CN104379746A

CN104379746A - Cotton ion channel protein and coding genes and use thereof

Info

Publication number: CN104379746A
Application number: CN201280004135.4A
Authority: CN
Inventors: 王建胜; 田大翠; 何云蔚; 崔洪志
Original assignee: Genesis Seed Industry Co ltd
Current assignee: Genesis Seed Industry Co ltd
Priority date: 2012-08-13
Filing date: 2012-08-13
Publication date: 2015-02-25
Anticipated expiration: 2032-08-13
Also published as: CN104379746B; WO2014026310A1

Abstract

Provided are an ion channel protein NHX1-1 from cotton and coding genes thereof, and the use in the incubation of transgenic plants with increased salt tolerance.

Description

Cotton ion channel protein and coding genes and use thereof

One cotton ion channel albuminoid and its encoding gene and application

The present invention relates to ion channel albuminoid and its encoding gene and application, the more particularly to one ion channel albuminoid NHX1-1 and its encoding gene from cotton, and its application in the genetically modified plants that salt tolerance is improved are cultivated for technical field.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. Sal t and drought stress s ingal transduct ion in plants. Annu. Rev. Plant Biol. 53： 1247-1273； Zhang ZL. 2011. Arabidops i s Floral Init iator SKBl Confers High Salt Tolerance by Regulat ing Transcript ion and Pre-mRNA Spl icing through Altering Hi stone H4R3 and Smal l Nuclear Ribonucleoprotein LSM4 Methylat ion. 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 has cloned an ion channel albuminoid for cotton using SSH with the RACE methods being combined

The DNA sequence dna of the encoding gene of (being named as NHX1-1 herein).And find to be conducted into after transfer-gen plant, the salt-resistance of transfer-gen plant is can obviously improve, 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, and it contains 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 is the rd29A-GhNHXl-l-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 tobacco.

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

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 tobacco.

Seventh aspect present invention provides the amino acid sequence of the gene code described in invention first aspect, such as SEQ ID Ν Ο:Shown in 1.Brief description of the drawings Fig. 1 is the structure flow of GhNHXl-1 plant expression vector (rd29A-GhNHXl-l-2300).

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

Fig. 3 is GhNHXl-1 Transgenic Tobacco Seeds and non-transgenic tobacco seed（Control）Growth result on regular MS media.On the left of culture dish（GM it is) to turn the growth result of tobacco gene seed；Right side（CK) to be non- Transgenic Tobacco Seeds（Control）Growth result.

Fig. 4 is GhNHXl-1 Transgenic Tobacco Seeds and non-transgenic tobacco seed（Control）Growth result on the MS culture mediums added with 100 mM NaCl.On the left of culture dish（GM it is) to turn tobacco gene seed（T_QH2 growth result)；Right side（CK) it is non-transgenic tobacco seed（Control）Growth result.

Fig. 5 is protein expression the results of the transgenosis T1 for tobacco plant and non-transgenic reference plant on transcriptional level.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 transgene tobacco T1 for plant.The present invention is further described with reference to non-limiting example for embodiment.Cotton SSH library constructions under the salt stress of embodiment 1：

Specific method is：

Subtracted library is built by Subtractive hybridization method using the method shown in the PCR-select cDNA Subtraction Kit kits of Clontech companies.MRNA using the root for the cotton seedling that 6h is handled through NaCl in experimentation is used as sample（), tester the mRNA using the root of untreated cotton seedling is used as control（driver).

(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 on sterilized vermiculite, is cultivated under the conditions of 25 °C, light dark period 16h/8h, 1/2MS cultures (9. 39 mM KN0 is poured weekly₃, 0. 625 mM KH2P0₄, 10. 3 mM NH^Oa, 0. 75 mM MgS0₄, 1. 5 mM CaCl₂, 50 μ Μ ΚΙ , 100 μ Μ Η₃Β0₃, 100 μM of MnS0₄, 30 μM of ZnS0₄, 1 μ M N¾Mo0₄, 0. 1 μ M CoCl₂, 100 μ Μ N EDTA, 100 μ Μ FeS0₄) once.It is used to test as the long up to 25-30 cm of seedling strain.

