CN105189535A

CN105189535A - Transporter HKT2 with high affinity for potassium ions and which is derived from cotton and the coding gene and use thereof

Info

Publication number: CN105189535A
Application number: CN201380074523.4A
Authority: CN
Inventors: 崔洪志; 王建胜; 王君丹; 刘捷
Original assignee: Genesis Seed Industry Co ltd
Current assignee: Genesis Seed Industry Co ltd
Priority date: 2013-06-24
Filing date: 2013-06-24
Publication date: 2015-12-23
Also published as: WO2014205597A1

Abstract

Disclosed in the present invention are a transporter HKT2 with a high affinity for potassium ions and which is derived from cotton and the coding gene thereof, and the use thereof in cultivating a genetically modified plant with an improved salt tolerance.

Description

Transporter HKT2 with high affinity for potassium ions and which is derived from cotton and the coding gene and use thereof

One grows cotton high affine kalium ion transport albumen HKT2 and its encoding gene and application

The present invention relates to vegetable protein and its encoding gene and application, more particularly to one high affine the kalium ion transport albumen HKT2 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 achieve many new developments, in terms of salt tolerant molecule mechanism especially in Land use models plant Arabidopsis thaliana to study plant, making the research in the field has breakthrough progress (the Salt and drought stress singal transduction in plants. Annu. Rev. Plant Biol. 53 of Zhu JK. 2002.: 1247-1273; Zhang ZL. 201 1. Arabidopsis Floral Initiator SKB 1 Confers High Salt Tolerance by Regulating Transcription and Pre-mRNA Splicing through Altering Histone H4R3 and Small Nuclear Ribonucleoprotein LSM4 Methylation. Plant Cell, 23 : 396-41 1 ) .Higher plant cell can experience the change of physico-chemical parameter in external environment by number of ways, so that extracellular signal is delivered into intracellular signal, stress signal finally is transferred into activating transcription factor in nucleus by the signal transduction of series.Activating transcription factor remakes the expression for starting induced gene in adversity for functional gene, so as 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 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 the one high affine kalium ion transport albumen of cotton（Be named as HKT2 herein) encoding gene, and determine its DNA sequence dna.And it was found that being conducted into plant by transgenic technology and making after its expression, the salt tolerance of transfer-gen plant can be significantly improved, and these characters can stablize heredity.

First aspect present invention provides the affine kalium ion transport albumen HKT2 one high of cotton encoding gene（GhHKT2 is named as herein), its sequence is SEQ ID NO: 2.

Second aspect of the present invention provides a kind of recombinant expression carrier, it contains the gene described in first aspect present invention, it is obtained by the way that the gene 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 recombinant expression carrier；Preferably, the expression vector is pCAMBIA2300_;Preferably, the recombinant expression carrier is the 35S-GhHKT2-2300 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 G r2 genes（35S-GhHKT2-2300) build flow（Scheme la-lb). Fig. 2 is the plant expression vector of G r2 genes（Plasmid figure 35S-GhHKT2-2300).Fig. 3 is salt tolerant experimental results of the T1 for Arabidopsis plant for turning GhHKT2 genes, and Tlg7 shows significant salt tolerance, and Tlgll, Tlgl3 result are similar with its, are not shown here.

Fig. 4 be using reverse transcription PCR to 1 in transgenic Arabidopsis plants and non-transgenic reference plant GhHKT2 genes transcriptional level progress molecular level detection result.M is DNA Ladder Marker (DL2000), and 1-8 is salt tolerant T1 for transgenic Arabidopsis plants（It is belonging respectively to tri- strains of Tlg7, Tlgll, Tlgl3）, 9 be 35S-GhHKT2-2300 plasmid PCR positive controls, and 10-13 is non-transgenic reference Arabidopsis plant.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 following examples is purchased from New England Biolabs companies.Cotton 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（Suppress subtracted library）.The mRNA extracted in experimentation using in the Levant Cotton Root of salt treatment is used as sample（Tester), the mRNA extracted using in untreated Levant Cotton Root is used as control（Driver) .Comprise the following steps that：

