CN104379746B

CN104379746B - One cotton ion channel albuminoid and its encoding gene and application

Info

Publication number: CN104379746B
Application number: CN201280004135.4A
Authority: CN
Inventors: 王建胜; 田大翠; 何云蔚; 崔洪志
Original assignee: Biocentury Seed Industry Co Ltd
Current assignee: Biocentury Seed Industry Co Ltd
Priority date: 2012-08-13
Filing date: 2012-08-13
Publication date: 2017-08-18
Anticipated expiration: 2032-08-13
Also published as: CN104379746A; WO2014026310A1

Abstract

There is provided a kind of ionophorous protein NHX1 1 and its encoding gene from cotton, and its application in the genetically modified plants that salt tolerance is improved are cultivated.

Description

One cotton ion channel albuminoid and its encoding gene and application

Technical field

The present invention relates to ion channel albuminoid and its encoding gene and application, more particularly to one from cotton Ion channel albuminoid NHX1-1 and its encoding gene, 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 earth is generally with sodium salt, calcium on salt marsh Based on salt or magnesium salts, as influence plant growth, cause the principal element of grain and the industrial crops underproduction.Saline-alkali soil in the world Area there are about 400,000,000 hectares, account for the 1/3 of irrigated farmland.In distribution in China extensively, existing saline alkali land area about 0.4 hundred million is public in salt-soda soil Hectare.As China human mortality increases, cultivated land area, the exploitation of saline alkali land resource have extremely important realistic meaning.And plant Thing salt resistance alkali, the raising of Drought resistance and suitable in saline and alkaline aerial and plant species with higher economy and the ecological value Or the seed selection of strain, then it is to utilize salt-soda soil economy, effective measures.For most crops, most plants pair Saline and alkaline, arid poor resistance, can only be grown on the soil that sodium chloride content is less than 0.3%, excessive Na in soil⁺Meeting Toxic action is produced to the normal growth metabolism of plant.Therefore crop yield how is improved under salt marsh environment just turns into complete The problem of particularly significant in world agriculture production.

The salt tolerance of plant is a sufficiently complex quantitative character, its Mechanisms of Salt Resistance is related to from plant to organ, tissue, Physiology and biochemistry is until each level of molecule.The scientist of various countries has also done substantial amounts of work for this, and achieves much newly to enter Exhibition, especially in terms of the salt tolerant molecule mechanism using high model plant arabidopsis to study plant, making the research in the field has Breakthrough progress (Zhu JK.2002.Salt and drought stress singal transduction in plants.Annu.Rev.Plant Biol.53：1247-1273：Zhang ZL.2011.Arabidopsis Floral Initiator SKBl Confers High Salt Tolerance by Regulating Transcription and Pre-mRNA Splicing through Altering Histone H4R3and Small Nuclear Ribonucleoprotein LSM4Methylation.Plant Cell, 23：396-411).Higher plant cell can have a variety of Approach experiences the change of physico-chemical parameter in external environment, so that extracellular signal is changed into intracellular signal, passes through the signal of series Stress signal is finally transferred in nucleus by conduction, 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 researcher never carried out and largely ground by ipsilateral Study carefully, but because its mechanism is sufficiently complex, many major issues in plant anti-salt still need to be explored.For example, the pass of plant anti-salt The key factor is not found yet；The molecular mechanism of plant salt tolerance is not fully aware of.

The content of the invention

The present inventor has cloned an ion channel albuminoid (this of cotton using SSH with the RACE methods being combined Text is named as NHX1-1) encoding gene DNA sequence dna.And find to be conducted into after transfer-gen plant, it can obviously improve and turn base Because of the salt-resistance of plant, and these characters can stablize heredity.

First aspect present invention provides an ion channel albuminoid NHX1-1 for cotton encoding gene, and its sequence is SEQ ID NO：2.

Second aspect of the present invention provides a kind of recombinant expression carrier, its contain the gene described in first aspect present invention and The nucleotide sequence of the gene is operably connected with the expression control sequence of the expression vector；Preferably, the carrier For the rd29A-GhNHX1-1-2300 carriers shown in accompanying drawing 2.

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

Fourth aspect present invention provides a kind of method of improvement plant salt endurance, including：By described in first aspect present invention Recombinant expression carrier described in gene or second aspect of the present invention imports plant or plant tissue and makes the gene expression；It is excellent Selection of land, the plant is tobacco.

Fifth aspect present invention provides a kind of method of prepare transgenosis plant, including：Effectively producing the condition of plant Lower plant or plant of the culture containing the recombinant expression carrier described in gene described in first aspect present invention, second aspect of the present invention Tissue；Preferably, the plant is tobacco.

Sixth aspect present invention provides the gene described in first aspect present invention, the restructuring table described in second aspect of the present invention It is used to improve plant salt endurance and use for plant breeding up to the recombinant cell described in carrier or third aspect present invention On the way；Preferably, the plant is tobacco.

Seventh aspect present invention provides the amino acid sequence of the gene code described in invention first aspect, such as SEQ ID NO： Shown in 1.

Brief description of the drawings

Fig. 1 is the structure flow of GhNHX1-1 plant expression vector (rd29A-GhNHX1-1-2300).

Fig. 2 is the plasmid figure of GhNHX1-1 plant expression vector (rd29A-GhNHX1-1-2300).

Fig. 3 is GhNHX1-1 Transgenic Tobacco Seeds and non-transgenic tobacco seed (control) on regular MS media Growth result.(GM) is the growth result for turning tobacco gene seed on the left of culture dish；Right side (CK) is non-transgenic tobacco seed The growth result of (control).

Fig. 4 is GhNHX1-1 Transgenic Tobacco Seeds and non-transgenic tobacco seed (control) added with 100mM NaCl MS culture mediums on growth result.(GM) is to turn tobacco gene seed (T on the left of culture dish₀H2 growth result)；Right side (CK) it is the growth result of non-transgenic tobacco seed (control).