(2) material process：

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

(3) Total RNAs extraction：

The 5g of Levant Cotton Root 0. for taking control and NaCl to handle respectively, uses plant RNA extraction kit（Invitrogen the total serum IgE of cotton) is extracted.Total serum IgE is determined in 260 nm with the ultraviolet specrophotometer U-2001 of HITACHI companies With 280 nm absorbance, 0D260/0D280 ratios are 1. 8-2 0, show that total serum IgE purity is higher, and total A integrality is detected 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（Purification of polyA+ RNA from total RNA) separation mRNA.

(4) suppressed subtractive hybridization：

In order to have increased access to EST (Expressed sequence tag, EST) (unigene) validity, it is to avoid gene is without restriction enzyme site and obtained sequence in non-translational region.This use for laboratory Rsal, Haelll (according to the scheme described in abatement kit, are obtained to double-strand cDNA by mRNA reverse transcriptions respectively）Digested, do two groups of suppression subtractives, other steps and method press the PCR_select of Clontech companies^TMMethod shown in cDNA Subtraction Kit kits carries 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

Second of PCR primer (QIAquick PCR Purification Kit are purified, purchased from Qiagen) of positive subtractive hybridization cDNA fragments (is purchased from Promega kits with pGEM_T Easy）Carrier is connected, and according to the program of pGEM_T Easy kits, is comprised the following steps that：Following ingredients are sequentially added with 200 l PCR pipes：The positive subtractive of purifying hybridizes 4 ligase buffer solution of second of PCR primer 3 μ 1, Τ 5 μ 1, pGEM-T Easy carriers 1 μ 1, the μ of T4 DNA ligases 1 of cDNA fragments, is stayed overnight in 4 °C of connections.10 μ L coupled reaction products are taken, are added in 100 μ L competence Escherichia coli JMI09 (being purchased from TAKARA), the min of ice bath 30, the s of heat shock 60, the min of ice bath 2 separately add 250 L LB nutrient solutions（1% Tryptone is purchased from 0X0ID, and 0. 5% Yeast Extract are purchased from 0X0ID, and 1% NaCl is purchased from traditional Chinese medicines）Put in 37 °C of water-baths, with the min of 225 r/min shaken cultivations 30, take 200 μ L bacterium solutions to plant in the LB containing 50 μ g/mL ampicillins (ibid）On/X-gal/IPTG (X-gal/IPTG is purchased from TAKARA) culture plate, 37 °C of 18 h of cultivation.Count the clear white and blue colonies number of the mm of diameter ＞ 1 in culture plate, random 300 white colonies of picking (numbering：Gh-S001 to Gh-S300).All white colonies are chosen in 96 porocyte culture plates (CORNING) of the LB fluid nutrient mediums containing 50 μ g/mL ampicillins, glycerol adding is saved backup to final concentration 20% in -80 °C after 37 °C of overnight incubations.With nest-type PRC primer Primer l and Primer 2R, (the PCR- select cDNA Subtraction Kit kits of Clontech companies are carried）Bacterium solution PCR amplifications are carried out, 211 positive colonies is obtained, Invitrogen is being sent to all positive colonies（Shanghai）The cDNA sequencing analysis of (6) differential cloning are sequenced in trade Co., Ltd：

After the cDNA that the DNA sequencing result of above-mentioned 211 differential clonings is removed to carrier and indefinite sequence and redundancy, 153 EST (unigene) are obtained.Find wherein 87 through BlastN has homologous sequence in GenBank, 29 Unknown Functions are hypothesis albumen, separately have 37 not obtain homologous matching, thus it is speculated that to be probably the shorter sequence in 3', 5' end non-translational region.The cotton ion channel class encoding gene GhNHXl-1 of embodiment 2 clone

Clone Gh-S037 sequences are SEQ ID No:3, sequence analysis shows that the amino acid sequence of the coding of the sequence belongs to ion channel albuminoid NHX1, and the clone Gh-S037 full-length genes encoded are named as into GhNHXl-1 herein, and the corresponding albumen of the gene is named as NHX1-1.

