CN104726476A - Soybean salt-tolerant gene GmCIPK2 and application thereof - Google Patents

Abstract

The invention discloses a soybean salt-tolerant gene GmCIPK2 and application thereof. A nucleotide sequence of cDNA of the soybean salt-tolerant gene GmCIPK2 is shown in SEQ ID No.1. According the application, a yeast two-hybrid system of Clontech is firstly utilized for constructing a yeast cDNA library of soybean 371005, soybean salt-tolerant gene GmCBL3 is cloned, a bait protein is constructed, and an interacting protein GmCIPK2 of a protein GmCBL3 is obtained by virtue of a yeast two-hybrid method; then a gateway is utilized for carrying out BP reaction on the gene GmCIPK2 to link the gene GmCIPK2 to pDONR221, massive cloning is carried out in escherichia coli DH5alpha by virtue of a conversion method, and then LR reaction is carried out to link the gene GmCIPK2 to an expression vector pK2GW7; the expression vector pK2GW7 is transferred into Arabidopsis by virtue of agrobacterium mediation, so that the salt tolerance of transgenic plants is obviously improved.

Description

Soybean salt-tolerance gene GmCIPK2 and application thereof

Technical field

The invention belongs to technical field of biological genetic engineering, particularly relate to soybean salt-tolerance genes involved GmCIPK2 and application thereof.

Background technology

China is saline soil distribution one of country comparatively widely, and the salinification in soil has become one of Main Barrier Factors of restriction China's agricultural development, particularly Soybean Industry.In the modification method of current soil, chemical modifying and biological modification are the emphasis of research at present.For chemical modifying, although instant effect, easily introduce new ion and cause secondary pollution, and fund input and technical requirements are all very high, implement more difficult to large-area land remediation.And biological modification can effectively reduce costs, be the most economical effective measures that salinization land recovers.

The ultimate aim of biological modification is also the restoration and reconstitution realizing vegetation, but improves the salt tolerance of drought of plant by conventional breeding methods art, time and effort consuming, and difficulty is large, has been difficult to cultivate real resistant variety.The separation of concerned plant saline-resisting and alkaline-resisting gene, extraction, clone possess some special knowledge, for the salt tolerance realizing improving soybean provides possibility.Understand the salt tolerance further its molecular mechanism of announcement of grasping soybean, to incubation soybean salt-tolerance new variety, expand the planting range of soybean, reduce the impact of the factors on soybean cultivations such as salt-affected soil and output, stablize the Soybean production of China, ensure Food Security in China and social stability important in inhibiting.Meanwhile, be also establish the own soybean brand of China, strengthen important prerequisite and the basis of China soybean competitive power in the international market.

The salt-tolerance character of plant is the quantitative character of a controlled by multiple genes, and the gene that a lot of anti-salt tolerant is correlated with is undiscovered, and this just determines the complicacy of plant salt tolerance mechanism.Salt stress energy inducing plant cell Ca ²⁺the change of concentration, and Ca ²⁺signal is accepted by receptor protein and conducts, and this proteinoid is calcineurin B-like proteins (Calcineurin B-like protein, CBL).CBL is the main regulatory gene of calcium channel intracellular signaling in plant stress-resistance gene regulatory network, in Arabidopis thaliana, 10 CBL genes are cloned at present, in other species, find (GeneBank) as also there being albuminoid in corn, potato and paddy rice.CBL1 is a member in CBL protein family, and the CBL1 in Arabidopis thaliana plays an important role in plant salt stress signal transmission path.CBL1 and CBL9 acting in conjunction can activate CIPK23 (CBL-interacting protein kinases, CIPK), and the latter is by phosphorylation K ⁺translocator AKT1 increases the K in born of the same parents ⁺concentration, reduces Na ⁺absorption, thus improve the salt tolerance of plant.But the CBL gene in soybean and report is rarely had to the research of plant salt endurance.

Soybean gene GmCIPK2 in the present invention belongs to soybean protein kinases, can by with soybean salt-tolerance gene GmCBL3 (see application for a patent for invention 201510116210.1, denomination of invention: soybean salt-tolerance gene GmCBL3 and application thereof, applicant: Shandong Crop Germplasm Resource Center, the applying date: 2015.3.17) interactions between protein regulate the expression of soybean GmCBL3, thus affect Ca in plant cell membrane ²⁺the conduction of signal, and then change plant to the adaptability of salt stress, be the important regulatory gene that plant salt coerces in lower calcium channel signal transduction path.Its process LAN in Arabidopis thaliana can significantly improve the salt tolerance of plant.The soybean gene GmCIPK2 that the present invention reports is aniso-in other members of its subfamily, has certain specificity.