(1) material to be tested：

African cotton（National Cotton mid-term storehouse, obtains unit Cotton research institute, Unified number：ZM-06838) it is seeded on sterilized vermiculite, is cultivated under the conditions of 25 °C, light dark period 16h/8h, 1/2MS fluid nutrient mediums are poured weekly（Containing 9.39 mMKN0₃, 0.625 mMKH2P0₄, 10.3 mMNH₄N0₃, 0.75 mMMgS0₄, 1.5 mMCaCl₂, 50 μ Μ Κ Ι, 100 μ Μ Η₃ΒΟ₃, 100 MMnSO₄, 30 MZnSO₄, 1 μ Μ Na₂Mo0₄, 0.1 MCoCl₂, 100 μ Μ Na₂EDTA, 100 MFeSO₄)-secondary.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, handles 6 hours.Timely two groups of childrens of clip after being disposed The root of seedling, after liquid nitrogen quick freeze, is preserved in -70 °C of refrigerators.

(3) Total RNAs extraction：

Control group and each 0.5 g of the Levant Cotton Root 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 companies_26Q/OD_28QRatio is 1.8-2.0, shows that total serum IgE purity is higher；The integrality of total serum IgE 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（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（Reverse transcription primer provides primer by kit）, obtain double-strand cDNA, then the progress subtractive hybridization using 2 μ g Tester cDNA and 2 μ g Driver cDNA as parent material.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 cDNA 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 is subjected to 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：Positive subtractive after the merging of purifying hybridizes second of μ 1 of inhibition PCR products 3, the μ 1 of 2 X T4 DNA ligases buffer solution 5, the μ 1 of pGEM-T Easy carriers 1, the μ 1 of 4 DNA ligases of Τ 1 of cDNA fragments, is stayed overnight in 4 °C of connections.Then 10 μ coupled reaction products are taken, the escherichia coli jm109 competent cells of 100 μ 1 are added to（Purchased from TAKARA) in, then ice bath 30 minutes, heat shock 60 seconds, ice bath 2 minutes add 250 l LB fluid nutrient mediums（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, then therefrom take the bacterium solutions of 200 μ 1 to be inoculated in containing 50 μ g/ml ampicillins, 40 g/mL X-gal (the chloro- 3- indoles-β-D- galactosides of 5- bromo- 4）, 24 g/mL IPTG (isopropyls - β-D- Thiogalactopyranosides）LB (ibid）On solid culture plate（X-gal and IPTG are purchased from TAKARA), 37 °C are cultivated 18 hours.Count diameter in culture plate>1 mm clear white and blue colonies, random 300 white colonies of picking（Numbering：Gh-S2-001 to Gh-S2-300).Institute's picking white colony is inoculated in 96 porocyte culture plates respectively（CORNING the LB fluid nutrient mediums containing 50 μ g/ml ampicillins in)（Ibid）In, glycerol adding to final glycerol concentration is 20% (volume ratio after 37 °C of overnight incubations）, then saved backup in -80 °C.Use the standing grain P Primer 2R of nest-type PRC primer Primer 1 (the PCR-select cDNA Subtraction Kit kits from Clontech companies）Enter performing PCR amplification checking respectively to the bacterium solution cultivated, obtain 231 positive colonies, 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, 203 effective expression sequence labels are obtained（Expressed Sequence Tag, EST ) ( Unigene) .The high affine kalium ion transport protein coding gene GhHKT2 of the cotton of embodiment 2 clone

The clone that numbering is Gh-S2-092 in the cotton SSH libraries identified is removed after redundant DNA, sequence is SEQ ID No:3, sequence analysis shows that the albumen of the sequential coding belongs to high affine kalium ion transport albumen.Herein by SEQ ID No:The corresponding total length encoding gene of 3 sequences is named as G r2, and its corresponding albumen is named as

HKT2。

SEQ ID No: 3：

102 1 AGTTTTCTTA AATCACTAGT GATTGT

The clone of GhHKT2 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.