The clone of GhNHXl-1 full-length genes

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

GhNHXl GSP1: SEQ ID NO: 4：

TGGATTGCTT AGTGCTTACA TTA

GhNHXl GSP2: SEQ ID NO: 5：

CAACGGATCG CGAGGTTGCT CTC

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 3' ends primer AUAP）, the amplification of first round PCR is carried out by template of the cDNA of mRNA reverse transcriptions.Comprise the following steps that：ExTaq is purchased from TAKARA, 50 lPCR reaction systems： 5 μ 1 Ι Ο Χ Ε χ Buffer, 3 μ 1 2. 5 mM dNTP, the cDNA of the mRNA reverse transcriptions of 2. 0 μ 1, the μ Μ of 1. 0 μ, 1 Ex Taq 10 primer SEQ ID NO:Each 2. 0 μ 1 of 4 and AUAP, and 35 μ 1 distilled water.PCR reaction conditions:94 °C of pre-degenerations 5 min, 94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of 2 min of extension, after 33 circulations, 72 °C of 10 min e of extension

The PCR primer of gained takes 2. 0 μ 1 as template after diluting 50 times with double distilled waters, with SEQ ID NO:5 carry out second with 3' ends primer AUAP takes turns PCR amplifications, comprises the following steps that：The PCR reaction systems of 50 μ 1：The first round PCR primer of 5 μ 1 Ι Ο Χ Ε χ Buffer, 3 μ 1 2. 5 mM dNTP, 2. 0 μ 1 dilution, the μ Μ of 1. 0 μ, 1 Ex Taq 10 primer SEQ ID NO:Each 2. 0 μ 1 of 5 and AUAP, and 35 μ 1 distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 min, 94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of extension 2min, after 33 circulations, 72 °C of 10 min of extension.It is about 1200bp bands that second of PCR primer, which reclaims fragment,（Gel Extraction Kit are purchased from OMEGA) pGEM_T Easy Vector are connected to, being transformed into e. coli jm109, (specific method is ibid）, random 10 white colonies of picking cultivate in the LB fluid nutrient mediums containing 50 μ g/mL ampicillins, and glycerol adding is to final concentration 20% after 37 °C of overnight incubations, and -80 °C save backup. SEQ ID NO :5 carry out bacterium solution PCR amplifications with 3' ends primer AUAP, obtain 5 positive colonies, send Invitrogen（Shanghai）Trade Co., Ltd's sequencing sequencing, obtains the cDNA of gene 3' ends.

According to the GhNHXl-1 genetic fragments obtained, three specific primers are designed, 5'RACE 3' end primers are used as.

GhNHXl-1 GSP3: SEQ ID NO : 6：

CCCTCGCAAC TGAGTATGGC CC

GhNHXl-1 GSP4: SEQ ID NO : 7：

TGCACCACGC ATAAGACCGG CC

GhNHXl-1 GSP5: SEQ ID NO : 8：

ACATGGAGCT TTCTTAGTCA AG

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 5' universal primers AAP）, with cDNA (the reverse transcription primer SEQ ID NO of mRNA reverse transcriptions:6) amplification of first round PCR is carried out for template, comprised the following steps that：Ex Taq are purchased from TAKARA, 50 μ PCR reaction systems：5 μ Ι Ο χ Ε χ Buffer, the 3 μ 2.5 mM μ Ex of dNTP, the cDNA of 2.0 μm of RNA reverse transcriptions, 1.0 Taq, 10 μ Μ primer SEQ ID NO:Each 2.0 μ 1 of 7 and AAP, and 35 μ distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 min, 94 °C of denaturation 30 s, 55 °C of annealing 30 s, 72 °C of extension 2min, after 33 circulations, 72 °C of 10 min of extension.The PCR primer of gained takes 2.0 μ as template after diluting 50 times with distilled water, with SEQ ID NO:8 carry out second with 3' ends primer AUAP takes turns PCR Amplification, is comprised the following steps that：The Ρ Ο reaction systems of 50 μ 1：The first round PCR primer of 5 μ lOxEx Buffer, 3 μ 2.5 mM dNTP, 2.0 μ 1 dilution, 1.0 l Ex Taq, 10 μ Μ primer SEQ ID NO:Each 2.0 μ 1 of 8 and AUAP, and 35 μ distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 min, 94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of 2 min of extension, after 33 circulations, 72 °C of 10 min of extension.It is about that 1200bp bands (Gel Extraction Kit are purchased from OMEGA) are connected to GEM-T Easy Vector that second of PCR, which reclaims fragment, it is transformed into JM109 (specific method is ibid), random 10 white colonies of picking are cultivated in the LB fluid nutrient mediums containing 50 g/mL ampicillins, glycerol adding is to final concentration 20% after 37 °C of overnight incubations, and -80 °C save backup. SEQ ID NO:8 carry out bacterium solution PCR amplifications with 3' ends primer AUAP（Reaction system and reaction condition are ibid）, 4 positive colonies are obtained, Invitrogen is sent（Shanghai）Trade Co., Ltd's sequencing sequencing, obtains the cDNA of gene 5' ends.