Fig. 5 is that transgenosis T1 verifies knot for the protein expression of tobacco plant and non-transgenic reference plant on transcriptional level Really.M is Marker, and 1-5 is control tobacco, 6-15 be salt tolerant transgene tobacco T1 for plant, 16-21 is not salt tolerant transgenosis cigarette Careless T1 is for plant.

Embodiment

The present invention is further described with reference to non-limiting example.

Cotton SSH library constructions under the salt stress of embodiment 1：

Specific method is：

Utilize the PCR-select of Clontech companies^TMMethod shown in cDNA Subtraction Kit kits passes through Subtractive hybridization method builds subtracted library.Made in experimentation with the mRNA of the root for the cotton seedling that 6h is handled through NaCl For sample (tester), control (driver) is used as using the mRNA of the root of untreated cotton seedling.

(1) material to be tested：

(National Cotton mid-term storehouse obtains unit Cotton research institute, Unified number to Ji cotton 14：ZM-30270) it is seeded into On sterilized vermiculite, cultivated under the conditions of 25 DEG C, light dark period 16h/8h, 1/2MS culture mediums (9.39mM KNO are poured weekly₃, 0.625mM KH2PO₄, 10.3mM NH₄NO₃, 0.75mM MgSO₄, 1.5mM CaCl₂, 50 μM of KI, 100 μM of H₃BO₃, 100 μM MnSO₄, 30 μM of ZnSO₄, 1 μM of Na₂MoO₄, 0.1 μM of CoCl₂, 100 μM of Na₂EDTA, 100 μM of FeSO₄) once.When seedling strain It is used to test during long up to 25-30cm.

(2) material process：

2 groups, every group 4 plants will be divided into for examination seedling.First group is control group, is cultivated under 25 DEG C, illumination, is placed into 1/ In 2MS fluid nutrient mediums.Second group is treatment group, 25 DEG C, cultivate under illumination, is placed into added with final concentration of 200mM NaCl 1/2MS fluid nutrient mediums in, handle 6 hours, the root of two groups of seedling of timely clip, uses liquid nitrogen quick freeze after being disposed Afterwards, preserved in -70 DEG C of refrigerators.

(3) Total RNAs extraction：

The Levant Cotton Root 0.5g for taking control and NaCl to handle respectively, is extracted with plant RNA extraction kit (invitrogen) The total serum IgE of cotton.Extinction of the total serum IgE in 260nm and 280nm is determined with the ultraviolet specrophotometer U-2001 of HITACHI companies Angle value, OD260/OD280 ratios are 1.8-2.0, show that total serum IgE purity is higher, detect total with 1.0% agarose gel electrophoresis RNA integrality, the brightness of 28S bands is about 2 times of 18S bands, shows that RNA integrality is good.Use Qiagen companies Oligotex mRNA purification kits (purification of polyA+RNA from total RNA) separation mRNA.

(4) suppressed subtractive hybridization：

In order to have increased access to the effective of EST (Expressed sequence tag, EST) (unigene) Property, it is to avoid gene is without restriction enzyme site and obtained sequence in non-translational region.This use for laboratory RsaI, HaeIII is respectively to double-strand CDNA (according to the scheme described in abatement kit, being obtained by mRNA reverse transcriptions) is digested, and does two groups of suppression subtractives, other Step and method press the PCR-select of Clontech companies^TMMethod shown in cDNA Subtraction Kit kits is carried out Suppressed subtractive hybridization, finally merges second of PCR primer of two groups of positive subtractive hybridization cDNA fragments.

(5) structure of cDNA subtractive libraries and preliminary screening, clone, identification

(QIAquick PCR Purification Kit are pure for second of PCR primer of positive subtractive hybridization cDNA fragments Change, purchased from Qiagen) it is connected with pGEM-T Easy (being purchased from Promega kits) carrier, according to pGEM-TEasy kits Program, is comprised the following steps that：Following ingredients are sequentially added with 200 μ l PCR pipes：The positive subtractive hybridization cDNA fragments of purifying Second of μ l of 1 μ l, T4DNA ligase of PCR primer 3 μ l, T4 ligase buffer solution, 5 μ l, pGEM-T Easy carriers 1, in 4 DEG C of companies Take over night.10 μ L coupled reaction products are taken, are added in 100 μ L competence Escherichia coli JMI09 (being purchased from TAKARA), ice bath 30min, heat shock 60s, ice bath 2min, separately adding 250 μ L LB nutrient solutions, (1%Tryptone is purchased from OXOID, 0.5%Yeast Extract is purchased from OXOID, and 1%NaCl is purchased from traditional Chinese medicines) put in 37 DEG C of water-baths, with 225r/min shaken cultivation 30min, take 200 μ L Bacterium solution is planted in LB (ibid)/X-gal/IPTG (X-gal/IPTG is purchased from TAKARA) culture containing 50 μ g/mL ampicillins On plate, 37 DEG C of cultivation 18h.The clear white and blue colonies number of diameter ＞ 1mm in culture plate is counted, random picking 300 is white Color bacterium colony (numbering：Gh-S001 to Gh-S300).All white colonies are chosen in the LB liquid containing 50 μ g/mL ampicillins In 96 porocyte culture plates (CORNING) of culture medium, glycerol adding is to final concentration 20% after 37 DEG C of overnight incubations, in -80 DEG C of guarantors Deposit standby.With nest-type PRC primer Primer1 and the Primer2R (PCR-select of Clontech companies^TMcDNA Subtraction Kit kits are carried) bacterium solution PCR amplifications are carried out, 211 positive colonies are obtained, all positive colonies are existed Invitrogen (Shanghai) Trading Co., Ltd. is sent to be sequenced

(6) the cDNA sequencing analysis of differential cloning：

After the cDNA that the DNA sequencing result of above-mentioned 211 differential clonings is removed to carrier and indefinite sequence and redundancy, altogether Obtain 153 EST (unigene).Find wherein 87 through BlastN has homologous sequence, 29 Unknown Functions in GenBank Or to assume albumen, separately there are 37 not obtain homologous matching, thus it is speculated that to be probably the shorter sequence in 3 ', 5 ' end non-translational regions Row.

