CN105073771A

CN105073771A - Tonoplast sodium-hydrogen antiport protein NHX1 from thellungiella halophila, and coding gene and application thereof

Info

Publication number: CN105073771A
Application number: CN201380074515.XA
Authority: CN
Inventors: 王建胜; 王君丹; 陈淼; 梁丽
Original assignee: Genesis Seed Industry Co ltd
Current assignee: Genesis Seed Industry Co ltd
Priority date: 2013-04-22
Filing date: 2013-04-22
Publication date: 2015-11-18
Anticipated expiration: 2033-04-22
Also published as: CN105073771B; WO2014172824A1

Abstract

A tonoplast sodium-hydrogen antiport protein NHX1 from thellungiella halophila, and a coding gene thereof, an application in breeding a transgenic plant with improved salt tolerance.

Description

Tonoplast sodium-hydrogen antiport protein NHX1 from thellungiella halophila, and coding gene and application thereof

The present invention relates to vegetable protein and its encoding gene and application with applied technical field by a kind of small salt mustard tonoplast sodium hydrogen antiporter protein NHX1 and its encoding gene, the more particularly to one tonoplast sodium hydrogen antiporter protein NHX1 and its encoding gene from small salt mustard, and its application in the genetically modified plants that salt tolerance is improved are cultivated.

Background technology salt stress is that world agriculture produces one of most important abiotic stress harm, and salt-affected soil as influence plant growth, causes the principal element of grain and the industrial crops underproduction generally based on sodium salt, calcium salt or magnesium salts.The area of saline-alkali soil there are about 400,000,000 hectares in the world, account for the 1/3 of irrigated farmland.Salt-soda soil is extensive in distribution in China, about 0. 4 hundred million hectares of existing saline alkali land area.As China human mortality increases, cultivated land area, the exploitation of saline alkali land resource have extremely important realistic meaning.Then it is to utilize salt-soda soil economy, effective measures and Genes For Plant Tolerance is saline and alkaline, Drought resistance raising and suitable in saline and alkaline aerial and plant species or the seed selection of strain with higher economy and the ecological value.For most crops, most plants can only be grown on the soil that sodium chloride content is less than 0. 3%, excessive Na in soil to saline and alkaline, arid poor resistance⁺Toxic action can be produced to the normal growth metabolism of plant.Therefore how the problem of crop yield just turns into particularly significant in whole world agricultural production is improved under salt marsh environment.

The salt tolerance of plant is a sufficiently complex quantitative character, and its Mechanisms of Salt Resistance is related to from plant to organ, tissue, Physiology and biochemistry are until each level of molecule.The scientist of various countries has also done substantial amounts of work for this, and achieves many new developments, especially in terms of the salt tolerant molecule mechanism using high model plant arabidopsis to study plant, makes the research in the field and has breakthrough progress（Zhu JK. 2002. Salt and drought stress singal transduction in plants. Annu. Rev. Plant Biol. 53： 1247-1273； Zhang ZL. 2011. Arabidopsi s Floral Initiator SKBl Confers High Salt Tolerance by Regulating Transcription and Pre-mRNA Spl icing through Altering Hi stone H4R3 and Smal l Nuclear Ribonucleoprotein LSM4 Methylation. Plant Cel l, 23 : 396 - 411 ) .Higher plant cell can have number of ways to experience the change of physico-chemical parameter in external environment, so as to which extracellular signal is changed into intracellular signal, finally stress signal is transferred in nucleus by the signal transduction of series, activating transcription factor, and activating transcription factor is remake for functional gene, start the expression of induced gene in adversity to improve the resistance of reverse of plant.Although never ipsilateral has carried out numerous studies to researcher, because its mechanism is sufficiently complex, many major issues in plant anti-salt still need to be explored.For example, the key factor of plant anti-salt is not found yet；The molecular mechanism of plant salt tolerance is simultaneously It is not fully aware of.

Content of the invention the present inventor utilizes SSH (Subtractive hybridizations）With RACE (cDNA ends rapid amplifyings）The method being combined has cloned a tonoplast sodium hydrogen antiporter protein for small salt mustard（Be named as NHX1 herein) encoding gene, and determine its DNA sequence dna.And it was found that being conducted into by transgenosis after plant, the salt tolerance of transfer-gen plant is can obviously improve, and these characters can stablize heredity.

First aspect present invention provides a tonoplast sodium hydrogen antiporter protein NHX1 for small salt mustard encoding gene (being named as 7 tt herein), and its sequence is SEQ ID NO: 2.

Second aspect of the present invention provides a kind of recombinant expression carrier, and it contains the gene described in first aspect present invention, and it is inserted into a kind of expression vector by the gene and obtained, it is preferable that the expression vector is

PCAMBIA2300;And the nucleotide sequence of the gene is operably connected with the expression control sequence of the recombinant expression carrier；Preferably, the recombinant expression carrier is the 35S-ThNHXl-2300 carriers shown in accompanying drawing 2.

Third aspect present invention provides a kind of recombinant cell, and it contains the recombinant expression carrier described in gene or second aspect of the present invention described in first aspect present invention；Preferably, the recombinant cell is restructuring agrobatcerium cell.

Fourth aspect present invention provides a kind of method of improvement plant salt endurance, including：Gene described in first aspect present invention or the recombinant expression carrier described in second aspect of the present invention are imported into plant or plant tissue and make the gene expression；Preferably, the plant is arabidopsis.

Fifth aspect present invention provides a kind of method of prepare transgenosis plant, including：The plant containing gene described in first aspect present invention or the recombinant expression carrier described in second aspect of the present invention or plant tissue are cultivated under conditions of plant is effectively produced；Preferably, the plant is arabidopsis.

Gene, the recombinant expression carrier described in second aspect of the present invention or the recombinant cell described in third aspect present invention that sixth aspect present invention is provided described in first aspect present invention are used to improve plant salt endurance and the purposes for plant breeding；Preferably, the plant is arabidopsis.

Seventh aspect present invention is provided as the protein of the gene code described in first aspect present invention, its amino acid sequence such as SEQ ID NO:Shown in 1.Brief description of the drawings

Fig. 1 is the plant expression vector of NHX1 genes（35S-ThNHXl-2300) build flow（Scheme la-lb).Fig. 2 is the plant expression vector of NHX1 genes（Plasmid figure 35S-ThNHXl-2300). Fig. 3 is the test plant arabidopsis of culture.Fig. 4 is salt tolerant experimental results of the T1 for plant of ThNHXl transgenic arabidopsis, and Tla4 shows obvious salt tolerance, and Tlal7, Tlal9 result are similar with its, are not shown here.