GhHKT2 GSP 1 : SEQ ID No : 4：

GAGGTGGTAA GTGCATATGG TAA

GhHKT2 GSP2 : SEQ ID No _:5：

TGGGTTATAG TTGCAAGCTA CGA experimental procedures are 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 that the mRNA reverse transcriptions extracted using salt treatment group cotton are obtained 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 2.0 μ 1, the Ex Taq of 1.0 μ 1 (being purchased from TAKARA), 10 μM of primer SEQ ID NO:4 standing grain mouthful each 2.0 μ of AUAP and 35 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 1 minute）, 72 °C extend 10 minutes.

2.0 μ are taken as template after the PCR primer of gained is diluted into 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 second and take turns the band that fragment is about 350 bp in PCR products（Gel Extraction Kit are purchased from

OMEGA), and pGEM-T Easy carriers are coupled with, be then transformed into escherichia coli jm109 competent cell（Specific method is ibid）, and the bacterium solution after conversion is coated on containing being screened on 50 ^lmL ampicillins, the g/mL IPTG of 40 ^glmL X-gaK 24 LB solid mediums.Random 10 white colonies of picking are inoculated in the LB fluid nutrient mediums containing 50 g/ml ampicillins respectively, 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:5 are carried out with universal primer AUAP Bacterium solution PCR amplification checkings, obtain 5 positive colonies, 4 positive colonies are delivered into Invitrogen（Shanghai）Trade Co., Ltd's sequencing sequencing, obtains the cDNA of gene 3' ends.

According to the G J2 genetic fragments obtained, following three specific primers are designed, 5'RACE 3' end primers are used as.

GhHKT2 GSP3: SEQ ID No_:6：

GCTAAAGCAA TTATGACTAA GA

GhHKT2 GSP4: SEQ ID No_:7：

TGTGAATAGG TTTAGTCCTT TGC

GhHKT2 GSP5: SEQ ID No_:8：

TTAACGTGCT TCGAGCACTT GAA

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 that the mRNA reverse transcriptions extracted with salt treatment group cotton are obtained:6, dCTP tailings）The amplification of first round PCR is carried out for template, is comprised 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 2.0 μ 1, the Ex Taq of 1.0 μ 1 (being purchased from TAKARA), 10 μM of primer SEQ ID NO:Each μ 1 of 2.0 μ 1 and 35 of 7 standing grain P 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.

Take 2.0 μ as template after the PCR primer of gained is diluted into 50 times with distilled water, use SEQ IDNO:8 carry out second with primer AUAP takes turns PCR amplifications, comprises the following steps that：

50 l PCR reaction systems：The first round PCR primer of the mM of 5 μ, 1 lOXEx Buffer, 3 μ 12.5 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 second and take turns the band that fragment is about 700 bp in PCR products（Gel Extraction Kit are purchased from OMEGA), and pGEM-TEasy carriers are coupled with, then it is transformed into escherichia coli jm109 competent cell（Specific method is ibid）, and the bacterium solution after conversion is coated on containing being screened on 50 ^lmL ampicillins, the g/mLIPTG of 40 ^glmL X-gaK 24 LB solid mediums.Random 10 white colonies of picking are inoculated with respectively In the LB fluid nutrient mediums containing 50 g/ml ampicillins, 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 verifies（Reaction system and reaction condition are ibid）, 6 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 5 ' the RACE product clonings sequencing of gained, by itself and above-mentioned 3'RACE products sequencing result and SEQ ID No:3 sequences are spliced, and obtain GhHKT2 full length cDNA sequence SEQ ID No: 9.