The cotton ion channel class encoding gene GhNHX1-1 of embodiment 2 clone

Clone Gh-S037 sequences are SEQ ID No：3, sequence analysis shows the amino acid sequence category of the coding of the sequence In ion channel albuminoid NHX1, the clone Gh-S037 full-length genes encoded are named as GhNHX1-1 herein, the gene pairs The albumen answered is named as NHX1-1.

SEQ ID No：3

1 ACATTGCTTG GGGTTTTGGC TGGATTGCTT AGTGCTTACA TTATTAAAAA GCTCTATTTC

61 GGAAGGCACT CAACGGATCG CGAGGTTGCT CTCATGATAC TCATGGCTTA CCTTTCATAT

121 ATGCTGGCTG AATTTTTCTA TTTAAGTGCG ATCCTCACTG TGTTCTTTTG TGGGATTGTT

181 ATGTCTCATT ATACATGGCA TAATGTTACC GAAAGTTCAA GAGTGACAAC CAAGCATGCT

241 TTTGCCACAC TATCATTTGT TGCTGAGATT TTCATCTTCC TCTATGTTGG TATGGATGCT

301 TTGGACATTG AGAAATGGAG AGTAGTTAGT GATAGCCCTG GGAAATCAGT TGGGGTGAGC

361 GCAATTCTAT TAGGCTTGAT TCTTGTTGGG AGAGCAGCCT TTGTTTTCCC TTTATCTTCC

421 ATTTCCAACT TGACTAAGAA AGCTCCATGT GACAAAATTG ATTTCAAACA GCAAGTTACC

481 ATTTGGTGGG CCGGTCTTAT GCGTGGTGCA GTTTCAATGG CACTTGCTTA TAATCAGTTT

541 ACCAGCTTGG GCCATACTCA GTTGCGAGGG AATGCAATGA TGATAACCAG CACAATCACA

601 GTTGT

Gram drop of GhNHX1-1 full-length genes

According to the Gh-S037 genetic fragments obtained, two specific primers are designed, 3 ' RACE 5 ' terminal specifics are used as Property primer.

GhNHX1GSP1：SEQ ID NO：4：

TGGATTGCTT AGTGCTTACA TTA

GhNHX1GSP2：SEQ ID NO：5：

CAACGGATCG CGAGGTTGCT CTC

Experimental procedure operates (3 ' RACE System for Rapid Amplification of by kit specification CDNA Ends kits are purchased from invitrogen companies).

With SEQ ID NO：4 and 3 ' end primer AUAP (kit is carried), are carried out by template of the cDNA of mRNA reverse transcriptions First round PCR is expanded.Comprise the following steps that：Ex Taq are purchased from TAKARA, 50 μ l PCR reaction systems：5μl10×Ex The cDNA of the μ l mRNA reverse transcriptions of Buffer, 3 μ l2.5mM dNTP, 2.0,1.0 μ l Ex Taq, 10 μM of primer SEQ ID NO：Each 2.0 μ l of 4 and AUAP, and 35 μ l distilled water.PCR reaction conditions：94 DEG C of pre-degeneration 5min, 94 DEG C are denatured 30s, 58 DEG C annealing 30s, 72 DEG C extension 2min, 33 circulation after, 72 DEG C extension 10min.

The PCR primer of gained takes 2.0 μ l as template after diluting 50 times with distilled water, with SEQ ID NO：5 draw with 3 ' ends Thing AUAP carries out second and takes turns PCR amplifications, comprises the following steps that：50 μ l PCR reaction systems：5 μ l10 × Ex Buffer, 3 μ The first round PCR primer of l2.5mM dNTP, 2.0 μ l dilution, 1.0 μ l Ex Taq, 10 μM of primer SEQ ID NO：5 Hes Each 2.0 μ l of AUAP, and 35 μ l distilled water.PCR reaction conditions：94 DEG C of pre-degenerations 5min, 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 2min, after 33 circulations, 72 DEG C of extension 10min.It is about 1200bp bands that second of PCR primer, which reclaims fragment, (Gel Extraction Kit are purchased from OMEGA) is connected to pGEM-T Easy Vector, is transformed into e. coli jm109 (tool Body method is ibid), random 10 white colonies of picking are cultivated in the LB fluid nutrient mediums containing 50 μ g/mL ampicillins, Glycerol adding is to final concentration 20% after 37 DEG C of overnight incubations, and -80 DEG C save backup.SEQ ID NO：5 and 3 ' end primer AUAP are carried out Bacterium solution PCR is expanded, and obtains 5 positive colonies, is sent Invitrogen's sequencing sequencing, is obtained the gene CDNA 3 ' ends.

According to the GhNHX1-1 genetic fragments obtained, three specific primers are designed, it is special as 5 ' RACE 3 ' ends Specific primer.

GhNHX1-1GSP3：SEQ IDNO：6：

CCCTCGCAAC TGAGTATGGC CC

GhNHX1-1GSP4：SEQ ID NO：7：

TGCACCACGC ATAAGACCGG CC

GhNHX1-1GSP5：SEQ ID NO：8：

ACATGGAGCT TTCTTAGTCA AG

Experimental procedure operates (5 ' RACE System for Rapid Amplification of by kit specification CDNA Ends kits are purchased from invitrogen companies).