Fig. 5 be using reverse transcription 1^1 to 1 in transgenic tobacco plant and non-transgenic reference plant ThNHXl genes transcriptional level progress molecular level detection result.M is DNA Ladder Marker (DL2000), and 1-4 is the control Arabidopsis plant of not salt tolerant, and 13 be plasmid PCR positive control（35S-ThNHXl-2300 plasmids）, 5-12 is salt tolerant T1 for transgenic Arabidopsis plants.

Embodiment provides following examples, to facilitate those skilled in the art to more fully understand the present invention.The embodiment is not intended to limit the scope of the present invention merely for exemplary purpose.

The restriction enzyme in the unreceipted source mentioned in example below is purchased from New England Biolabs companies.Small salt mustard SSH library constructions under the salt stress of embodiment 1.：

Specific method is：

According to the PCR-select of Clontech companies^TMMethod shown in cDNA Subtraction Kit kit specifications builds SSH libraries by Subtractive hybridization method（Subtracted library）.The mRNA extracted in experimentation using in growth course in the small salt mustard tissue of salt treatment is used as sample（Tester), the mRNA extracted using in untreated small salt mustard tissue is used as control（Driver) .Comprise the following steps that：

(1) material to be tested：

Small salt mustard（TheUungieUa halophila, purchased from inner mongolia Ba Yan Nor City carex meyeriana green plants garden halophytes Breeding Center）It is seeded on the vermiculite of sterilizing, in 22 °C, 12 hours photoperiods illumination/12 hour dark（Light intensity 3000-4000 Lx) under the conditions of cultivate, 1/2MS culture mediums are poured weekly（Contain 9.39 mM KN0₃ , 0.625 mM KH₂P0₄, 10.3 mM NH4NO3,0.75 mM MgS0₄, 1.5 mM CaCl₂, 50 μ Μ KI, 100 μ Μ H₃B0₃, 100 μ Μ MnS0₄, 30 μ Μ ZnS0₄, 1 μ Μ Na₂Mo0₄, 0.1 μ Μ CoCl₂, 100 μ Μ Na₂EDTA, 100 μΜ FeS0₄)-secondary.It is used to test when plant diameter reaches 5-6 cm.

(2) material process：

2 groups, every group of 4 basins, per 3 plants of basin will be divided into for examination plant.First group is control group, is normally poured with 1/2MS；Second group is salt treatment group, the 1/2MS solution containing 300 mM NaCl is poured, by two groups of plants in 22 °C, 12 hours photoperiods illumination/12 hour dark（Light intensity 3000-4000 Lx) under the conditions of culture processing 10 days, then in time collect two groups of plant（Root is cleaned with leavened water is steamed）, after liquid nitrogen quick freeze, preserved in -70 °C of refrigerators. (3) Total RNAs extraction：

Control group and the small g of salt mustard 3.0 of salt treatment group are taken respectively, use plant RNA extracts kit（Purchased from Invitrogen) extract total serum IgE.Gained total serum IgE is determined in 260 nm and 280 nm absorbance, OD with the ultraviolet specrophotometer U-2001 of HITACHI companies₂₆。/OD₂₈.Ratio is 1.8-2.0, shows that total serum IgE purity is higher, detects the integrality of total serum IgE with 1.0% agarose gel electrophoresis, the brightness of 28S bands is about 2 times of 18S bands, shows that RNA integrality is good.Use the Oligotex mRNA purification kits of Qiagen companies（PolyA+ RNA are purified from total serum IgE) separation mRNA.

(4) Subtractive hybridization：

By the PCR-select of Clontech companies^TMMethod shown in cDNA Subtraction Kit kit specifications carries out Subtractive hybridization.Driver mRNA and Tester mRNA are first distinguished into reverse transcription, double-strand cDNA, then the progress subtractive hybridization using 2 μ g Tester cDNA and 2 μ g Driver cDNA as parent material is obtained.Then the Tester cDNA after digestion are divided into joints different in two equal portions, connection by Tester cDNA and Driver cDNA Rsa I digestions 1.5 hours respectively under 37 °C of water-baths, and Driver cDNA are not connected to head.Two kinds of Tester cDNA for being connected with different joints are mixed with excessive Driver respectively, carry out positive subtractive hybridization for the first time.The products of two kinds of positive subtractives hybridization for the first time are mixed, then second of positive subtractive is carried out with the Driver cDNA that are newly denatured and are hybridized, pass through the fragment of the amplification enrichment difference expression genes of inhibition PCR twice（Before PCR is carried out, second of positive subtractive hybrid product carries out end-filling）.

(5) structure of subtracted library and preliminary screening, clone, identification

According to pGEM-T Easy kits（Purchased from Promega) specification, described second positive subtractive is hybridized to second of inhibition pcr amplification product of cDNA fragments（Purified using QIAquick PCR Purification Kit, purchased from Qiagen) it is connected with pGEM-T Easy carriers, it is comprised the following steps that：Following ingredients are sequentially added in 200 l PCR pipes：Second μ 1 of PCR primer 3, the μ 1 of 2 Χ Τ, 4 ligase buffer solutions, 5 μ pGEM-T Easy carriers 1, the μ 1 of T4 DNA ligases 1 are purified, is stayed overnight in 4 °C of connections.Then the coupled reaction products of 10 μ 1 are taken, it is added in the competence e. coli jm109s of 100 μ 1 (being purchased from TAKARA), ice bath 30 minutes, 42 °C of heat shocks 60 seconds, ice bath 2 minutes, separately plus the LB fluid nutrient mediums of 250 μ 1（Contain 1% tryptone（Tryptone, purchased from OXOID), 0.5% yeast extract（Yeast Extract, purchased from OXOID) 1% NaCl of standing grain P (are purchased from traditional Chinese medicines））After be placed in 37 °C of shaking tables, with 225 rpm shaken cultivations 30 minutes, the bacterium solutions of 200 μ 1 are then therefrom taken to be inoculated in LB (ibid)/X-gal (the chloro- 3- indoles-β-D- galactosides of 5- bromo- 4)/IPTG (isopropyl-β-D- Thiogalactopyranosides containing 50 g/ml ampicillins）On (X-gal/IPTG is purchased from TAKARA) culture plate, 37 °C are cultivated 18 hours.Count diameter in culture plate>1 mm clear white and blue colonies, random 450 white colonies of picking（Numbering：Th-S001 to Th-S450).Institute picking white colony clone is inoculated in 96 porocyte culture plates（CORNING add after the LB fluid nutrient mediums containing 50 μ g/ml ampicillins in), 37 °C of overnight incubations Glycerine to final glycerol concentration is 20% (volume ratio）, then saved backup in -80 °C.To the colony clone cultivated with nest-type PRC primer Primer 1 and Primer 2R (the PCR-select cDNA Subtraction Kit kits from Clontech companies）Bacterium solution PCR amplification checkings are carried out, 342 positive colonies is obtained, then send Invitrogen by all positive colonies（Shanghai）Trade Co., Ltd is sequenced.

