performance that improve enzymic activity, protein output and improve protein (enzyme).Its principle is the nucleotide sequence random fragmentation that a group is closely related, utilize no primer PCR that these random fragments are re-assemblied then and obtain the nucleotide sequence of total length, introducing suddenlys change and different sudden changes is recombinated widely in this process, thereby finish rapid evolution to the purpose nucleotide sequence, with the function that improves nucleotide sequence or its encoded protein (Stemmer W P.DNA shuffling byrandom fragmentation and reassembly:in vitro recombination for molecular evolution.Proc Natl Acad Sci USA.1994,91:10747-10751).

The DNA shuffling technology is since being born, successfully several genes such as industrial enzymes, antibody and some important protein etc. have been carried out orthogenesis, specificity, protein function, thermostability that makes activity, substrate specificity and the antibody of enzyme etc. obtained significant raising (Crameri A, Raillard S, Bermudez E, StemmerW P.DNA shuffling of a family of genes from diverse species acceleratesdirected evolution.Nature.1998,391:288).A series of achievements in research have been obtained as this technology of application such as Stemmer, obtain enzyme work and improved 32000 times β-Nei Xiananmei (Stemmer WP.Rapid evolution of a protein in vitro by DNA shuffling.Nature.1994,370:389～391), fluorescence intensity surpasses green fluorescent protein (the Crameri A of 45 times of wild-types, Whitehorn EA, Tate E, Stemmer W P.Improved green fluorescent protein by molecularevolution using DNA shuffling.Nat Biotechnol.1996,14:315～319).By DNA reorganization method, Crameri etc. have successfully obtained the arsenate resistance and have improved 40 times bacterial strain (Crameri A, Dawes G, Rodriguez E J, Silver S, Stemmer W P.Molecular evolution of anarsenate detoxification pathway by DNA shuffling.Nat Biotechnol.1997,15:436～438).But, use this technology and improving Na ⁺/ H ⁺Reverse transport protein gene and proteinic aspect of performance thereof there is no report both at home and abroad.

Summary of the invention

The object of the invention is to obtain the strong resistant gene of salt AtNHXS1 of a kind of new plant, and this gene can be encoded and be had the active Na of ion transport ⁺/ H ⁺Reverse transport protein.

Another object of the present invention is to provide the Na of this genes encoding ⁺/ H ⁺Reverse transport protein, the Na of its ion transport specific activity wild-type ⁺/ H ⁺Reverse transport protein is stronger.

The present invention also provides the recombinant vectors that contains said gene.

Another aspect of the present invention also provides the host cell that contains above-mentioned recombinant plasmid.

Another aspect of the present invention also provides the methods such as structure, transgenic plant of recombinant vectors, cultivates the stronger transgenic plant new variety of salt tolerance to use said gene and proteins encoded.

Na ⁺/ H ⁺Reverse transport protein (Na ⁺/ H ⁺Antiporter) in plant salt tolerance, play an important role the salt tolerance of its active affects plant.The present invention is by the DNA shuffling technology, to coding Na ⁺/ H ⁺The wild-type resistant gene of salt AtNHX1 of reverse transport protein carries out external molecular evolution, to obtain the higher Na of coding ion transport activity ⁺/ H ⁺The new gene of reverse transport protein.The Na of this new coded by said gene ⁺/ H ⁺Reverse transport protein can be with more Na ⁺Separating be in vacuole from tenuigenin, thereby the new gene transformation that will obtain can be cultivated the stronger transgenic plant new variety of salt resistance ability in target plant.

Specifically, by the Na of external DNA shuffling technology to wild-type ⁺/ H ⁺The reorganization that suddenlys change of reverse transport protein gene is by the method for having complementary functions, at Na ⁺/ H ⁺The active significantly enhanced Na of directed screening ion transport in the yeast mutant of reverse transport protein genetically deficient ⁺/ H ⁺The reverse transport protein new gene that suddenlys change.

The present invention relates to a kind of new gene A tNHXS1 that can improve the plant anti-salt ability, its nucleotide sequence such as SEQ ID NO:1 also comprise the nucleotide sequence of aminoacid sequence shown in the coding SEQ ID NO:2.

The present invention relates to a kind of albumen that can improve the plant anti-salt ability, its aminoacid sequence such as SEQ IDNO:2.

Technical scheme of the present invention is as follows:

Na ⁺/ H ⁺The clone of reverse transport protein gene: can adopt several different methods such as pcr amplification method, recombination method or synthetic method to obtain Na ⁺/ H ⁺Reverse transport protein Nucleotide full length sequence or its fragment.Such as, based on Na ⁺/ H ⁺Reverse transport protein gene order design polynucleotide probes screens goal gene from cDNA library or genomic library, also can directly amplify relevant sequence from cDNA or genome with the pcr amplification method.