Summary of the invention

The object of this invention is to provide a kind of resistant gene of salt-GmCIPK2 that separation obtains from salt tolerant, low-temperature sensitive type soybean germplasm 371005 (Shandong Coastal Area germ plasm resource collects numbering), this gene is forwarded in model plant Arabidopis thaliana, obtains the transfer-gen plant that salt tolerance improves.

Soybean salt-tolerance gene GmCIPK2 of the present invention, is characterized in that, the nucleotide sequence of its gene cDNA is as shown in SEQID No.1.

Present invention also offers a kind of plant expression vector pK2GW7-GmCIPK2 containing above-mentioned soybean salt-tolerance gene GmCIPK2.

The invention also discloses above-mentioned soybean salt-tolerance gene GmCIPK2 and improve the purposes in plant salt endurance.

Wherein: described plant is Arabidopis thaliana; Further, described Arabidopis thaliana is Col-0 wild-type.

First the present invention utilizes the yeast two-hybrid system of Clontech, constructs the yeast cDNA library of soybean 371005; Then in soybean 371005 plant, be cloned into soybean salt-tolerance gene GmCBL3 (as shown in SEQ ID No.2), construct bait protein, obtained the interact protein GmCIPK2 of GmCBL3 albumen by the method for yeast two-hybrid; Then GmCIPK2 gene is carried out BP reaction forming to pDONR221 by sharp gateway system of crossing, cloned in a large number in bacillus coli DH 5 alpha by the method transformed, then carry out LR reaction this gene GmCIPK2 is connected on expression vector pK2GW7, and express in bacillus coli DH 5 alpha; Then screening transgenic intestinal bacteria, and extract transform plastids, finally transform plastids is proceeded in agrobacterium strains GV3101; Transformation Agrobacterium bacterial strain is proceeded in Arabidopis thaliana and soybean, thus the function of the GmCIPK2 of checking expression.

Beneficial effect of the present invention: utilize existing plant gene engineering technology, the present invention clones first and obtains soybean salt-tolerance gene GmCIPK2 and express in Arabidopis thaliana, and render transgenic Arabidopis thaliana obtains the ability of the raising salt tolerance not available for non-transgenic Arabidopis thaliana.Gene of the present invention can be widely used in the plant variety of cultivating salt tolerant.

Accompanying drawing explanation

Fig. 1 is that LD-PCR detects amplified production.1, without Template-negative controls; 2,1 μ g mouse liver positive control; 3, LD-PCR amplified production; M, Marker; Result shows this experiment and obtains the cDNA with positive control a great deal of, and LD-PCR increases successfully;

Fig. 2 is carrier pGBKT7 schematic diagram;

Fig. 3 is carrier pDONR221 schematic diagram;

Fig. 4 is plant expression vector pK2GW7 schematic diagram;

Fig. 5 is wild-type is the normal salt tolerance functional verification figure cultivating 10 days on the 1/2MS substratum of 0.5% in NaCl concentration with the Arabidopis thaliana transgenic positive plant turning GmCIPK2 gene.

Embodiment

Embodiment 1: the yeast cDNA library of soybean 371005 builds

The collection of salt tolerant soybean germplasm: the present inventor to collect in Qi Jia village of Ming Ji township, Dongying City Lijin County in October, 2009 and filters out soybean germplasm (the soil salt concentration (‰): 1.5 of a kind of salt tolerant, low-temperature sensitive type, latitude/°: 37.59, longitude/°: 118.22, height above sea level/m:10).According to Shandong Province's numbering with 37 beginnings, the 1005th part of resource, therefore called after 371005 (Shandong Coastal Area germ plasm resource collects numbering); Its relative salt gypsum rock: 18.00.

The extraction of 1.1 soybean 371005 plant total serum IgE:

(1) mortar of precooling put into by soybean 371005 material, adds liquid nitrogen and grinds to form rapidly uniform powder;

(2) proceeded to rapidly in the centrifuge tube of precooling by material after liquid nitrogen volatilization, every 50-100mg organization material adds 1mlTRIZOL solution, after mixing, places 5min, 12000r/min, 2-8 DEG C of centrifugal 10min go precipitation in room temperature;

(3) add 0.2ml chloroform in every 1ml TRIZOL solution, vibration 15sec fully mixes, and places 5min, 12000r/min, 2-8 DEG C of centrifugal 15min in room temperature;

(4) get supernatant, every 1ml TRIZOL adds the Virahol of 0.5ml, mixing, and room temperature places 10 ~ 20min;

(5) the 2-8 DEG C of centrifugal 10min of 12000r/min, abandons supernatant;

(6) add 1ml 75% washing with alcohol and precipitate 2 times, 4 DEG C are no more than the centrifugal 5min of 7500r/min, abandon supernatant;

(7), after room temperature is placed and dried, the distilled water adding 15 μ l DEPC process dissolves RNA.