(6) the cDNA sequencing analysis of differential cloning：

After the cDNA that DNA sequencing result is removed to carrier and indefinite sequence and redundancy, 301 effective expression sequence labels (Expressed sequence tag, EST) (Unigene) are obtained.

The small salt mustard tonoplast sodium hydrogen antiporter gene ThNHXl of embodiment 2 clone

Clone from bacterium colony Th-S211 in the small salt mustard SSH libraries of the identification is removed after redundant DNA, sequence is SEQ ID No:3, sequence analysis shows that the albumen of the sequential coding belongs to tonoplast sodium hydrogen antiporter protein.Herein by SEQ ID No:The corresponding total length encoding gene of 3 sequences is named as 7 NHJW, and its corresponding albumen is named as NHX1.

SEQ ID No: 3：

1 ACCTCTTCTT CACTAGCACA ATCCTCGGAA TTGCTGTGGG GTTAGTAACG TCTTATGTCC 61 TGAAAACCTT GTATTTTGGA AGACACTCAA CTACACGTGA ACTCGCGATC ATGGTTCTTA

121 TGGCATACCT TTCATATATG TTAGCTGAGC TCTTCTCATT AAGTGGGATT CTCACCGTTT

181 TCTTCTGTGG CGTTTTAATG TCGCATTATG CATCTTATAA TGTGACAGAA AGTTCAAGAA

241 TCACTTCAAG GCATGTGTTT GCAATGTTGT CCTTTATAGC GGAGACGTTC ATATTTTTAT

301 ATGTTGGAAC AGATGCTCTT GATCTTACAA AGTGGAAGAC AAGCAGCTTA AGCGTTGGGG 361 GT

The clone of ThNHXl total length encoding genes

According to the SEQ ID No obtained:3 sequences, design following two specific primers, are used as 3 ' RACE 5 ' end primers.

ThNHXl GSP1 : SEQ ID No: 4：

TCTTCTGTGG CGTTTTAATG TC

ThNHXl GSP2: SEQ ID No: 5：

TCACTTCAAG GCATGTGTTT GCA

Experimental procedure is operated by kit specification（3 ' RACE System for Rapid Amplification of cDNA Ends kits are purchased from Invitrogen companies）.

With SEQ ID NO:4 (kit is carried with universal primer AUAP）, the cDNA of the mRNA reverse transcriptions extracted using the small salt mustard of salt treatment group is template progress first round PCR amplification.Comprise the following steps that：

The PCR reaction systems of 50 μ 1：The mM of 5 μ, 13 μ of Ι Ο Χ Ε χ Buffer 1 2.5 dNTP, the cDNA of the mRNA reverse transcriptions of 2.0 μ 1, the Ex Taq of 1.0 μ 1 (being purchased from TAKARA), 10 μ Μ primer SEQ ID NO:Each μ distilled waters of 2.0 μ 1 and 35 of 4 and AUAP.PCR reaction conditions：94 °C of pre-degenerations 5 minutes, 33 circulations（94 °C are denatured 30 seconds, and 58 °C are annealed 30 seconds, and 72 °C extend 1 minute）, 72 °C extend 10 minutes.The PCR primer of gained takes 2.0 μ as template after diluting 50 times with distilled water, with SEQ ID NO:5 carry out second with universal primer AUAP takes turns PCR amplifications, comprises the following steps that：

50 y l PCR reaction systems：The first round PCR primer of the mM of 5 μ, 13 μ of lO X Ex Buffer 1 2.5 dNTP, 2.0 μ 1 dilution, 10 μM of 1.0 μ, 1 Ex Taq primer SEQ ID NO:Each μ 1 of 2.0 μ 1 and 35 of 5 standing grain P AUAP distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 minutes, 33 circulations（94 °C are denatured 30 seconds, and 58 °C are annealed 30 seconds, and 72 °C extend 1 minute）, 72 °C extend 10 minutes.Reclaim the band that fragment in second of PCR primer is about 800 bp（Gel Extraction Kit are purchased from OMEGA), and pGEM-T Easy carriers are coupled with, then it is transformed into escherichia coli jm109 competent cell（Specific method is ibid）, and screened on the LB solid mediums containing 50 g/mL ampicillins.Random 10 white colonies of picking are inoculated in the LB fluid nutrient mediums containing 50 g/ml ampicillins, and glycerol adding is to (the volume ratio of final concentration 20% after 37 °C of overnight incubations）, -80 °C save backup.With SEQ ID NO:5 carry out bacterium solution PCR amplifications with universal primer AUAP, obtain 6 positive colonies, 4 positive colonies are delivered into Invitrogen（Shanghai）Trade Co., Ltd is sequenced, and obtains the cDNA of the gene 3 ' ends.

According to the 77 NHJW genetic fragments obtained, following three specific primers are designed, 5 ' RACE 3 ' end primers are used as.

ThNHXl GSP3： SEQ ID No: 6：

AGTGATTCTT GAACTTTCTG TC

ThNHXl GSP4: SEQ ID No: 7：

AACATATATG AAAGGTATGC CA

ThNHXl GSP5 : SEQ ID No: 8：

GATCGCGAGT TCACGTGTAG TTG

Experimental procedure is operated by kit specification（5 ' RACE System for Rapid Amplification of cDNA Ends kits are purchased from Invitrogen companies）.

With SEQ ID NO:7 (kit is carried with universal primer AAP）, cDNA (the reverse transcription primer SEQ ID NO of the mRNA reverse transcriptions extracted with the small salt mustard of salt treatment group:6) amplification of first round PCR is carried out for template, comprised the following steps that：

The PCR reaction systems of 50 μ 1：The mM of 5 μ, 13 μ of lO X Ex Buffer 1 2.5 dNTP, the cDNA of the mRNA reverse transcriptions of 2.0 μ 1, the Ex Taq of 1.0 μ 1 (being purchased from TAKARA), 10 μ Μ primer SEQ ID NO:Each μ of 2.0 μ 1 and 35 of 7 and AAP distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 minutes, 33 circulations（94 °C are denatured 30 seconds, and 55 °C are annealed 30 seconds, and 72 °C extend 1 minute）, 72 °C extend 10 minutes.