After the 5'RACE product clonings sequencing of gained, splice with 3'RACE products sequencing result.Obtain GhNHXl_l full length 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 CAT T GAG AAA TGGAGAGTAG T TAG T GAT AG 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 AG AG AT C AC A 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

It is as follows that 2041 ATTTTATTGA TTGTATATCT TTTTATTAAA AAAAAAAAAA AAAAAAAAAA design pair of primers according to GhNHXl-1 full length cDNA sequences：

GhNHXl-lF: SEQ ID NO : 9：

ATGGCAATCG GGATATTAAG CACT

GhNHXl-lR: SEQ ID NO : 10：

TCACTGCCAT TGTTGATCAT TTTG

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

Using TaKaRa PrimeSTAR HS archaeal dna polymerases, performing PCR reaction is entered by template of the cDNA of cotton.The PCR reaction systems of 50 μ 1：10 μ 15 X PS Buffer, 3 μ 125 mM dNTP, 2. 0 μ 1 cDNA, 1. 0 μ 1 PrimeSTAR, 10 μ Μ primer SEQ ID NO:9 and SEQ ID NO:10 each 20 μ 1, and 30 μ distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 min, 94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of extension 2min, after 33 circulations, 72 °C of 10 min of extension.

Pcr amplification product adds A tails：PCR primer adds 2. 5 times of absolute ethyl alcohol, and _ 20 °C are placed 10 minutes, and centrifugation is removed supernatant, dried, and is dissolved with 21 μ distilled waters.Add 2. 5 μ 1 lO X Ex Buffer, 0. 5 μ 15 mM dATP, the lO X Ex Taq of 2. 5 μ 1.Reaction condition：70 °C are reacted 30 minutes.The DNA fragmentation for obtaining about 1600 bp is reclaimed（Omega QIAquick Gel Extraction Kits）, it is connected on pGEM T-easy carriers, converts JM109 (methods Ibid）, random 10 white colonies of picking cultivate in the LB fluid nutrient mediums containing 50 μ g/mL ampicillins, and glycerol adding is to final concentration 20% after 37 °C of overnight incubations, and -80 °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）, 4 positive colonies are obtained, Invitrogen is delivered to（Shanghai）Trade Co., Ltd is sequenced, and sequence is SEQ ID NO: 2.

The amino acid sequence of NHX1- -1 albumen： SEQ]

1 MAIGILSTLL AKSETILGSG

21 HSSWSMNLF VALLCGCIVI

41 GHLLEESRWM NESITALAIG

61 LCTGIV works: LLT TGGKSSHLLV

81 FSEDLFFNYL LPP work works FNAGF

101 QVKKKQFFRN FMTIMLFGAV

121 GTLISFAIIS LGAIH TKKM

141 SIGNLKIGDY LAIGAIFSAT

161 DSVCTLQVLN QDDTPLLYSL

181 VFGEGWNDA

201 SFDLPHINTT works ALQFVGNFL

221 YLFISSTLLG VLAGLLSAYI

241 work KKLYFGRHS TDREVALMIL

261 MAYLSYMLAE TYLSAILTV

281 TCGIVMSHY TWHNVTESSR

301 VTTKHAFATL SFVAEIFIFL

321 YVGMDALDIE KWRWSDSPG

341 KSVGVSAILL GLILVGRAAF

361 VFPLSSISNL TKKAPCDKID

The beautiful A GLMRGAVSMA of 381 FKQQVT works

401 LAYNQFTSLG HTQLRGNAMM

421 ITSTITWLF STWFGLMTK

441 PLIRILLPSP KNRMLSSEPS

461 TPKSLTVPLL TNGHDLEAYE

481 SDRNLITRPT SLRMFLSTPS

501 NTVHYYWRKF DNAFMRPVFG

521 GRGFVPFVPG SPIEQNDQQW 541

The nucleotide sequence of GhNHXl-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

The GhNHXl-1 gene plant expression vector establishments of embodiment 3

Select plant binary expression vector PCAMBIA2300 (public purchased from the prosperous biotechnology Limited Liability of Beijing ancient cooking vessel state Department）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.Inducible promoter rd29A and Tnos is selected as the promoter and terminator of GhNHXl_l genes.