With SEQ ID NO：7 and 5 ' universal primer AAP (kit is carried), with the cDNA of mRNA reverse transcriptions, (reverse transcription is drawn Thing SEQ ID NO：6) amplification of first round PCR is carried out for template, comprised the following steps that：Ex Taq are purchased from TAKARA, 50 μ l PCR Reaction system：The cDNA of 5 μ l10 × Ex μ l mRNA reverse transcriptions of Buffer, 3 μ l2.5mM dNTP, 2.0,1.0 μ l Ex Taq, 10 μM of primer SEQ ID NO：Each 2.0 μ l of 7 and AAP, and 35 μ l distilled water.PCR reaction conditions：94 DEG C of pre-degenerations 5min, 94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 2min, after 33 circulations, 72 DEG C of extension 10min.The PCR of gained Product takes 2.0 μ l as template after diluting 50 times with distilled water, with SEQ ID NO：8 and 3 ' end primer AUAP carry out second and taken turns PCR is expanded, and is comprised the following steps that：50 μ l PCR reaction systems：5 μ l10 × Ex Buffer, 3 μ l2.5mM dNTP, 2.0 μ l is dilute The first round PCR primer released, 1.0 μ l Ex Taq, 10 μM of primer SEQ ID NO：Each 2.0 μ l of 8 and AUAP, and 35 μ l Distilled water.PCR reaction conditions：94 DEG C of pre-degenerations 5min, 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 2min, 33 are followed After ring, 72 DEG C of extension 10min.It is about that (Gel Extraction Kit are purchased from 1200bp bands that second of PCR, which reclaims fragment, OMEGA pGEM-T Easy Vector) are connected to, JM109 (specific method is ibid), random 10 white colonies of picking is transformed into Cultivated in the LB fluid nutrient mediums containing 50 μ g/mL ampicillins, glycerol adding is to final concentration after 37 DEG C of overnight incubations 20%, -80 DEG C save backup.SEQ ID NO：8 and 3 ' end primer AUAP carry out bacterium solution PCR amplifications (reaction system and reaction bar Part is ibid), 4 positive colonies are obtained, Invitrogen's sequencing sequencing is sent, obtains the cDNA of the gene 5 ' end.

After 5 ' the RACE product clonings sequencing of gained, splice with 3 ' RACE products sequencing results.Obtain GhNHX1-1 total lengths CDNA sequence SEQ ID NO：19.