The PCR primer of gained takes 2.0 μ as template after diluting 50 times with distilled water, with SEQ ID NO:8 with Primer AUAP carries out second and takes turns PCR amplifications, comprises the following steps that：

50 y l PCR reaction systems：The first round PCR primer of the mM of 5 μ, 13 μ of lO X Ex Buffer 1 2.5 dNTP, 2.0 μ 1 dilution, 10 μM of 1.0 μ, 1 Ex Taq primer SEQ ID NO:Each μ 1 of 2.0 μ 1 and 35 of 8 standing grain P AUAP distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 minutes, 33 circulations（94 °C are denatured 30 seconds, and 58 °C are annealed 30 seconds, and 72 °C extend 1 minute）, 72 °C extend 10 minutes.Reclaim the band that fragment in second of PCR primer is about 800 bp（Gel Extraction Kit are purchased from OMEGA), and pGEM-T Easy carriers are coupled with, then it is transformed into escherichia coli jm109 competent cell（Specific method is ibid）, and screened on the LB solid mediums containing 50 g/mL ampicillins.Random 10 white colonies of picking are inoculated in the LB fluid nutrient mediums containing 50 g/ml ampicillins, and glycerol adding to final glycerol concentration is 20% (volume ratio after 37 °C of overnight incubations）, -80 °C save backup.With SEQ ID NO:8 carry out bacterium solution PCR amplifications with primer AUAP（Reaction system and reaction condition are ibid）, 7 positive colonies are obtained, wherein 4 clones is chosen and delivers to Invitrogen（Shanghai）Trade Co., Ltd is sequenced, and obtains the cDNA of the gene 5 ' ends.After the 5'RACE product clonings sequencing of gained, by itself and 3'RACE products sequencing result and SEQ ID No:3 sequences are spliced.Obtain 7 full length cDNA sequence SEQ ID No: 9：

1 GTGTCTACTT TGTTTGCGAT TTGTGTTGTA TTAAGGATCT AGAATCGACG AAAGTGAAAA

61 ACATTTTTCG AAAGTCTTTT TTTGTTGGTC GTGAAAATGG GTGTTGGATT TACAGAGTTT

121 TTTTCGATAC ACCTAGCCAC GGAGCATCCT CAGGTGATAC CAATCTCGGT GTTCATCGTC

181 ATTCTCTGCC TCTGTTTAGT TATCGGCCAC TTGCTTGAAG AGAACCGATG GGTCAATGAA

241 TCCATTACCG CCATTTTAGT CGGAGCAGTA TCAGGAACGG TTATATTGCT TATTAGTAGA

301 GGAAAGAGTT CTCACATTTT GGTGTTTGAT GAAGAACTCT TCTTCATATA CCTTCTTCCT

361 CCAATCATTT TCAATGCTGG ATTCCAAGTC AAGAAAAAGA AGTTTTTTCA CAACTTTTTA

421 ACCATCATGT CGTTTGGTGT GATTGGTGTT TTCATCTCCA CTGTCATTAT TTCGTTTGGC

481 ACTTGGTGGC TTTTTCCCAA GTTGGGATTT AAGGGATTGA GTGCTCGAGA CTATCTTGCC

541 ATCGGAACGA TTTTCTCGTC AACTGATACC GTTTGCACTC TACAGATTCT TCATCAAGAT

601 GAAACTCCAT TGCTATACAG CTTAGTCTTT GGAGAAGGAG TAGTGAATGA TGCAACCTCA

661 GTTGTGCTGT TCAACGCCGT GCAGAAGATT CACTTCGAAA GCCTCAACGG TTGGACGGCT

721 CTGCGAGTCT TTGGAAACTT TTTGTACCTC TTCTTCACTA GCACAATCCT CGGAATTGCT

781 GTGGGGTTAG TAACGTCTTA TGTCCTGAAA ACCTTGTATT TTGGAAGACA CTCAACTACA

841 CGTGAACTCG CGATCATGGT TCTTATGGCA TACCTTTCAT ATATGTTAGC TGAGCTCTTC

901 TCATTAAGTG GGATTCTCAC CGTTTTCTTC TGTGGCGTTT TAATGTCGCA TTATGCATCT

961 TATAATGTGA CAGAAAGTTC AAGAATCACT TCAAGGCATG TGTTTGCAAT GTTGTCCTTT

1021 ATAGCGGAGA CGTTCATATT TTTATATGTT GGAACAGATG CTCTTGATCT TACAAAGTGG

1081 AAGACAAGCA GCTTAAGCGT TGGGGGTACT TTGGGTGTCT CCGGTGTCAT AACCACATTA

1141 GTATTGCTTG GACGAGCAGC GTTTGTGTTT CCACTCTCGG TTTTTACAAA TTTCATGAAT

1201 AGAAACACTG AAAGAAGCGA GACTATCACA TTTAAGCACC AGGTGATTAT TTGGTGGGCT

1261 GGGCTTATGC GAGGTGCTGT CTCAATTGCT CTGGCTTTCA AACAGTTCAC ATACTCTGGT

1321 GTTACATTGG ATCCTGTGAA TGCTGCCATG GTCACAAACA CCACTATCGT TGTCCTCTTT

1381 ACTACACTGG TCTTTGGTTT CCTCACAAAG CCACTTGTGA ACTATCTGCT TCCTCATGAT

1441 GCAAATCAAA ACACTGGAGG AAATGGAGGT AAACACACTG CGCCAGGTTC CCCGAGGGAA

1501 GATGCGACGC TTCCTCTCCT CTCCTTTGAC GAGTCTGCTT CCACCAACTT CAACAGAGCT

1561 AAAGATAGTA TCTCCCTTCT GATGGAACAG CCTGTTTACA CTATCCACCG CTACTGGAGG 1621 AAGTTCGACG ATACATACAT GAGACCTATC TTTGGTGGAC CTCGTCGAGA TAATCAACCA

1681 GAATGCTAGT TTATGATTTA GGTTCTCCTC AGGGAATGCA TGATGAGTTA GTTTTTTTGG

1741 ATAGTGGATG TAGTGAAAAA CCAGTATATG TTATAGTTTT CTTTAATGTA TAGAACAAGG

1801 TTCTTCTATA TACACAAGGT GATCGAAGAT CATTTCAAAA AAAAAAAAAA AAA

According to SEQ ID NO:9 sequences Design pair of primers are as follows:

SEQ ID No: 10：

ATGGGTGTTGGATT TACAGAGTT SEQ ID No: 11：

CTAGCATTCT GGTTGATTAT CTC pass through SEQ ID NO:10 and SEQ ID NO:11 clone ThNHXl total length encoding genes.