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

SEQ ID No: 10：

ATGTGGTGTA TGAAGATGAA CC SEQ ID No: 11：

TTAAGAAAAC TTCCAAACCA TG pass through SEQ ID NO:10 and SEQ ID NO:11 clone GhHKT2 total length encoding genes.

Using TAKARA PrimeSTAR HS archaeal dna polymerases, performing PCR reaction is entered by template of the cDNA of above-mentioned cotton.50 l PCR reaction systems：10 l 5XPS Buffer, the mM of 3 μ 12.5 dNTP, the PrimeSTAR HS archaeal dna polymerases of 2.0 μ, 1 cDNA, 1.0 μ 1,10 μM of primer SEQ ID NO:10 and 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, 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.

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, then precipitated with 21 μ distilled waters dissolving gained.Then the mM of 2.5 μ, 1 lOXEx Buffer, 0.5 μ 15 dATP, 1.0 lExTaq are 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 and obtain GhHKT2-pGEM plasmids, then connection product is transformed into escherichia coli jm109 competent cell（Method is ibid）, and the bacterium solution after conversion is coated on containing being screened on 50 g/mL ampicillins, the g/mLIPTG of 40 g/mL X-gaK 24 LB solid mediums.Random 10 white colonies of picking are inoculated in the LB fluid nutrient mediums containing 50 g/ml ampicillins respectively, 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:10 and SEQ ID NO:11 carry out bacterium solution PCR amplification checkings（Reaction system and reaction condition are ibid）, 7 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 HKT2 albumen： SEQIDNO: 1

1 MWCMK NQYC SCFHKK PKF

21 LVSLSYSSSK LTRLCHVLSV

41 NPFWTRILYF IFLSLLGFWG

61 LNVLEPRTDS FKPRKFDLFF 81 TSVSATTVSS MSTVEMEILS

101 NPQLI IMTIL MFIGGEVFTS

121 MAGLFFSNFI SEKNNVIDDG

141 EIELGNRKPE NLNPEQLAQN

The beautiful GEH LLYHS IMFLG of 161 QGEAF

181 FVILGYLLIV NILGSAMVFL

201 YVSFVSSARS TLKSKGLNLF

221 TFS I FTTISS FTNCGFI PTN

241 ENMWFNKNS GLLLVI IALA

261 LLGNPLFPMC LRFLIWVMGK

281 CVNKVGDYCD YLLKNTREIG

301 FHHLFTTRRS CCWGTALVF

321 VWQALLFFA MEWNSASLKE

341 LNPFERTIGV LFQSVNTSQA

361 GETIVNLPAM STVILWITI

381 IMYFPPYTS I PFVKDEKKEQ

401 QNQEKRKGKT AKELLTQLAS

421 ICI FVFLICI TERKNMKEDP

441 FNFTPFNFLF EWSAYGNVG

461 YSMGYSCKLR LKDEANCVDK

481 SYGFVGRWSD AGKTVLIWM

501 MLGRLKRYNM VWKFS *

781

841

901

961

1021

1081

1141

1201

1261

1321

1381

1441

The GhHKT2 gene plant expression vector establishments of 1501 ATGCTTGGTA GACTAAAGAG ATACAACATG GTTTGGAAGT TTTCTTAA embodiments 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 GhHKT2 genes, build flow chart as shown in Figure 1.

Use primer SEQIDNO:12 and SEQIDNO:13, with plant expression vector pBI121 plasmids（Purchased from ocean Science and Technology Ltd. of Beijing China）For template amplification Pnos, using TAKARA PrimeSTAR HS archaeal dna polymerases.50 lPCR reaction systems：10 l5XPSBuffer, the mM of 3 μ 12.5 dNTP, the plasmids of 1.0 μ, 1 ρ Β Ι 121, the PrimeSTAR HS archaeal dna polymerases of 1.0 μ 1,10 μM of primer SEQ ID NO:12 and SEQIDNO:13 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 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 and SEQ ID NO:15 using pBI121 plasmids as template amplification Tnos, using TAKARA PrimeSTAR HS archaeal dna polymerases.The PCR reaction systems of 50 μ 1： 10 μ 1 5XPS The mM of 3 μ of Buffer 1 2.5 dNTP, the plasmids of 1.0 μ, 1 ρ Β Ι 121, the PrimeSTAR HS archaeal dna polymerases of 1.0 μ 1,10 μM of primer SEQ ID NO:14 standing grain P SEQ ID NO:15 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 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：