SEQ ID NO：19：

1 CTCTTAGCAA AGACTTCAAA ATCATTAAAG TTTTGTTCGT TAACACAATG GCAATCGGGA

61 TATTAAGCAC TCTTTTAGCA AAATCAGAGA CGATTTTAGG GTCTGGTCAC AGCTCAGTAG

121 TTTCAATGAA TTTATTCGTT GCTCTTCTTT GCGGGTGTAT CGTGATCGGT CATTTGCTAG

181 AGGAAAGCCG ATGGATGAAT GAATCCATTA CTGCCCTTGC CATTGGGCTG TGCACTGGAA

241 TTGTAATTTT GCTTACAACA GGAGGAAAAA GCTCTCATCT TTTAGTTTTC AGTGAAGATT

301 TGTTCTTTAA TTATTTGCTT CCACCTATTA TTTTCAATGC AGGATTCCAG GTGAAAAAGA

361 AGCAATTTTT CCGCAACTTT ATGACCATAA TGCTGTTTGG TGCAGTTGGC ACTTTAATAT

421 CCTTTGCCAT CATATCTCTA GGTGCCATAC ATTTTTTCAA GAAAATGAGT ATTGGTAATC

481 TCAAGATAGG GGATTATCTT GCCATTGGGG CAATATTTTC AGCAACAGAC TCTGTTTGCA

541 CTTTGCAAGT GCTTAATCAG GACGATACGC CTTTATTGTA TAGTCTGGTT TTCGGGGAGG

601 GAGTTGTGAA TGATGCCACA GCTGTTGTTC TTTTCAAAGC AATCCAAAGC TTTGACCTTC

661 CTCACATCAA CACCACCATT GCTTTGCAAT TTGTTGGGAA CTTTTTATAT TTATTCATCT

721 CGAGTACATT GCTTGGGGTT TTGGCTGGAT TGCTTAGTGC TTACATTATT AAAAAGCTCT

781 ATTTCGGAAG GCACTCAACG GATCGCGAGG TTGCTCTCAT GATACTCATG GCTTACCTTT

841 CATATATGCT GGCTGAATTT TTCTATTTAA GTGCGATCCT CACTGTGTTC TTTTGTGGGA

901 TTGTTATGTC TCATTATACA TGGCATAATG TTACCGAAAG TTCAAGAGTG ACAACCAAGC

961 ATGCTTTTGC CACACTATCA TTTGTTGCTG AGATTTTCAT CTTCCTCTAT GTTGGTATGG

1021 ATGCTTTGGA CATTGAGAAA TGGAGAGTAG TTAGTGATAG CCCTGGGAAA TCAGTTGGGG

1081 TGAGCGCAAT TCTATTAGGC TTGATTCTTG TTGGGAGAGC AGCCTTTGTT TTCCCTTTAT

1141 CTTCCATTTC CAACTTGACT AAGAAAGCTC CATGTGACAA AATTGATTTC AAACAGCAAG

1201 TTACCATTTG GTGGGCCGGT CTTATGCGTG GTGCAGTTTC AATGGCACTT GCTTATAATC

1261 AGTTTACCAG CTTGGGCCAT ACTCAGTTGC GAGGGAATGC AATGATGATA ACCAGCACAA

1321 TCACAGTTGT ACTTTTCAGC ACTGTGGTTT TTGGCTTGAT GACTAAACCA TTGATTAGAA

1381 TCTTGCTTCC TTCACCGAAA AATCGAATGC TCTCTTCCGA ACCGTCTACA CCTAAGTCAC

1441 TCACCGTGCC GCTTCTCACC AATGGCCACG ACTTAGAGGC TTACGAAAGC GACCGAAATC

1501 TAATAACCCG GCCAACAAGC TTAAGGATGT TCTTGAGCAC TCCTTCCAAC ACCGTGCACT

1561 ACTATTGGAG AAAATTCGAC AACGCGTTCA TGCGACCTGT ATTCGGAGGA AGGGGTTTCG

1621 TACCCTTCGT TCCGGGATCG CCCATCGAAC AAAATGATCA ACAATGGCAG TGAATAATGA

1681 AGAGATCACA GATACCAGGT ACTAAAATAC TACACTAATT TTTATAAACT TGTAGGATAG

1741 TGGAATGAAA ACCGGCTTTT CAGTGTGAAG TTTGCTAATA TATACGTGAG AGTTATGTTA

1801 CTTGGACTCG GGTTTGGGTT TTAAATATGG GTATATGTCT GACTTGGATA TATTCATAAA

1861 AAAAATTAAA ACAAAATAGA AGGACACAAA AGTGAGAGTT TGGCCGGTTG ATTTGATTTT

1921 AACAATTTTT TTCCAGCGTT TGCTGTTTAA TGTTTGATTG TATCTTACAT TGAATGCTGC

1981 TTTGGAGTTT TTTTTTCTTT TTTCTTTTTT TTTGTGAGGG TTTTTTTTGT AAAAGGTTTA

2041 ATTTTATTGA TTGTATATCT TTTTATTAAA AAAAAAAAAA AAAAAAAAAA

Pair of primers is designed according to GhNHX1-1 full length cDNA sequences as follows：

GhNHX1-1F：SEQ ID NO：9：

ATGGCAATCG GGATATTAAG CACT

GhNHX1-1R：SEQ ID NO：10：

TCACTGCCAT TGTTGATCAT TTTG

Pass through SEQ ID NO：9 and SEQ ID NO：10 clone GhNXH1-1 total lengths.

Using TaKaRa PrimeSTAR HS archaeal dna polymerases, performing PCR reaction is entered by template of the cDNA of cotton.50μl PCR reaction systems：The 10 μ l5 × PS μ l of Buffer, 3 μ l2.5mM dNTP, 2.0 cDNA, 1.0 μ l PrimeSTAR, 10 μM Primer SEQ ID NO：9 and SEQ ID NO：10 each 2.0 μ l, and 30 μ l distilled water.PCR reaction conditions：94 DEG C of pre-degenerations 5min, 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 2min, after 33 circulations, 72 DEG C of extension 10min.

Pcr amplification product adds A tails：PCR primer adds 2.5 times of absolute ethyl alcohol, and -20 DEG C are placed 10 minutes, and centrifugation is gone Clearly, dry, dissolved with 21 μ l distilled waters.Add 2.5 μ l10 × Ex Buffer, 0.5 μ l5mM dATP, 2.5 μ l10 × Ex Taq.Reaction condition：70 DEG C are reacted 30 minutes.The DNA fragmentation for obtaining about 1600bp is reclaimed into (Omega QIAquick Gel Extraction Kits), connection To pGEM T-easy carriers, JM109 (method is ibid) is converted, random 10 white colonies of picking are in containing 50 μ g/mL ammonia benzyls Cultivated in the LB fluid nutrient mediums of penicillin, glycerol adding is to final concentration 20% after 37 DEG C of overnight incubations, and -80 DEG C save backup.SEQ ID NO：9 and SEQ ID NO：10 carry out bacterium solution PCR amplifications (reaction system and reaction condition are ibid), obtain 4 positive colonies, Invitrogen's sequencing is delivered to, sequence is SEQ ID NO：2.