Using TaKaRa PrimeSTAR HS archaeal dna polymerases, performing PCR reaction is entered by template of the cDNA of the small salt mustard of salt treatment group.50 y l PCR reaction systems：The X PS Buffer of 10 y l 5, the mM of 3 μ 1 2.5 dNTP, the cDNA of 2.0 μ 1, the PrimeSTAR of 1.0 μ 1,10 μ Μ primer SEQ ID NO:10 standing grain mouthful SEQ ID NO:

11 each μ 1 of 2.0 μ 1 and 30 distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 minutes, (94 °C are denatured 30 seconds, and 58 °C are annealed 30 seconds, and 72 °C extend 2 minutes for 33 circulations）, 72 °C extend 10 minutes.

Pcr amplification product adds A tails：The absolute ethyl alcohol of 2.5 times of volumes is added in PCR primer, -20 °C are placed 10 minutes, centrifugation is removed supernatant, dried, and is then dissolved with 21 μ distilled waters.Then the mM of 2.5 0.5 μ of μ Ι Ο Χ Ε χ Buffer 15 dATP, the Ex Taq of 1.0 μ 1 is added thereto.Reaction condition：70 °C are reacted 30 minutes.The DNA fragmentation of about 1600 obtained bp is reclaimed（Omega QIAquick Gel Extraction Kits）, and be connected on pGEM T-easy carriers（Obtain ThNHXl-pGEM plasmids）, then (method is ibid in conversion escherichia coli jm109 competent cell）, and screened on the LB solid mediums containing 50 g/mL ampicillins.Random 10 white colonies of picking are inoculated in the LB fluid nutrient mediums containing 50 g/ml ampicillins, and glycerol adding is to (the volume ratio of final concentration 20% after 37 °C of overnight incubations）, -80 °C save backup.With SEQ ID NO:10 and SEQ ID NO:11 carry out bacterium solution PCR amplifications（Reaction system and reaction condition are ibid）, 5 positive colonies are obtained, wherein 4 positive colonies is chosen and delivers to Invitrogen（Shanghai）Trade Co., Ltd is sequenced, and gained sequence is SEQ ID NO:2, the amino acid sequence of its protein encoded is SEQ ID NO: 1.