AAGGGTACCGAATTTCCCCGATCGTTCAAA SEQ ID NO: 15：

TCAGAATTCCCAGTGAATTCCCGATCTAGTA primer SEQ ID NO:16 and SEQ ID NO:17 using pCAMBIA2300 plasmids as template amplification 35S promoters.Using TAKARA PrimeSTAR HS archaeal dna polymerases.50 y l PCR reaction systems：The mM of 10 μ, 153 μ of X PS Buffer 1 2.5 dNTP, the pCAMBIA2300 plasmids of 1.0 μ 1, the PrimeSTAR HS archaeal dna polymerases of 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 product 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 amplification G r2 encoding genes（Template is that embodiment 2 obtains positive GhHKT2-pGEM plasmids）, using TAKARA PrimeSTAR HS archaeal dna polymerases.50 μ PCR reaction systems：The X PS Buffer of 10 l 5, the mM of 3 μ 1 2.5 dNTP, the GhHKT2-pGEM plasmids of 1.0 μ 1, the PrimeSTAR HS archaeal dna polymerases of 1.0 μ 1,10 μ Μ primer SEQ ID NO:18 and SEQ ID NO:19 each distilled waters of 2.0 μ, 1 and 31 μ 1.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 PCR primer of Sall, Kpnl digestion by obtained by（Connection method is ibid）To pCAMBIA2300-3, plant expression vector 35S-GhHKT2-2300 (Fig. 2) is obtained after empirical tests. SEQ ID NO: 18

ACTGTCGAC ATGTGGTGTA TGAAGATGAA CC SEQ ID NO: 19

The 35S-GhHKT2-2300 expression vectors of ACTGGTACC TTAAGAAAAC TTCCAAACCA TG embodiments 4 convert Agrobacterium

The preparation of Agrobacterium LBA4404 (being purchased from Biovector Science Lab, Inc) competent cell：Agrobacterium LBA4404 is drawn to single spot inoculation on the LB solid mediums containing 50 g/ml rifampins and 50 g/ml streptomysins, 28 °C are cultivated 1 to 2 day.Picking single bacterium colony is inoculated in 5 ml containing 50 μ_§The rifampins of/ι η 1 and 50 μ_§In the LB fluid nutrient mediums of the streptomysins of/ι η 1, overnight incubation is shaken under 28 °C（About 12-16 hours）To OD₆..It is worth for 0.4, formation seed bacterium solution.Take the bacterium solution after 5 ml culture activation（1 :20 ratio）100 ml are inoculated in containing 50 μ_§The rifampins of/ι η 1 and 50 μ_§In the LB fluid nutrient mediums of the streptomysins of/ι η 1,28 °C of shakes cultivate 2-2.5 hours to OD₆..=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）Glycerine 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-GhHKT2-2300 of 1 μ embodiments 3 acquisition, ice bath about 10 minutes after mixing are added into the 40 μ competent cell.The mixture of competent cell after ice bath and 35S-GhHKT2-2300 plasmids is transferred to the electric shock cup of the 0.1cm specifications of ice precooling with micropipettor（Purchased from Bio-Rad) in, rapping makes suspension reach electric shock cup bottom（It 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.MicroPulser (being purchased from Bio-Rad) program is set to " Agr ", 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 micropipettor.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 g/ml rifampins, 50 μ_§The streptomysins of/ι η 1,50 μ_§The kanamycins of/ι η 1）, 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.Using the cm of diameter 9 flowerpot, 20-30 arabidopsis seed is sowed per basin（Colombia's type, the arabidopsis Biological Resource Center from Ohio State Univ-Columbus USA）.The film in flowerpot upper cover later is sowed, the growth to plant provides the environment of a moistening.The V of constant temperature 22, intensity of illumination 3500-4000 lx, periodicity of illumination are 12 hours dark/12 hours illumination cultivations, pour a 1/2MS fluid nutrient medium within every 7 days.After culture 30 days, retain 4-5 plant per basin, 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.The leaching conversion of the thaliana flower of embodiment 6