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

1 MAIGILSTLL AKSETILGSG

21 HSSVVSMNLF VALLCGCIVI

41 GHLLEESRWM NESITALAIG

61 LCTGIVILLT TGGKSSHLLV

81 FSEDLFFNYL LPPIIFNAGF

101 QVKKKQFFRN FMTIMLFGAV

121 GTLISFAIIS LGAIHFFKKM

141 SIGNLKIGDY LAIGAIFSAT

161 DSVCTLQVLN QDDTPLLYSL

181 VFGEGVVNDA TAVVLFKAIQ

201 SFDLPHINTT IALQFVGNFL

221 YLFISSTLLG VLAGLLSAYI

241 IKKLYFGRHS TDREVALMIL

261 MAYLSYMLAE FFYLSAILTV

281 FFCGIVMSHY TWHNVTESSR

301 VTTKHAFATL SFVAEIFIFL

321 YVGMDALDIE KWRVVSDSPG

341 KSVGVSAILL GLILVGRAAF

361 VFPLSSISNL TKKAPCDKID

381 FKQQVTIWWA GLMRGAVSMA

401 LAYNQFTSLG HTQLRGNAMM

421 ITSTITVVLF STVVFGLMTK

441 PLIRILLPSP KNRMLSSEPS

461 TPKSLTVPLL TNGHDLEAYE

481 SDRNLITRPT SLRMFLSTPS

501 NTVHYYWRKF DNAFMRPVFG

521 GRGFVPFVPG SPIEQNDQQW

541 Q^*

The nucleotide sequence of GhNHX1-1 encoding genes：SEQ ID NO：2

1 ATGGCAATCG GGATATTAAG CACTCTTTTA GCAAAATCAG AGACGATTTT AGGGTCTGGT

61 CACAGCTCAG TAGTTTCAAT GAATTTATTC GTTGCTCTTC TTTGCGGGTG TATCGTGATC

121 GGTCATTTGC TAGAGGAAAG CCGATGGATG AATGAATCCA TTACTGCCCT TGCCATTGGG

181 CTGTGCACTG GAATTGTAAT TTTGCTTACA ACAGGAGGAA AAAGCTCTCA TCTTTTAGTT

241 TTCAGTGAAG ATTTGTTCTT TAATTATTTG CTTCCACCTA TTATTTTCAA TGCAGGATTC

301 CAGGTGAAAA AGAAGCAATT TTTCCGCAAC TTTATGACCA TAATGCTGTT TGGTGCAGTT

361 GGCACTTTAA TATCCTTTGC CATCATATCT CTAGGTGCCA TACATTTTTT CAAGAAAATG

421 AGTATTGGTA ATCTCAAGAT AGGGGATTAT CTTGCCATTG GGGCAATATT TTCAGCAACA

481 GACTCTGTTT GCACTTTGCA AGTGCTTAAT CAGGACGATA CGCCTTTATT GTATAGTCTG

541 GTTTTCGGGG AGGGAGTTGT GAATGATGCC ACAGCTGTTG TTCTTTTCAA AGCAATCCAA

601 AGCTTTGACC TTCCTCACAT CAACACCACC ATTGCTTTGC AATTTGTTGG GAACTTTTTA

661 TATTTATTCA TCTCGAGTAC ATTGCTTGGG GTTTTGGCTG GATTGCTTAG TGCTTACATT

721 ATTAAAAAGC TCTATTTCGG AAGGCACTCA ACGGATCGCG AGGTTGCTCT CATGATACTC

781 ATGGCTTACC TTTCATATAT GCTGGCTGAA TTTTTCTATT TAAGTGCGAT CCTCACTGTG

841 TTCTTTTGTG GGATTGTTAT GTCTCATTAT ACATGGCATA ATGTTACCGA AAGTTCAAGA

901 GTGACAACCA AGCATGCTTT TGCCACACTA TCATTTGTTG CTGAGATTTT CATCTTCCTC

961 TATGTTGGTA TGGATGCTTT GGACATTGAG AAATGGAGAG TAGTTAGTGA TAGCCCTGGG

1021 AAATCAGTTG GGGTGAGCGC AATTCTATTA GGCTTGATTC TTGTTGGGAG AGCAGCCTTT

1081 GTTTTCCCTT TATCTTCCAT TTCCAACTTG ACTAAGAAAG CTCCATGTGA CAAAATTGAT

1141 TTCAAACAGC AAGTTACCAT TTGGTGGGCC GGTCTTATGC GTGGTGCAGT TTCAATGGCA

1201 CTTGCTTATA ATCAGTTTAC CAGCTTGGGC CATACTCAGT TGCGAGGGAA TGCAATGATG

1261 ATAACCAGCA CAATCACAGT TGTACTTTTC AGCACTGTGG TTTTTGGCTT GATGACTAAA

1321 CCATTGATTA GAATCTTGCT TCCTTCACCG AAAAATCGAA TGCTCTCTTC CGAACCGTCT

1381 ACACCTAAGT CACTCACCGT GCCGCTTCTC ACCAATGGCC ACGACTTAGA GGCTTACGAA

1441 AGCGACCGAA ATCTAATAAC CCGGCCAACA AGCTTAAGGA TGTTCTTGAG CACTCCTTCC

1501 AACACCGTGC ACTACTATTG GAGAAAATTC GACAACGCGT TCATGCGACC TGTATTCGGA

1561 GGAAGGGGTT TCGTACCCTT CGTTCCGGGA TCGCCCATCG AACAAAATGA TCAACAATGG

1621 CAGTGA

Embodiment 3GhNHX1-1 gene plant expression vector establishments

Select plant binary expression vector pCAMBIA2300 (being purchased from Beijing DingGuo ChangSheng Biology Technology Co., Ltd) As plant expression vector, the 35S promoter that NPTII genes contain double enhancers is replaced with Pnos promoters, to reduce NPTII eggs Expression in plant in vain.Inducible promoter rd29A and Tnos is selected as the promoter and terminator of GhNHX1-1 genes.

With primer SEQ ID NO：11 and SEQ ID NO：12 (are purchased from Beijing China ocean with plant expression vector PBI121 Science and Technology Ltd.) it is template amplification Pnos, using TaKaRa PrimeSTAR HS archaeal dna polymerases.50 μ l PCR reactants System：The 10 μ l5 × PS μ l of Buffer, 3 μ l2.5mM dNTP, 1.0 PBI121,1.0 μ l PrimeSTAR, 10 μM of primer SEQ ID NO：11 and SEQ ID NO：12 each 2.0 μ l, and 31 μ l distilled water.PCR reaction conditions：94 DEG C of pre-degeneration 5min, 94 DEG C denaturation 30s, 56 DEG C annealing 30s, 72 DEG C extension 30s, 33 circulation after, 72 DEG C extension 10min.Pass through EcoRI, BglII enzyme Cut and be connected to pCAMBIA2300 (promega, T4 ligase box) acquisitions pCAMBIA2300-1.

SEQ ID NO：11：

GCACGAATTCATACAAATGGACGAACGGAT

SEQ ID NO：12：

ATCCAGATCTAGATCCGGTGCAGATTATTTG

SEQ ID NO：13 and SEQ ID NO：14 using PBI121 as template amplification Tnos, using TaKaRa's PrimeSTAR HS archaeal dna polymerases.50 μ l PCR reaction systems：10 μ l5 × PS Buffer, 3 μ l2.5mM dNTP, 1.0 μ l PBI121,1.0 μ l PrimeSTAR, 10 μM of primer SEQ ID NO：13 and SEQ ID NO：14 each 2.0 μ l, and 31 μ l Distilled water.PCR reaction conditions：94 DEG C of pre-degenerations 5min, 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 30s, 33 are followed After ring, 72 DEG C of extension 10min.PCAMBIA2300-1 (promegaT4 ligases box) is connected to by SacI, EcoRI digestion to obtain Obtain pCAMBIA2300-2

SEQ ID NO：13：

AAG GAGCTCGAATTTCCCCGATCGTTCAAA

SEQ ID NO：14：

TCA GAATTCCCAGTGAATTCCCGATCTAGTA

SEQ ID NO：15 and SEQ ID NO：16 with arabidopsis, (Colombia's type, is purchased from Www.arabidopsis.org) DNA be template amplification arabidopsis rd29A promoters (with reference to Zeng J., et L.2002, Preparation of total DNA from " recalcit rant plant taxa ", Acta Bot.Sin., 44 (6)： Method in 694-697 extracts arabidopsis DNA).Using TaKaRa PrimeSTAR HS archaeal dna polymerases.50 μ l PCR react System：The 10 μ l5 × PS μ l arabidopsis of Buffer, 3 μ l2.5mM dNTP, 1.0 DNA, 1.0 μ l PrimeSTAR, 10 μM draw Thing SEQ ID NO：15 and SEQ ID NO：16 each 2.0 μ l, and 31 μ l distilled water.PCR reaction conditions：94 DEG C of pre-degenerations 5min, 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 30s, after 33 circulations, 72 DEG C of extension 10min.Pass through HindIII, PstI digestion are connected to (connection method is ibid) pCAMBIA2300-2 and obtain pCAMBIA2300-3