The amino acid sequence of NHX1 albumen： SEQ ID NO: 1

1 MGVGFTEFFS IHLATEHPQV

21 IPISVFIVIL CLCLVIGHLL

41 EENRWVNESI TAILVGAVSG

61 TVILLISRG SSHILVFDEE

81 LFFIYLLPPI IFNAGFQV

101 FFHNFLTI MSFGVIGVFI

121 STVIISFGTW WLFP LGF G 141 LSARDYLAIG TIFSSTDTVC

161 TLQILHQDET PLLYSLVFGE

181 GVVNDATSVV LFNAVQKIHF

201 ESLNGWTALR VFGNFLYLFF

221 TSTILGIAVG LVTSYVL TL

241 YFGRHSTTRE LAIMVLMAYL

261 SYMLAELFSL SGILTVFFCG

281 VLMSHYASYN VTESSRITSR

301 HVFAMLSFIA ETFIFLYVGT

321 DALDLT W T SSLSVGGTLG

341 VSGVITTLVL LGRAAFVFPL

361 SVFTNFMNRN TERSETITF

381 HQVI IWWAGL MRGAVSIALA

401 F QFTYSGVT LDPVNAAMVT

421 NTTIVVLFTT LVFGFLT PL

441 VNYLLPHDAN QNTGGNGG legs

461 TAPGSPREDA TLPLLSFDES

481 ASTNFNRAKD SISLLMEQPV

501 YTIHRYWR F DDTY put PIFG

521 GPRRDNQPEC *

The nucleotide sequence SEQ ID NO of ThNHXl genes:： 2

1 ATGGGTGTTG GATTTACAGA GTTTTTTTCG ATACACCTAG CCACGGAGCA TCCTCAGGTG

61 ATACCAATCT CGGTGTTCAT CGTCATTCTC TGCCTCTGTT TAGTTATCGG CCACTTGCTT

121 GAAGAGAACC GATGGGTCAA TGAATCCATT ACCGCCATTT TAGTCGGAGC AGTATCAGGA

181 ACGGTTATAT TGCTTATTAG TAGAGGAAAG AGTTCTCACA TTTTGGTGTT TGATGAAGAA

241 CTCTTCTTCA TATACCTTCT TCCTCCAATC ATTTTCAATG CTGGATTCCA AGTCAAGAAA

301 AAGAAGTTTT TTCACAACTT TTTAACCATC ATGTCGTTTG GTGTGATTGG TGTTTTCATC

361 TCCACTGTCA TTATTTCGTT TGGCACTTGG TGGCTTTTTC CCAAGTTGGG ATTTAAGGGA

421 TTGAGTGCTC GAGACTATCT TGCCATCGGA ACGATTTTCT CGTCAACTGA TACCGTTTGC

481 ACTCTACAGA TTCTTCATCA AGATGAAACT CCATTGCTAT ACAGCTTAGT CTTTGGAGAA

541 GGAGTAGTGA ATGATGCAAC CTCAGTTGTG CTGTTCAACG CCGTGCAGAA GATTCACTTC

601 GAAAGCCTCA ACGGTTGGAC GGCTCTGCGA GTCTTTGGAA ACTTTTTGTA CCTCTTCTTC

661 ACTAGCACAA TCCTCGGAAT TGCTGTGGGG TTAGTAACGT CTTATGTCCT GAAAACCTTG

721 TATTTTGGAA GACACTCAAC TACACGTGAA CTCGCGATCA TGGTTCTTAT GGCATACCTT

781 TCATATATGT TAGCTGAGCT CTTCTCATTA AGTGGGATTC TCACCGTTTT CTTCTGTGGC

841 GTTTTAATGT CGCATTATGC ATCTTATAAT GTGACAGAAA GTTCAAGAAT CACTTCAAGG

901 CATGTGTTTG CAATGTTGTC CTTTATAGCG GAGACGTTCA TATTTTTATA TGTTGGAACA

961 GATGCTCTTG ATCTTACAAA GTGGAAGACA AGCAGCTTAA GCGTTGGGGG TACTTTGGGT

1021 GTCTCCGGTG TCATAACCAC ATTAGTATTG CTTGGACGAG CAGCGTTTGT GTTTCCACTC

1081 TCGGTTTTTA CAAATTTCAT GAATAGAAAC ACTGAAAGAA GCGAGACTAT CACATTTAAG

1141 CACCAGGTGA TTATTTGGTG GGCTGGGCTT ATGCGAGGTG CTGTCTCAAT TGCTCTGGCT

1201 TTCAAACAGT TCACATACTC TGGTGTTACA TTGGATCCTG TGAATGCTGC CATGGTCACA

1261 AACACCACTA TCGTTGTCCT CTTTACTACA CTGGTCTTTG GTTTCCTCAC AAAGCCACTT

1321 GTGAACTATC TGCTTCCTCA TGATGCAAAT CAAAACACTG GAGGAAATGG AGGTAAACAC

1381 ACTGCGCCAG GTTCCCCGAG GGAAGATGCG ACGCTTCCTC TCCTCTCCTT TGACGAGTCT

1441 GCTTCCACCA ACTTCAACAG AGCTAAAGAT AGTATCTCCC TTCTGATGGA ACAGCCTGTT The plant expression vector construction of the ThNHXl genes of 1501 1561 GGACCTCGTC GAGATAATCA ACCAGAATGC TAG embodiments of TACACTATCC ACCGCTACTG GAGGAAGTTC GACGATACAT ACATGAGACC TATCTTTGGT 3

Plant binary expression vector pCAMBIA2300 is selected (to be purchased from Beijing DingGuo ChangSheng Biology Technology Co., Ltd）As plant expression vector, the 35S promoter that Ν Ρ Τ Π genes contain double enhancers is replaced with Pnos promoters, to reduce expression of the Ν Ρ Τ Π albumen in plant.Select 35S promoter and Tnos terminators respectively as the promoter and terminator of ThNHXl genes, build flow chart as shown in Figure 1.

With primer SEQ ID NO:12 and SEQ ID NO:13 (are purchased from ocean Science and Technology Ltd. of Beijing China with plant expression vector pBI121）For template amplification Pnos, using TaKaRa PrimeSTAR HS archaeal dna polymerases.50 μ PCR reaction systems：The mM of 10 μ, 53 μ of X PS Buffer 1 2.5 dNTP, the ρ Β Ι 121 of 1.0 μ 1, the μ Μ of 1.0 μ, 1 PrimeSTAR 10 primer SEQ ID NO:12 standing grain P SEQ ID NO:13 each μ 1 of 2.0 μ 1 and 31 distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 minutes, 33 circulations（94 °C are denatured 30 seconds, and 56 °C are annealed 30 seconds, and 72 °C extend 30 seconds）, 72 °C extend 10 minutes.Kit explanation is pressed as PCR primer of EcoRI, Bglll digestion by obtained by（Promega, T4 connect enzyme reagent kit）It is connected to pCAMBIA2300 and obtains pCAMBIA2300-l.

SEQ ID NO: 12

GCACGAATTC ggcgggaaac gacaatctga

SEQ ID NO: 13

ATCCAGATCTAGATCCGGTGCAGATTATTTG

With primer SEQ ID NO:14 standing grain P SEQ ID NO:15 using pBI121 as template amplification Tnos, using TaKaRa PrimeSTAR HS archaeal dna polymerases.50 l PCR reaction systems：The X PS Buffer of 10 μ 15, the mM of 3 μ 1 2.5 dNTP, the Prime STAR of 1.0 μ, 1 pBI121,1.0 μ 1,10 μ Μ primer SEQ ID NO:14 and SEQ ID NO:15 each 2.0 μ and 31 μ distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 minutes, 33 circulations（94 °C are denatured 30 seconds, and 58 °C are annealed 30 seconds, and 72 °C extend 30 seconds）, 72 °C extend 10 minutes.Connected as PCR primer of Kpnl, EcoRI digestion by obtained by（Promega T4 connection enzyme reagent kits）PCAMBIA2300-2 is obtained to pCAMBIA2300-l.

SEQ ID NO: 14：

AAGGGTAACGAATTTCCCCGATCGTTCAAA SEQ ID NO: 15：

TCAGAATTCCCAGTGAATTCCCGATCTAGTA primer SEQ ID NO:16 and SEQ ID NO:17 open by template amplification 35S of pCAMBIA2300 Mover.Using TaKaRa PrimeSTAR HS archaeal dna polymerases.The PCR reaction systems of 50 μ 1：The mM dNTP of 10 μ, 153 l of X PS Buffer 2.5, the Prime STAR of 1.0 μ, 1 pCAMBIA2300,1.0 μ 1,10 μM of primer SEQ ID NO:16 standing grain P SEQ ID NO:17 each 2.0 μ and 31 μ distilled waters.PCR reaction conditions：94 °C of pre-degenerations 5 minutes, 33 circulations（94 °C are denatured 30 seconds, and 58 °C are annealed 30 seconds, and 72 °C extend 30 seconds）, 72 °C extend 10 minutes.Connected as PCR primer of HindIII, Sail digestion by obtained by（Connection method is ibid）PCAMBIA2300-3 is obtained to pCAMBIA2300-2.

SEQ ID NO: 16：

ACTAAGCTTTAGAGCAGCTTGCCAACATGGTG SEQ ID NO: 17：

TGAGTCGACAGAGATAGATTTGTAGAGAGAGACT primer SEQ ID NO:18 and SEQ ID NO:The full length sequence of 19 7 tt encoding genes of amplification（Template is that embodiment 2 obtains positive ThNHXl-pGEM plasmids）, using TaKaRa PrimeSTAR HS DNA polymerases.50 l PCR reaction systems：The X PS Buffer of 10 l 5, the mM of 3 μ 1 2.5 dNTP, the PrimeSTAR of 1.0 μ, 1 ThNHXl-pGEM, 1.0 μ 1,10 μ Μ primer SEQ ID NO:18 standing grain P SEQ ID NO:19 each 2.0 μ and 31 μ distilled waters.PCR reaction conditions：94 °C of pre-degenerations 5 minutes, 33 circulations（94 °C are denatured 30 seconds, and 58 °C are annealed 30 seconds, and 72 °C extend 2 minutes）, 72 °C extend 10 minutes.Connected as the PCR primer of Sal l, Kpn l digestions by obtained by（Connection method is ibid）To pCAMBIA2300-3, plant expression vector 35S-ThNHXl-2300 (Fig. 2) is obtained.