The LBA4404 Agrobacterium bacterium solutions for having converted 35 S-GhHKT2-2300 expression vectors that embodiment 4 is obtained are seeded to containing 50 μ_§The kanamycins of/ι η 1（Kan overnight incubation in LB fluid nutrient mediums), the next morning presses 1:50 are seeded to containing 50 μ_§The new LB culture mediums of the kanamycins of/ι η 1（In 1L), about 8 hours are cultivated, to agrobacterium liquid OD₆..Between 1.0 to 1.2.The rpm of room temperature 5000 is centrifuged 5 minutes, abandons supernatant, and Agrobacterium precipitation is suspended in into dip-dye culture medium（1/2MS fluid nutrient mediums, and contain 5% sucrose；PH5.7 is adjusted to KOH;0.02% Silwet L-77) in, make OD₆..0.8 or so.The top of the arabidopsis for being used to convert prepared by embodiment 5 slowly, is spirally immersed in the dip-dye culture medium containing Agrobacterium, gently rocks clockwise, about 2 minutes, is covered tightly to keep humidity with blister pack, be put into greenhouse and stay 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, seed is collected after drying in 1-2 week, transformant screening is carried out, while taking the arabidopsis Fruit pod of unconverted processing as control.The screening of the arabidopsis transgenic positive transformant of embodiment 7

Seed disinfection：First soaked 10 minutes with 70% ethanol, and make seed suspension every now and then；Then washed four times with sterile, and make seed suspension every now and then.Then, the seed after processing is uniformly coated on containing 50 μ_§On the 1/2MS solid screening and culturing primary surfaces of the kanamycins of/ι η 1（The plate of one piece of 150 mm diameter at most 1500 seeds of sowing）, 4 °C of vernalization 2 days, then culture 7-10 days in the case where 22 °C of constant temperature, intensity of illumination 3500-4000 k, periodicity of illumination are 12 hours dark/12 hours illumination conditions.After transgenic seed is sprouted 2 weeks on the screening and culturing medium, it is possible to sprout and the plant of normal growth is transferred to soil continuation culture.Described in clip can on screening and culturing medium each plant of normal growth 1-2 blade, its DNA is extracted as template, with SEQ ID NO:18 and SEQ ID NO:19 enter performing PCR as primer Detection（Reaction system and condition are ibid）, the negative plant of PCR are removed, the seed for collecting PCR positive plants is numbered (T0gl-T0g25) and preserved respectively.Embodiment 8 is overexpressed GhHKT2 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.Will numbering

T0gl-T0g25 every kind of transformant and non-transgenic reference arabidopsis seed respectively 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 fluid nutrient medium within every 7 days.After culture 25 days, 1-2 blade of every plant of clip simultaneously extracts its DNA as template, with SEQ ID NO:18 and SEQ ID NO:19 enter performing PCR detection as primer（Reaction system and condition are ibid）.The negative plant of PCR are removed, the positive seedlings of 7-8 PCR are retained per basin, are continued after cultivating 10 days, salt tolerant experiment is carried out per 5-7 more consistent transgenic arabidopsis of basin reservation size or non-transgenic reference arabidopsis seedling.The transgenic arabidopsis T1 that embodiment 9 is overexpressed GhHKT2 is tested for the salt tolerant of plant