SEQ ID NO：15：

ACTAAGCTTCCTTCTTGACATCATTCAATTTTA

SEQ ID NO：16：

TGA CTGCAGTCCAAAGATTTTTTTCTTTCCAATAG

SEQ ID NO：17 and SEQ ID NO：18 amplification GhNHX1-1 (template is that embodiment 2 obtains GhNHX1-1), Using TaKaRa PrimeSTAR HS archaeal dna polymerases.50 μ l PCR reaction systems：10 μ l5 × PS Buffer, 3 μ l2.5mM DNTP, 1.0 μ l GhNHX1-1-pGEM, 1.0 μ lPrimeSTAR, 10 μM of primer SEQ ID NO：17 and SEQ ID NO： 18 each 2.0 μ l, and 31 μ l distilled water.PCR reaction conditions：94 DEG C of pre-degenerations 5min, 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 2min, after 33 circulations, 72 DEG C of extension 10min.It is connected to that (connection method is same by PstI, SacI digestion On) pCAMBIA2300-3, obtain plant expression vector rd29A-GhNHX1-1-2300.

SEQ ID NO：17：

TGAC TGCAGATGGCAATCGGGATATTAAGCACT

SEQ ID NO：18：

AAGGAGCTCTCACTGCCATTGTTGATCATTTTG

The rd29A-GhNHX1-1-2300 expression vectors of embodiment 4 convert Agrobacterium

It is prepared by Agrobacterium LBA4404 (being purchased from Biovector Science Lab, Inc) competence：1-2 days in advance by agriculture Bacillus LBA4404 draws single spot inoculation, 28 DEG C of trainings on the LB solid mediums containing 50 μ g/ml rifampins and 50 μ g/ml streptomysins Support 1 to 2 day.Picking single bacterium colony is inoculated in LB fluid nutrient mediums of the 5ml containing 50 μ g/ml rifampins and 50 μ g/ml streptomysins, and 28 It is 0.4 that overnight incubation (about 12-16h) to 0D600 values are shaken at DEG C, forms seed bacterium solution.Take the bacterium solution (1: 20 after 5ml activation Ratio) be inoculated in the LB fluid nutrient mediums of the same concentration antibiotic of 100ml, 28 DEG C shake culture 2-2.5h to OD₆₀₀= 0.8.Ice bath bacterium solution 10min, shakes up once every 3min, makes bacterium even into resting state.Centrifuged in 4000g at 4 DEG C 10min, abandons supernatant；Add 4000g at certain glycerine resuspension thalline of head for precooling 10%, 4 DEG C and centrifuge 10min, collect precipitation； Repeated to wash 3-4 times with 10% glycerine；Adding 10% glycerine of appropriate ice bath precooling, suspended bacterial is precipitated again, will with 40 μ l/ pipes It is dispensed, and is saved backup in -70 DEG C.

Convert Agrobacterium：Melt competent cell on ice, 1 μ l plasmid is added into 40 μ l competent cell, mix Even rear ice bath about 10min.Competence and DNA mixture are transferred in the electric shock of precooling cup with rifle, rapping arrives suspension Up to bottom, bubble has been careful not to.Electric shock cup (being purchased from bio-rad) is put on the slideway of electroporation chamber, promotes slideway to shock by electricity Cup is put to electroporation chamber base electrode.Using 0.1cm electric shock cup when, MicroPulser (be purchased from bio-rad) program It is set to " Agr ", electric shock is once.Electric shock cup is taken out immediately, adds the LB culture mediums of 28 DEG C of preheatings.It is quick and it is soft will with rifle Cell is beaten.Suspension is transferred to 1.5ml centrifuge tube, 28 DEG C, 225rpm cultures 1h.Take 100~200 μ l bacterium solution be coated with (LB solid mediums, containing 50 μ g/ml rifampins, 50 μ g/ml streptomysins, 50 μ g/ml on corresponding resistance screening culture medium flat plate Kanamycins), 28 DEG C of cultures.

Embodiment 5 obtains transgene tobacco using Agrobacterium-medialed transformation method

With 75% alcohol-pickled tobacco seed, (countries tobacco mid-term storehouse obtains unit：Compile in tobacco institute of the Chinese Academy of Agricultural Sciences, storehouse Number I5A00660) 30s, is washed twice with sterilizing distilled water.8min is soaked with 0.1% mercuric chloride again, is washed twice with sterilizing distilled water, it is complete Into surface sterilizing.The tobacco seed of surface sterilizing is placed in MS (18.78mMKNO₃, 1.25mM KH₂PO₄, 20.6mM NH₄NO₃, 1.5mM MgSO₄, 3.0mM CaCl₂, 50 μM of KI, 100 μM of H₃BO₃, 100 μM of MnSO₄, 30 μM of ZnSO₄, 1 μM of Na₂MoO₄, 0.1μM CoCl₂, 100 μM of Na₂EDTA, 100 μM of FeSO₄, 7.4g/L agar, sucrose 30g/L) under aseptic condition germinate, Prepare aseptic seedling.Take tests for sterility to be cut into the leaf dish of 5mm × 5mm sizes, contain expression vector with exponential phase Rd29A-GhNHX1-1-2300 During Agrobacterium leaf dish 10min, blots bacterium solution, and 2 days are co-cultured under dark condition, and (MS is trained Support base).Blade is gone into differential medium, and (the MS+1mg/L basic elements of cell division (RA)+0.1mg/L methyl α-naphthyl acetates (NAA)+50mg/L blocks That mycin+500mg/L cephalosporins) on, cultivated 45 days or so under illumination condition, cut after bud is grown up and be transferred to culture of rootage Culture 30 days or so in base (MS+50mg/L kanamycins+500mg/L cephalosporins), after being transferred to seedling only after well developed root system Preservation is numbered on MS culture mediums added with 500mg/L cephalosporins.