Na ⁺/ H ⁺The DNA reorganization of reverse transport protein gene: the substrate that is used for DNA reorganization can be the Na of organism Different Individual, strain system or kind ⁺/ H ⁺The reverse transport protein gene also can be the Na of the different mutant forms produced by ordinary methods such as fallibility PCR, rite-directed mutagenesises ⁺/ H ⁺The reverse transport protein gene.

To Na ⁺/ H ⁺The key step that the reverse transport protein gene carries out DNA reorganization is: at first use DNase I or restriction enzyme to Na ⁺/ H ⁺The reverse transport protein gene carries out random fragmentation, and the small segment that reclaims wherein is an overlapping fragments.Then under the situation that does not add primer, with archaeal dna polymerase carry out Primerless PCR make small segment each other primer and template increase, thereby different sudden changes is recombinated widely., PrimerPCR obtains containing reorganization Na after increasing ⁺/ H ⁺The recombination storehouse of reverse transport protein gene fragment.

Contain reorganization Na ⁺/ H ⁺The recombination storehouse of reverse transport protein gene fragment can be transferred in a kind of cell or the organism and screen as bacterium, yeast or vegetable cell.For example, the recombination storehouse that the present invention can be obtained is connected among any Yeast expression carrier such as the pYPGE15, utilizes method well known in the art such as transformed yeast Na such as lithium acetate technology, electric commentaries on classics method ⁺/ H ⁺Among reverse transport protein mutant such as the W303 Δ ena1-4 Δ nhx1, construction expression recombination storehouse.Yeast transformant being coated in the APG selectivity flat board that contains high density NaCl with high salt is that selective pressure is carried out high flux screening, obtains the mutant strain that the salt tolerance significantly improves.

From the yeast mutant that the salt tolerance that screens significantly improves, extract plasmid, obtain new anti-high salt Na wherein by checking order ⁺/ H ⁺The nucleotide sequence of reverse transport protein gene, the biosoftware that utilization is conventional such as DNA Star, Clustalx and TMpred etc. are to anti-high salt Na ⁺/ H ⁺Reverse transport protein gene and wild-type Na ⁺/ H ⁺The reverse transport protein gene carries out bioinformatic analysis such as nucleotide sequence comparison, aminoacid sequence comparison and wetting ability hydrophobicity analysis.The result shows, Na ⁺/ H ⁺Reverse transport protein AtNHXS1 is than not reorganizing Na ⁺/ H ⁺The reverse transport protein gene has stronger ion transport activity, can be with more Na ⁺Separating from kytoplasm in vacuole, make the host cell that imports this gene have better salt tolerance.

Anti-high salt Na ⁺/ H ⁺The new gene A tNHXS1 of reverse transport protein is as goal gene, can be structured in the Ti-plasmid binary vector of any expression of plants such as on the pBISN1, the T-DNA 25bp tumor-necrosis factor glycoproteins that LB and RB are wherein arranged, mannosaminic acid synthase promoter (Pmas), polyA no terminator, also have the neomycin phosphotransferase II (nptII) that uses as selective marker in eukaryote, the microbiotic that is used to select is a kantlex.Promotor in the plant expression vector can be any constitutive promoter, tissue-specific promoter or environmental induction type promotor, as cauliflower mosaic virus (CAMV) 35S promoter, Ubiqutin promotor.Enhanser in the carrier both can be a transcriptional enhancer, also can be translational enhancer.For the ease of transgenic plant cells or plant being identified and screening, the marker (as kantlex, weedicide etc.) that the alternative mark (gus gene, luciferase gene etc.) of plant also should be arranged in the carrier or have resistance such as microbiotic.

By research method well known in the art such as freeze-thaw method, electric shocking method etc. above-mentioned recombinant vectors is imported in the Agrobacterium, carry out Agrobacterium-mediated Transformation.Can pass through conventional biotechnological meanss such as microinjection, particle gun, agriculture bacillus mediated, pollen tube channel method with anti-high salt Na ⁺/ H ⁺The new gene A tNHXS1 of reverse transport protein changes in the plant, cultivates the kind that anti-salt is best in quality and the improved plant of biological character is new.By the plant transformed host both can be that monocotyledons also can be a dicotyledons, as: Arabidopis thaliana, tobacco, tomato, rape, paddy rice, wheat, corn, soybean, vegetables, trees, flowers, herbage and turfgrass etc.Gene of the present invention is for cultivating the salt-resistant plant kind, and improve crop yield, improve the ecological environment etc. has great importance.

Transgenosis recombinant vectors of the present invention can be used as the kind, strain of commercial use or directly uses on producing or carry out agro-ecology breeding and transgenic plant to improve the salt resistance of crop as genetic resources.