SEQ ID NO: 18

ACTGTCGACATGGGTGTTGGATT TACAGAGTTT

SEQ ID NO: 19

ACTGGTACCCTAGCATTCT GGTTGATTAT CTCG

The 35S-ThNHXl-2300 expression vectors of embodiment 4 convert Agrobacterium

The preparation of Agrobacterium LBA4404 (being purchased from Biovector Science Lab, Inc) competent cell：Agrobacterium LBA4404 was drawn to single spot inoculation on the LB solid mediums containing 50 μ g/ml rifampins and 50 μ g/ml streptomysins in 1-2 days in advance, 28 °C are cultivated 1 to 2 day.Picking single bacterium colony is inoculated in LB fluid nutrient mediums of 5 ml containing 50 μ g/ml rifampins and 50 μ g/ml streptomysins, and overnight incubation is shaken under 28 °C（About 12-16 hours）To OD_6QQIt is worth for 0.4, formation seed bacterium solution.Take the bacterium solution after 5 ml culture activation（1 :20 ratio）It is inoculated in LB fluid nutrient mediums of 100 ml containing 50 μ g/ml rifampins and 50 μ g/ml streptomysins, 28 °C of shakes cultivate 2-2.5 hours to OD_6QQ=0.8.Ice bath bacterium solution 10 minutes, shook up once every 3 minutes, made the bacterium even into resting state.Centrifuged 10 minutes in 4000 g under 4 °C, abandon supernatant；Add 10% (volume ratio of 1 ml ice precoolings）It is sweet Oily resuspension thalline, 4000 g are centrifuged 10 minutes under 4 °C, collect precipitation；Ice-cold 10% (volume ratio）Glycerine repeats to wash 3-4 times；Then 10% (volume ratio of appropriate ice precooling is added）Again suspended bacterial is precipitated glycerine, that is, LBA4404 competent cells are made, is dispensed, is saved backup in -70 °C with 40 μ/pipe.

Convert Agrobacterium：Melt described LBA4404 competent cells on ice, the plasmid 35S-ThNHXl-2300 of the embodiments 3 of 1 μ 1 acquisition, ice bath about 10 minutes after mixing are added into 40 μ 1 competent cell.The mixture of competent cell after ice bath and 35S-ThNHXl-2300 plasmids is transferred in the electric shock of ice precooling cup (being purchased from Bio-Rad) with liquid-transfering gun, rapping makes suspension reach electric shock cup bottom, has been careful not to bubble.The electric shock cup is put on the slideway of electroporation chamber, promotes slideway to put electric shock cup to electroporation chamber base electrode.Using 0.1 cm specifications electric shock cup when, MicroPulser (be purchased from Bio-Rad) program is set to " Agr ", and electric shock is once.Electric shock cup is taken out immediately, adds 200 μ Ι Β culture mediums of 28 °C of preheatings.It is quickly soft to be beaten competent cell with liquid-transfering gun.Suspension is transferred to 1.5 ml centrifuge tube, 225 rpm shake culture 1 hour under 28 °C.100-200 μ bacterium solution is taken to be coated on corresponding resistance screening culture medium flat plate（LB solid mediums, containing 50 y g/ml rifampins, 50 y g/ml streptomysins, 50 g/ml kanamycins）, 28 °C of cultures.Positive transformants clone is screened, and its bacterium solution is saved backup in -70 °C.The acceptor material arabidopsis culture of embodiment 5

Good water absorption is selected, the soft vermiculite of soil property coordinates Nutrition Soil（1 :1) as arabidopsis planting soil.The cm of diameter 9 flowerpot, 20-30 is sowed per basin.The film in flowerpot upper cover later is sowed, the growth to plant provides the environment of a moistening.22 °C of constant temperature, intensity of illumination 3500-4000 lx, periodicity of illumination is 12 hours dark, 12 hours illumination cultivations, pour a 1/2MS within every 7 days, after culture 30 days, retain 4_5 plant, periodicity of illumination is adjusted to 8 hours dark, 16 hours illumination cultivations, after treating most of plant all boltings, whole main tongue is cut in inflorescence base portion, its apical dominance is removed, 4-6 newborn side tongue is grown after about 1 week at axillary bud position, when side tongue inflorescence formation bud and part are bloomed or form 1-2 silique, just available for converting（Fig. 3).The leaching conversion of the thaliana flower of embodiment 6：

The Agrobacterium bacterium solution for having converted expression vector that embodiment 4 is obtained is seeded to containing 10-50 μ g/ml kanamycins（Kan overnight incubation in LB culture mediums), the next morning presses 1：50 are seeded in the new LB culture mediums containing antibiotic（1L), about 8 hours, agrobacterium liquid 0D are cultivated₆..Should be between 1. 0 to 1. 2.The rpm of room temperature 5000 is centrifuged 5 minutes, abandons supernatant, and Agrobacterium precipitation is suspended in osmotic medium（1/2MS;5% sucrose；PH5. 7 is adjusted to K0H;0. 02%Si lwet L-77), make 0D₆..0. 8 or so.The top of the arabidopsis for being used to convert prepared by embodiment 5 slowly, in spiral immersion inoculation medium, gently rock clockwise, about 2 minutes, with saturating Bright plastic jacket covers tightly to keep humidity, is put into greenhouse and stays overnight.Plastic, transparent cover is removed after 24 hours, is irrigated with water.2-3 weeks afterwards, it is ensured that plant moisture is sufficient.When plant stops blooming, and first Fruit pod maturation turns yellow, entangled with paper bag, after all Fruit pods in paper bag turn yellow, stop watering, laboratory is fetched after drying in 1-2 weeks, transformant selection is carried out, while taking the arabidopsis Fruit pod of unconverted processing as control.The screening of the arabidopsis positive transformant of embodiment 7：