The transgenic tobacco leaf of acquisition is taken, DNA (arabidopsis DNA extraction method in be the same as Example 3) is extracted, uses SEQ ID NO：9：With SEQ ID NO：10 (50 μ l PCR reaction systems：5 μ l10 × Ex Buffer, 3 μ l2.5mM dNTP, 2.0 μ l DNA, 1.0 μ l Ex Taq, 10 μM of primer SEQ ID NO：9 and SEQ ID NO：10 each 2.0 μ l, and 35 μ l distilled water. PCR reaction conditions：94 DEG C of pre-degenerations 5min, 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 2min, after 33 circulations, 72 DEG C extension 10min), PCR identification, preserve positive plant T is numbered₀H1-T₀H20。

Embodiment 6 is overexpressed GhNHX1-1 transgene tobaccos T1 salt tolerant simulated experiment and Function Identification

T₀H1-T₀H20 tobacco seeds, non-transgenic reference tobacco seed sterilizing distilled water immersion 30min, use 75% wine Essence immersion 30s, is washed twice with sterilizing distilled water.8min is soaked with 0.1% mercuric chloride again, is washed with sterilizing distilled water twice, completes table Face sterilizes.Do two groups of experiments：First group：By the T of surface sterilizing₀H1-T₀H20 tobacco seeds, control tobacco seed are placed in MS solids In germination under aseptic condition on culture medium.Second group：By the T of surface sterilizing₀H1-T₀H20 tobacco seeds, control tobacco seed are put In on the MS solid mediums added with 100mM NaCl under aseptic condition germinate.25 DEG C, 10 hours optical culture/14 hour it is dark Culture circulation, result is observed after 10 days.First group of result of the test shows that T0 is sent out for the seed of each strain in MS solid mediums Bud rate is 90%, and growth conditions are good, with compareing no significant difference (Fig. 3)；Second group of result of the test is found, containing 100mM In NaCl MS culture mediums, T₀H1-T₀H20 tobacco seeds and control seed can be sprouted, but adjoining tree growth is substantially pressed down System.T1 shows for the Salt-Tolerance Identification of transfer-gen plant (plant that T0 grows up to for the seed of genetically modified plants), T₁H2、T₁H5、 T₁H7、T₁H13、T₁H15、、T₁H16、T₁H19、T₁The growth of H20 transfer-gen plants is overall to be higher than adjoining tree, shows obvious Salt tolerance is (referring to Fig. 4, with T₁Exemplified by H2, T₁H5、T₁H7、T₁H13、T₁H15、、T₁H16、T₁H19、T₁H20 and T₁H2 result class Seemingly, it is not shown here).

Embodiment 7 verifies NHX1-1 protein expressions on transcriptional level

Take respectively control tobacco, not salt tolerant transgene tobacco T1 for plant, salt tolerant transgene tobacco T1 for plant (growth shape Condition is good) 100mM NaCl handle 14 days leaf 0.05g, with plant RNA extraction kit (invitrogen) extract it is total RNA.Absorbance of the total serum IgE in 260nm and 280nm is determined with the ultraviolet specrophotometer U-2001 of HITACHI companies, is calculated Each RNA concentration.Using invitrogen reverse transcription reagent box SuperScript III Reverse Transcriptase simultaneously Carrying out reverse transcription according to method in specification, (2 μ g total serum IgEs are used as template, reverse transcription primer SEQ ID N0：10).Pass through SEQ ID NO：9 and SEQ ID NO：10 amplification GhNHX1-1, detect GhNHX1-1 albumen relative expression's situations.Using TaKaRa's PrimeSTAR HS archaeal dna polymerases, performing PCR reaction is entered by template of the cDNA of reverse transcription.50 μ l PCR reaction systems：10μl5 × PS μ the l of Buffer, 3 μ l2.5mM dNTP, 2.0 cDNA, 1.0 μ l PrimeSTAR, 10 μM of primer SEQ ID NO：9 Hes SEQ ID NO：10 each 2.0 μ l, and 30 μ l distilled water.PCR reaction conditions：94 DEG C of pre-degenerations 5min, 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 1min, after 29 circulations, 72 DEG C of extension 10min.Product electrophoresis result is as shown in Figure 5：M is DNA Ladder Marker (DL2000, purchased from Shenzhen Rui Zhen Bioisystech Co., Ltd), 1-5 are control tobacco, and 6-15 is resistance to Salt transgene tobacco T1 is not salt tolerant transgene tobacco T1 for plant for plant, 16-21.Stripe size shown in figure with GhNHX1-1 genes it is in the same size.As a result show to compare the mRNA that tobacco does not transcribe GhNHX1-1, salt tolerant transgene tobacco T1 is stronger for plant pair GhNHX1-1 transcription, and salt tolerant transgene tobacco T1 does not transcribe or transcribed for plant very weak.

Claims

1. an ion channel albuminoid encoding gene for cotton, its sequence is SEQ ID NO：2.

2. a kind of recombinant expression carrier, it contains nucleotide sequence and the institute of the gene described in claim 1 and the gene The expression control sequence for stating expression vector is operably connected.

3. a kind of recombinational agrobacterium cell, it contains the recombination expression described in gene or claim 2 described in claim 1 Carrier.

4. a kind of method of improvement plant salt endurance, including：By described in the gene or claim 2 described in claim 1 Recombinant expression carrier imports plant or plant tissue and makes the gene expression, wherein the plant is tobacco.

5. a kind of method of prepare transgenosis plant, including：Culture contains claim 1 institute under conditions of plant is effectively produced The plant for the recombinant expression carrier described in gene or claim 2 stated or plant tissue, wherein the plant is tobacco.

6. the recombinant expression carrier described in gene, claim 2 described in claim 1 or the restructuring described in claim 3 Cell is used to improve plant salt endurance and the purposes for plant breeding, wherein the plant is tobacco.

7. the albumen of the gene code described in claim 1, its sequence such as SEQ ID NO：Shown in 1.