Description of drawings

The AtNHXS1 gene that Fig. 1 obtains for reorganization with do not reorganize the AtNHX1 gene in the two mutant strain W303-1B Δ ena1-4 Δ nhx1 of yeast function complementation experiment relatively, promptly following cell at pH5.5, contain 0, growing state on the APG flat board of 50mM, 75mM, 100mM and 125mM NaCl:

Wherein I is the wild-type W303-1B that contains the PYPGE15 plasmid, II is the W303-1B Δ ena1-4::HIS3 that contains the PYPGE15 plasmid, III is the W303-1B Δ ena1-4::HIS3 Δ nhx1::TRP1 that contains the PYPGE15 plasmid, IV is the W303-1B Δ ena1-4::HIS3 Δ nhx1::TRP1 that contains the AtNHX1-PYPGE15 recombinant plasmid, and V is the W303-1B Δ ena1-4::HIS3 Δ nhx1::TRP1 of AtNHXS1-PYPGE15 recombinant plasmid.

Embodiment

Embodiment 1. Arabidopis thaliana Na ⁺/ H ⁺The clone of reverse transport protein gene

With TRizol reagent total RNA of extracting Arabidopis thaliana from the Arabidopis thaliana spire, be that masterplate carries out reverse transcription with total RNA, synthetic cDNA first chain.With synthetic cDNA is masterplate, with reference to the cDNA sequence of AtNHX1, designs following two sections primers (5 ' end comprises SmaI and Sal I restriction enzyme site respectively):

AtR(5’-GC GTCGACTCAAGCCTTACTAAGATCAGGAGG-3’)

AtF(5’-TCC CCCGGGATGTTGGA?TTCTCTAGTG-3’)

Obtain Arabidopis thaliana Na by pcr amplification ⁺/ H ⁺Reverse transport protein gene A tNHX1 is connected in the pGM-T carrier, and changes escherichia coli DH5a over to, and the sequence of the AtNHX1 of being cloned into is definite by checking order.

The DNA reorganization of embodiment 2.AtNHX1 gene

1) preparation of parent material is masterplate with pGM-T-AtNHX1, with the Taq archaeal dna polymerase AtNHX1 is carried out pcr amplification, the parent material of reorganizing as DNA behind the purifying.

2) DNase I at random enzyme cut the AtNHX1 of 10 μ g purifying, join (10mM Tris-HCl, pH7.4,50mM MnCl in the 50 μ l endonuclease reaction systems ₂), 15 ℃ are reacted 10min down.Add DNase I0.15U, mixing, 15 ℃, 2min, 90 ℃, the 10min. product downcuts the segmental gel and the recovery that contain 100-200bp by 2.5% agarose gel electrophoresis.

3) no primer PCR is got the small segment that 1 μ g reclaims, and joins (10 * Pfu buffer, each 0.2mM of dNTP, 0.6U/ μ l Pfu polysaccharase) in the 50 μ l reaction systems.The PCR program is a reaction conditions: 94 ℃ of pre-sex change 60s, and 94 ℃ of sex change 30s, 50 ℃ of annealing 30s, 72 ℃ are extended 30s, are total to 40cycles, and last 72 ℃ are extended 5min.

4) there is primer PCR to get product that 1 μ l do not have primer PCR, the AtNHX1 full length gene sequence of reorganization carried out pcr amplification with AtR and two primers of AtF as template.The PCR reaction system is (10 * Pfu damping fluid, each 0.2mM of dNTP, every kind of each 30 μ M of primer, a 0.6U Pfu polysaccharase), the PCR response procedures is: 94 ℃ of pre-sex change 60s, 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 30s, are total to 30cycles, and last 72 ℃ are extended 5min.

By DNA reorganization, obtained to contain reorganization Na ⁺/ H ⁺The recombination storehouse of reverse transport protein gene fragment.

Embodiment 3. expresses the structure and the anti-high salt Na in reorganization library ⁺/ H ⁺The screening of reverse transport protein gene

The recombinant products that embodiment 2 is obtained with Sal I and Sma I carries out enzyme and cuts, be connected to behind the purifying among the Yeast expression carrier pYPGE15 that same enzyme cuts, the expression reorganization library that will make up with the lithium acetate method imports among the yeast mutant W303-1B Δ ena1-4::HIS3 Δ nhx1::TRP1, being applied to the APG that contains 100mM NaCl (not containing uridylic) selects on the substratum, 30 ℃, cultivate 2d, carry out anti-high salt Na ⁺/ H ⁺The high flux screening of reverse transport protein gene.Finally obtain yeast mutation of a normal growth, contained the strong resistant gene of salt AtNHXS1 of new plant.

The sequential analysis of embodiment 4. anti-high salt gene AtNHXS1

The plasmid that extracts in halophilic yeast mutation with yeast plasmid extraction test kit carries out dna sequencing.With softwares such as DNA Star, Clustalx and TMpred to anti-high salt Na ⁺/ H ⁺Reverse transport protein gene and wild-type Na ⁺/ H ⁺The reverse transport protein gene carries out nucleotide sequence comparison, aminoacid sequence comparison and wetting ability hydrophobicity analysis.