With primer SEQ ID NO:11 and SEQ ID NO:12 (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.The PCR reaction systems of 50 μ 1：10 μ 15XPS Buffer, 3 μ 12 5 mM dNTP, 1.0 μ, 1 PBI121,1.0 μ 1 PrimeSTAR, 10 μ Μ primer SEQ ID NO:11 and SEQ ID NO:12 each 2.0 μ 1, and 31 μ distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 min, 94 °C of denaturation 30 s, 56 °C of annealing 30 s, 72 °C of 30 s of extension, after 33 circulations, 72 °C of 10 min of extension.PCAMBIA2300 (promega, T4 ligase boxes are connected to by EcoRI, Bglll digestion）Obtain pCAMBIA2300_l.

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.The PCR reaction systems of 50 μ 1：10 μ 15XPS Buffer, 3 μ 12.5 mM dNTP, the PrimeSTAR of 1.0 μ, 1 PBI121,1.0 μ 1,10 μ Μ primer SEQ ID NO:13 and SEQ ID NO:14 each 2.0 μ 1, and 31 μ 1 distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5,94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of 30 s of extension, after 33 circulations, 72 °C of 10 mine of extension are connected to pCAMBIA2300-l (promega T4 ligase boxes by Sacl, EcoRI digestion）Obtain PCAMBIA2300-2

SEQ ID NO: 13：

AAG dOTAATTTCCCCGATCGTTCAAA

SEQ ID NO: 14：

TCA GAA M AGTGAATTCCCGATCTAGTA

SEQ ID NO:15 and SEQ ID NO:16 with arabidopsis（Colombia's type, 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.The PCR reaction systems of 50 μ 1：10 μ 5XPS Buffer, 3 μ 1 2.5 mM dNTP, 1.0 μ 1 arabidopsis DNA, 1.0 μ 1 PrimeSTAR, 10 μ Μ primer SEQ ID NO:15 and SEQ ID NO:16 each 2.0 μ 1, and 31 μ distilled water.PCR reaction conditions：94 °C of min of pre-degeneration 5,94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of 30 s of extension, after 33 circulations, 72 °C of 10 min of extension.It is connected to by HindIII, Pstl digestion（Connection method is ibid）PCAMBIA2300-2 obtains pCAMBIA2300-3

SEQ ID NO : 15：

ACTAAGCTTCCTTCTTGACATCATTCAATTTTA SEQ ID NO : 16：

TGAO^ CTCCAAAGATTTTTTTCTTTCCAATAG

SEQ ID NO :17 and SEQ ID NO:18 amplification GhNHXl_l (template is that embodiment 2 obtains GhNHXl_l), using TaKaRa PrimeSTAR HS archaeal dna polymerases.The PCR reaction systems of 50 μ 1：10 μ 15 X PS Buffer, 3 μ 1 2. 5 mM dNTP, 1. 0 μ 1 GhNHXl- 1- pGEM, 1. 0 μ 1 PrimeSTAR, 10 μ Μ primer SEQ ID NO:17 and SEQ ID NO:18 each 2. 0 μ 1, and 31 μ 1 distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 min, 94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of extension 2min, after 33 circulations, 72 °C of 10 min of extension.It is connected to by Pstl, Sad digestion（Connection method is ibid）PCAMBIA2300-3, obtains plant expression vector rd29A_ GhNHXl-l_2300.

SEQ ID NO : 17：

TGAC 7¾ i7ATGGCAATCGGGATATTAAGCACT

SEQ ID NO : 18：

The rd29A-GhNHXl-l-2300 expression vectors of AAGGUCTCACTGCCATTGTTGATCATTTTG embodiments 4 convert Agrobacterium

It is prepared by Agrobacterium LBA4404 (being purchased from Biovector Science Lab, Inc) competence：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 h) to 0D600 values be 0. 4, formed seed bacterium solution.Take the bacterium solution after 5 ml activation（1 :20 ratio）In the LB fluid nutrient mediums for being inoculated in the same concentration antibiotic of 100 ml, 28 °C are shaken the culture h of 2-2. 5 to 0D₆..=0. 8.The min of ice bath bacterium solution 10, shakes up once every 3 min, makes bacterium even into resting state.10 min are centrifuged in 4000 g under 4 °C, supernatant is abandoned；Add 4000 g under certain glycerine resuspension thalline of head for precooling 10%, 4 °C and centrifuge 10 min, collect precipitation；Repeated to wash 3-4 times with 10% glycerine；Adding 10% glycerine of appropriate ice bath precooling, suspended bacterial is precipitated again, is dispensed, is saved backup in -70 °C with the pipes of 40 μ 1/.