Transgenic arabidopsis, control arabidopsis in embodiment 8 are respectively retained into a basin plant not deal with, it is normal to pour

1/2MS fluid nutrient mediums, a basin plant is respectively taken to pour the 1/2MS fluid nutrient mediums containing 150 mM NaCl simultaneously, 22 °C of constant temperature, intensity of illumination 3500-4000 k, optical culture/12 hour light culture circulation in 12 hours, observation experiment result after 14 days.T1 is for transfer-gen plant（The plant that TO grows up to for the seed of transfer-gen plant）Salt-Tolerance Identification show that T1 shows significant salt tolerance for tri- strains of transfer-gen plant Tlg7, Tlgl l, Tlgl3（See Fig. 3, with Tlg7, Tlgl l, Tlgl3 result with it is similar, be not shown here）.Embodiment 10 verifies the expression of G r2 genes on transcriptional level

By the T1 of good salt tolerance in embodiment 9 for randomly selecting 8 in transfer-gen plant（It is belonging respectively to tri- salt-resistance strains of above-mentioned Tlg7, Tlgl l and Tlgl3）, adjoining tree randomly selects 4, each clip salt in embodiment 9（150 mM NaCl) the processing g of blade 0.05 of 14 days, 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.Method shown in neat Ll boxes Superscript III Reverse Transcriptase is tried according to Invitrogen reverse transcriptions and carries out reverse transcription, takes 1 total serum IgE as template, reverse transcription primer is SEQ ID NO: 11.Use primer SEQ ID NO:10 standing grain mouthful SEQ ID NO: 20 ( SEQ ID NO: 20：ACTTCCAAAC CATGTTGTAT C) amplification GhffK fragments, detect its transcription situation. Using TAKARA PrimeSTAR HS archaeal dna polymerases, performing PCR reaction is entered by template of the cDNA obtained by above-mentioned reverse transcription.The Ρ Ο reaction systems of 50 μ 1： 10 μΐ 5 ><PS Buffer, 3 μ 2.5 mM dNTP, 2.0 μ cDNA, 1.0 μ PrimeSTAR HS archaeal dna polymerases, 10 μ Μ primer SEQ ID NO:10 standing grain P SEQ ID NO:20 each 2.0 μ 1, and 30 μ 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 2 minutes）, 72 °C extend 10 minutes.

PCR primer electrophoresis result is as shown in Figure 4：M is DNA Ladder Marker (DL2000, purchased from Shenzhen Rui Zhen Bioisystech Co., Ltd）, 1-8 is salt tolerant T1 for transgenic Arabidopsis plants（It is belonging respectively to tri- strains of Tlg7, Tlgl l, Tlgl3）, 9 be 35S-GhHKT2-2300 plasmid PCR positive controls, and 10-13 is non-transgenic control Arabidopsis plant.Stripe size shown in figure and positive control it is in the same size（About 1500 bp).As a result show, salt tolerant T1 has in notable transcription, non-transgenic reference Arabidopsis plant for G J2 in transgenic Arabidopsis plants does not have GhHKT2 transcription.

Claims

Claims

1. the one high affine kalium ion transport albumen of cotton, its sequence is SEQ ID N0: 1.

2. encoding the gene of the high affine kalium ion transport albumen of claim 1, its sequence is SEQ ID NO: 2.3. a kind of recombinant expression carrier, it is obtained by the way that the gene described in 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, it is preferable that the expression vector is pCAMBIA2300.

4. the carrier described in claim 3, it is the 35S-GhHKT2-2300 carriers shown in accompanying drawing 2.

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

6. a kind of method of improvement plant salt endurance, 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：The plant containing the recombinant expression carrier described in the gene or claim 3 or 4 described in claim 2 or plant tissue are cultivated under conditions of plant is effectively produced.

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

9. the recombinant expression carrier described in gene, claim 3 or 4 described in claim 2 or the recombinant cell described in claim 5 are used to improve plant salt endurance and the purposes for plant breeding.

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