Seed disinfection：First soaked 10 minutes with 70% ethanol, to make seed suspension every now and then in above-mentioned processing；Then washed four times with sterile, preferably also make seed suspension every now and then in this step processing.Seed after processing is uniformly coated on vernalization 2 days on the 1/2MS solid screening and culturing primary surfaces containing 50 g/ml kan（The at most sowing 1500 of the plate of one piece of 150 mm diameter）, 22 °C of constant temperature, intensity of illumination 3500-4000 k, periodicity of illumination is 12 hours dark, 12 hours illumination cultivations, is cultivated 7-10 days.Transgenic seed is determined whether according to upgrowth situation.More than 4 true leaves can be gone out by normal growth on resistance culture base by being successfully transferred to the seed of recombinant plasmid.Non-transgenic seed is unable to normal growth, is only capable of growing 2 cotyledons, the growth of root is also heavily suppressed, general to sprout death later in 10 days.After transgenic seed is sprouted 2 weeks on MS+kan flat boards, positive plant is transferred to soil and continues to cultivate, transgenic arabidopsis SEQ ID NO:18 and SEQ ID NO:19 do PCR detections, remove negative plant, collect positive plant seed, label： T0al-T0a25.Embodiment 8 is overexpressed ThNHXl plantations of the transgenic arabidopsis T1 for plant

Good water absorption is selected, the soft vermiculite of soil property coordinates Nutrition Soil（1 :1) as arabidopsis planting soil. TO_aThe each transformants of l-TOa20 sow 2 basins, and control arabidopsis sows 2 basins, 20-30 seed is sowed per basin.The film in flowerpot upper cover later is sowed, the growth to plant provides the environment of a moistening.22 °C of constant temperature, intensity of illumination 3500-4000 lx, periodicity of illumination is 12 hours dark, 12 hours illumination cultivations, pours a 1/2MS within every 7 days, after cultivating 25 days, transgenic arabidopsis SEQ ID NO:18 and SEQ ID NO:19 do PCR detections, remove negative plant, retain the positive seedling of 12-14, continue after cultivating 10 days, choose transgenic arabidopsis of the same size, control arabidopsis and do salt tolerant experiment, per 7-9 more consistent seedling of basin reservation size.The transgenic arabidopsis T1 that embodiment 9 is overexpressed ThNHXl is tested for the salt tolerant of plant

Transgenic arabidopsis, control each basin of arabidopsis are not dealt with, it is normal to pour 1/2MS, transgenic arabidopsis, each basin of control arabidopsis pour the 1/2MS containing 150 Mm NaCl, constant temperature 22V, intensity of illumination 3500-4000 lx, optical culture/12 hour light culture circulation in 12 hours.Observation experiment result after 10 days：T1 is for the transfer-gen plant (plant that T0 grows up to for the seed of transfer-gen plant）Salt-Tolerance Identification show that T1 shows obvious salt tolerance for tri- strains of transfer-gen plant Tla4, Tlal7, Tlal9（See Fig. 4, with Tla4, Tla7, Tlal9's As a result it is similar with its, it is not shown here）Embodiment 10 verifies in the expression embodiments 9 of 7 tt genes the good T1 of salt tolerant for randomly selecting 8 in transfer-gen plant on transcriptional level（It is belonging respectively to above three salt-resistance strain）, adjoining tree randomly selects 4 in embodiment 9, and each g of the clip salt treatment blade of 14 days 0.05 uses plant RNA extraction kit（Invitrogen total serum IgE) is extracted.Total serum IgE calculates each RNA concentration in 260 nm and 280 nm absorbance obtained by ultraviolet spectrophotometry.Reverse transcription is carried out according to method shown in Invitrogen reverse transcription reagent box Superscript III Reverse Transcriptase（1 μ g total serum IgEs are used as template, reverse transcription primer SEQ ID NO: 11 ) .Pass through SEQ ID NO:10 and SEQ ID NO: 20 ( SEQ ID NO : 20：ATTCCGAGGA TTGTGCTAGT GA) amplification ThNHXl, detects its transcription situation.Using TaKaRa PrimeSTAR HS archaeal dna polymerases, performing PCR reaction is entered by template of the cDNA of above-mentioned reverse transcription.50 y l PCR reaction systems：The X PS Buffer of 10 μ 15, the mM of 3 μ 1 2.5 dNTP, the cDNA of 2.0 μ 1,1.0 μ l PrimeSTAR, 10 μM of primer SEQ ID NO:10 standing grain P SEQ ID NO:20 each μ of 2.0 μ 1 and 30 distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 minutes, 32 circulations（94 °C are denatured 30 seconds, and 58 °C are annealed 30 seconds, and 72 °C extend 1 minute）, 72 °C extend 10 minutes.Product electrophoresis result is as shown in Figure 5：M is DNA Ladder Marker (DL2000, purchased from Shenzhen Rui Zhen Bioisystech Co., Ltd）, 1-4 is plasmid PCR positive control for not salt tolerant control Arabidopsis plant, 13（35S-ThNHXl-2300 plasmids）, 5-12 is to quote thing Arabidopsis plant in salt tolerant T1 generations.Stripe size shown in figure and positive control it is in the same size（About 700 bp).As a result show, salt tolerant T1 is stronger for the transcription of ThNHXl in transgenic Arabidopsis plants there is no ThNHXl transcription in salt tolerant control Arabidopsis plant.

Claims

Claims

1. a tonoplast sodium hydrogen antiporter protein for small salt mustard, its amino acid sequence such as SEQ ID NO:Shown in 1.

2. encode the gene of the tonoplast sodium hydrogen antiporter protein of claim 1, its nucleotide sequence such as SEQ ID

NO:Shown in 2.

3. a kind of recombinant expression carrier, it is obtained by the way that the gene of claim 2 is inserted into a kind of expression vector, and the nucleotide sequence of the gene is operably connected with the expression control sequence of the expression vector, preferably, the expression vector is pC AMBI A2300.

4. the recombinant expression carrier of claim 3, it is the 35S-ThNHXl-2300 carriers shown in accompanying drawing 2.

5. the recombinant expression carrier of a kind of recombinant cell, its gene for containing claim 2 or claim 3 or 4；Preferably, the recombinant cell is restructuring agrobatcerium cell.

6. a kind of method of improvement plant salt tolerance, including：Recombinant expression carrier described in gene or claim 3 or 4 described in claim 2 is imported into plant or plant tissue and makes the gene expression；Preferably, the plant is arabidopsis.

7. a kind of method of prepare transgenosis plant, including：Plant or the plant tissue of the recombinant expression carrier containing the gene described in claim 2 or claim 3 or 4 are cultivated under conditions of plant is effectively produced.

8. the method described in claim 7, wherein the plant is arabidopsis.

9. the recombinant cell described in gene, the recombinant expression carrier or claim 4 of claim 3 or 4 described in claim 2 is used to improve plant salt endurance and the purposes for plant breeding.

10. the purposes described in claim 9, wherein the plant is arabidopsis.