Convert Agrobacterium：Melt competent cell on ice, 1 μ 1 plasmid is added into 40 μ 1 competent cell, competence and DNA mixture are transferred in the electric shock of precooling cup by the min o of ice bath about 10 with rifle after mixing, and rapping makes suspension reach bottom, has been careful not to bubble.To be shocked by electricity cup（Purchased from bio-rad) it is put into electroporation chamber On slideway, slideway is promoted to put electric shock cup to electroporation chamber base electrode.Using 0. lcm 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 the LB culture mediums of 28 °C of preheatings.It is quickly soft to be beaten cell with rifle.Suspension is transferred to 1.5 ml centrifuge tube, 28 °C, 225 rpm cultivate 1 h.Take 100 200 μ 1 bacterium solution be coated with corresponding resistance screening culture medium flat plate（LB solid mediums, containing 50 μ g/ml rifampins, 50 μ g/ml streptomysins, 50 μ g/ml kanamycins）, 28 °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：Tobacco institute of the Chinese Academy of Agricultural Sciences, storehouse numbering I5A00660) 30 s, are washed twice with sterilizing distilled water.8 min are soaked with 0. 1% mercuric chloride again, is washed with sterilizing distilled water twice, completes surface sterilizing.The tobacco seed of surface sterilizing is placed in MS (18.78 mMKN0₃, 1.25 mM KH₂P0₄, 20.6 mM NH4NO3, 1.5 mM MgS0₄, 3.0 mM CaCl₂, 50 μ M KI, 100 μΜ H₃B0₃, 100 μM of MnS0₄ , 30 μ M ZnS0₄, 1 μM of Na₂Mo0₄, 0. 1 μΜ CoCl₂, 100 μ M Na₂EDTA, 100 μΜ FeS0₄, 7.4 g/L agar, the g/L of sucrose 30) under aseptic condition germinate, prepare aseptic seedling.Take tests for sterility to be cut into the leaf dish of 5 mmX5 mm sizes, with the min of During Agrobacterium leaf dish 10 of the rd29A-GhNHXl-l-2300 containing expression vector in exponential phase, blot bacterium solution, co-cultured 2 days under dark condition（MS culture mediums）.Blade is gone into differential medium（The MS+1 mg/L basic elements of cell division（BA) the mg/L cephalosporins of+0. 1+50 mg/L kanamycins of mg/L methyl α-naphthyl acetate (Surface+500）On, cultivated 45 days or so under illumination condition, cut after bud is grown up and be transferred to root media（The mg/L cephalosporins of MS+50 mg/L kanamycins+500）Middle culture 30 days or so, preservation is numbered after being transferred to seedling after well developed root system on the MS culture mediums only added with 500 mg/L cephalosporins.

Take the transgenic tobacco leaf of acquisition, extract DNA (arabidopsis DNA extraction methods in be the same as Example 3）, with SEQ ID NO: 9：With SEQ ID NO:10 (the PCR reaction systems of 50 μ 1：5 μ 1 Ι Ο Χ Ε χ Buffer, 3 μ 125 mM dNTP, 10 μM of 1 Ex Taq of 2.0 μ 1 DNA, 1.0 μ primer SEQ ID NO:9 and SEQ ID NO:10 each 2.0 μ 1, and 35 μ 1 distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 min, 94 °C of denaturation 30 s, 58 °C of 30 s of annealing, 72V extends 2 min, after 33 circulations, 72 °C of 10 min of extension), PCR identifications preserve positive plant and T are numbered.H1_T.H20.Embodiment 6 is overexpressed GhNHXl-1 transgene tobaccos T1 salt tolerant simulated experiment and Function Identification