The result shows, AtNHXS1 gene and wild-type Na ⁺/ H ⁺Between the reverse transport protein gene A tNHX1 sudden change of 7 Nucleotide is arranged, and the disappearance of 34 Nucleotide is arranged, this disappearance causes the premature termination of AtNHXS1 gene translation.AtNHXS1 proteins encoded and wild-type Na ⁺/ H ⁺4 amino acid whose changes are arranged between the reverse transport protein, and 296 amino acid whose disappearances are arranged at C-terminal.As sequence table SEQ ID NO:1 and SEQ ID NO:2.

The structure of embodiment 5. recombinant vectorss and host cell

With Sal I and Sma I the recombinant plasmid pGM-T-AtNHXS1 that obtains among the embodiment 3 being carried out enzyme cuts, be connected in the Yeast expression carrier PYPGE15 that same enzyme is cut after reclaiming, with the lithium acetate method AtNHXS1-PYPGE15 recombinant plasmid that makes up is imported among the yeast double-mutant strain W303-1B Δ ena1-4::HIS3 Δ nhx1::TRP1, in not containing the APG selective medium of uridylic, screen, obtain positive transformant.

The comparative experiments that has complementary functions of embodiment 6. yeast

Wild-type yeast W303-1B is a kind of yeast with salt tolerance, its mutant strain W303-1B Δ ena1-4::HIS3 Δ nhx1::TRP1 salt tolerance is very low, the recombinant vectors that will contain the AtNHXS1 gene imports this yeast mutant, and comparative experiments can have complementary functions.

With Sal I and Sma I recombinant plasmid pGM-T-AtNHX1 being carried out enzyme cuts, be connected in the Yeast expression carrier PYPGE15 that same enzyme is cut after reclaiming, with the lithium acetate method recombinant plasmid that makes up is imported among the yeast double-mutant strain W303-1B Δ ena1-4::HIS3 Δ nhx1::TRP1, Yeast expression carrier PYPGE15 with sky imports to respectively among wild-type yeast W303-1B, yeast mutant W303-1B Δ ena1-4::HIS3 and the W303-1B Δ ena1-4::HIS3 Δ nhx1::TRP1 simultaneously, screens in not containing the APG selective medium of uridylic.The positive transformant that screens and the anti-high salt yeast strain that screens be inoculated into respectively in the APG liquid nutrient medium cultivate, with OD ₆₀₀All adjust to 0.5, carry out 5 times of dilutions respectively, respectively get 5 μ l dibblings to the APG flat board of the different salt concn of pH5.5 (0,50mM, 75mM, 100mM, 125mM NaCl), 30 ℃, cultivate 2d, observe growing state.

The result lacks Na as shown in Figure 1 ⁺/ H ⁺The yeast mutant W303-1B Δ ena1-4::HIS3 Δ nhx1::TRP1 (III) of reverse transport protein gene than the yeast strain W303-1B (I) of wild-type to salt-sensitive many, the yeast mutant W303-1B Δ ena1-4::HIS3 Δ nhx1::TRP1 (IV) that expresses AtNHX1 can the complementary zymic salt tolerance of part, and the AtNHXS1 gene can be given the stronger salt tolerance of yeast mutant (V), and this shows the Na that this reorganization obtains ⁺/ H ⁺Reverse transport protein AtNHXS1 is than not reorganizing Na ⁺/ H ⁺Reverse transport protein has stronger ion transport activity, can be with more Na ⁺Separating from kytoplasm in vacuole.

Sequence table

Claims (6)

1. the strong resistant gene of salt AtNHXS1 of new plant is characterized in that, its gene order is the polynucleotide of protein sequence shown in the SEQ ID NO:2 in the code sequence tabulation.

2. the strong resistant gene of salt AtNHXS1 of the described new plant of claim 1 is characterized in that, its gene order is shown in the SEQ ID NO:1 in the sequence table.

3. the proteins encoded of the strong resistant gene of salt AtNHXS1 of new plant is for having the active Na of ion transport ⁺/ H ⁺Reverse transport protein is characterized in that, its aminoacid sequence is shown in SEQ ID NO:2 in the sequence table.

4. the recombinant vectors that contains claim 1 or 2 described genes.

5. the host cell that contains claim 1 or 2 described genes.

6. claim 1 or the 2 described a kind of new application of the strong resistant gene of salt AtNHXS1 of plant aspect the cultivation salt-resistant plant, it is characterized in that, the strong resistant gene of salt AtNHXS1 of described plant can change in the plant by microinjection, particle gun, method agriculture bacillus mediated, pollen tube channel, cultivates the improved new variety of plant of salt tolerance and biological character.

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