T.H1-T.H20 tobacco seeds, non-transgenic reference tobacco seed sterilizing distilled water immersion 30min, with 75% alcohol-pickled 30 s, are washed twice with sterilizing distilled water.8 min are soaked with 0. 1% mercuric chloride again, is washed with sterilizing distilled water twice, completes surface sterilizing.Do two groups of experiments：First group：By the T of surface sterilizing.H1-T.H20 tobacco seeds, control tobacco Seed is placed on MS solid mediums in germination under aseptic condition.Second group：By the T of surface sterilizing.H1-T.H20 tobacco seeds, control tobacco seed are placed on the MS solid mediums added with lOOmMNaCl in germination under aseptic condition. 25 .C, optical culture/14 hour light culture circulation in 10 hours, result is observed after 10 days.First group of result of the test shows that TO is 90% for the percentage of seedgermination of each strain in MS solid mediums, and growth conditions are good, with compareing no significant difference（Fig. 3);Second group of result of the test is found, in the MS culture mediums containing lOOmMNaCl, T.H1_T.H20 tobacco seeds and control seed can be sprouted, but adjoining tree growth is substantially suppressed.T1 is for transfer-gen plant（The plant that T0 grows up to for the seed of genetically modified plants）Salt-Tolerance Identification show, Η 2, Η 5, Η 7, Η 13, Η 15, Η 16, Η 19, the growth of the transfer-gen plants of Η 20 is overall is higher than adjoining tree, show obvious salt tolerance（Referring to Fig. 4, by taking Η 2 as an example, Η 5, Η 7, Η 13, Η 15, Η 16, Η 19, Η 20 it is similar with Η 2 result, be 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（Upgrowth situation is good）LOOmMNaCl handles 14 days leaf 0.05g, uses plant RNA extraction kit（Invitrogen) the total serum IgE extracted.Total serum IgE is determined in 260 nm and 280 nm absorbance with the ultraviolet specrophotometer U-2001 of HITACHI companies, calculates each RNA concentration.Reverse transcription is carried out using invitrogen reverse transcription reagent box Superscript III Reverse Transcriptase and according to method in specification（The total R A of 2 μ g are used as template, reverse transcription primer SEQ ID NO: 10).Pass through SEQ ID N0:9 and SEQ ID NO:10 amplification GhNHXl_l, detect GhNHXl-1 albumen relative expression's situations.Using TaKaRa PrimeSTAR HS archaeal dna polymerases, performing PCR reaction is entered by template of the cDNA of reverse transcription.The PCR reaction systems of 50 μ 1：10 μ 5 X PS Buffer, 3 μ 1 2.5 mM dNTP, 2.0 μ 1 cDNA, 1.0 μ 1 PrimeSTAR, 10 μ Μ primer SEQ ID N0:9 and SEQ ID NO:10 each 2.0 μ 1, and 30 μ 1 distilled water.PCR reaction conditions：94 °C of pre-degenerations 5 min, 94 °C of denaturation 30 s, 58 °C of annealing 30 s, 72 °C of extension lmin, after 29 circulations, 72 °C of 10 mine product electrophoresis results of extension are as shown in Figure 5：M is DNA Ladder Marker (DL2000, purchased from Shenzhen Rui Zhen Bioisystech Co., Ltd）, 1-5 is control tobacco, 6-15 be salt tolerant transgene tobacco T1 for plant, 16_21 is not salt tolerant transgene tobacco T1 for plant.Stripe size shown in figure is in the same size with GhNHXl-1 genes.As a result show to compare the mRNA that tobacco does not transcribe GhNHXl-1, salt tolerant transgene tobacco T1 is stronger for plant pair GhNHXl-1 transcription, and salt tolerant transgene tobacco T1 does not transcribe or transcribed for plant very weak.

Claims

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 the gene described in claim 1 and the nucleotide sequence of the gene is operably connected with the expression control sequence of the expression vector.

3. the carrier described in claim 2, it is the rd29A-GhNHXl-l-2300 carriers shown in accompanying drawing 2.

4. a kind of recombinant cell, it contains the recombinant expression carrier described in gene or Claims 2 or 3 described in claim 1；Preferably, the recombinant cell is restructuring agrobatcerium cell.

5. a kind of method of improvement plant salt endurance, including：Recombinant expression carrier described in gene or claim 2 or 3 described in claim 1 is imported into plant or plant tissue and makes the gene expression；Preferably, the plant is tobacco.

6. a kind of method of prepare transgenosis plant, including：The plant containing the recombinant expression carrier described in the gene or Claims 2 or 3 described in claim 1 or plant tissue are cultivated under conditions of plant is effectively produced.

7. the method described in claim 6, wherein the plant is tobacco.

8. the recombinant expression carrier described in gene, Claims 2 or 3 described in claim 1 or the recombinant cell described in claim 4 are used to improve plant salt endurance and the purposes for plant breeding.

9. the purposes described in claim 8, wherein the plant is tobacco.

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