1.2 utilize Clontech yeast two-hybrid library kits reverse transcription to synthesize the first chain cDNA

(1) in centrifuge tube, following reagent is mixed:

1 – 2 μ l RNA sample

1.0 μ l primers (5 '-ATTCTAGAGGCCGAGGCGGCCGACATG-d (T) 30VN-3 ' SEQ IDNo.3)

1 – 2 μ l deionized water

4.0 μ l cumulative volumes;

(2) 72 DEG C, hatch 2min;

(3) cooled on ice 2min, then 14000rpm, centrifugal 10s;

(4) mix following reagent, mixed solution is added in the sample that above-mentioned steps (3) obtains, pat mixing;

2.0 μ l 5 × First-Strand damping fluids

1.0μl DTT

1.0 μ l dNTP mixtures

1.0 μ l SMART MMLV ThermoScript II

9.0 μ l cumulative volumes;

(5) 42 DEG C, 10min, hatches;

(6) add 1 μ l SMART III-modified oligo (5 '-AAGCAGTGGTATCAACGCAGAGTGGCCATTATGGCCGGG-3 ' SEQ ID No.4), mixing, 42 DEG C, 1hr is hatched;

(7) 75 DEG C, 10min, stops the first chain synthesis reaction;

(8) room temperature cooling, adds 1 μ l RNA enzyme H;

(9) 37 DEG C, 20min, hatches;

(10) LD-PCR amplification is proceeded.

1.3 use long range PCR (LD-PCR) to increase ds cDNA

(1) prepare: the first chain cDNA, the PCR instrument of preheating;

(2) set up the amplification system of two pipe 100ul, one is experimental group, and another is control group:

2 μ l first chain cDNA

70 μ l distilled water

10 μ l 10 × 2PCR damping fluid

2 μ l 50 × dNTP mixtures

2 μ l 5 ' PCR primer (5 '-TTCCACCCAAGCAGTGGTATCAACGCAGAGTGG-3 ' SEQID No.5)

2 μ l 3 ' PCR primer (5 '-GTATCGATGCCCACCCTCTAGAGGCCGAGGCGGCCGACA-3 ' SEQ ID No.6)

10 μ l 10 × lysates (test kit carries composition)

2 μ l 50 × Advantage 2 polymerase mixture

100 μ l cumulative volumes;

(3) amplification program: 95 DEG C of 30sec, 36Cycles; 95 DEG C of 10sec; 68 DEG C of 6min ^b, 68 DEG C of 5min; Amplification program is often crossed a circulation time and is extended time increase 5sec;

(4) agarose electrophoretic analysis (as Fig. 1);

(5) after column purification, by stand-by for ds cDNA Bao Cun Yu – 20 DEG C.

1.4 operation purifying ds cDNA

(1) prepare: LD-PCR amplification ds cDNA

Sodium-acetate (3M)

Ice ethanol (95 – 100%);

(2) the cDNA sample of every 93ul uses a CHROMA SPIN TE-400 purification column

Put upside down purification column for several times, resuspended gel matrix, until evenly,

Remove top cap,

Break the break away (spacer plate) of disconnected column bottom,

Post is placed on the collection tube of 2ml;

(3) 700rpm, 5min are centrifugal, abandon collection tube and balance liquid, relief matrix (matrix) half-dried;

(4) centrifugal column is placed in another second collection tube, then, adds the sample of 93ul to the surface that gel matrix is smooth;

(5) 700rpm, 5min are centrifugal;

(6) by the purifying sample collection of twice in a centrifuge tube, and carry out precipitation ds cDNA with ethanol:

Add the 3M sodium-acetate of 1/10 volume;

Add the ice ethanol (95 – 100%) of 2.5 times of volumes;

– 20 DEG C, 1hr places precipitation;

14,000rpm, the centrifugal 20min of room temperature;

Abandon supernatant;

14,000rpm brief centrifugation, removes residual supernatant liquor;

Dry air 10min;

The resuspended ds cDNA of (7) 20 μ l deionized water.

1.5 prepare competent yeast cells

(1) reagent

(with deionized-distilled water preparation, membrane filtration is degerming for 1M LiCl; If desired with the dilution of sterilization deionized water) 50%PEG3350 (Sigma P3640 deionized-distilled water is prepared, and membrane filtration is degerming, the bottle packing with tighter lid)

2mg/ml salmon sperm DNA/TE (10mM Tris-Cl, pH8.0,1.0mM EDTA)-20 DEG C preservation;

(2) preparation of competence pichia yeast

Inoculation yeast bacterial classification in 50ml YPD substratum, 30 DEG C shake bacterium spend the night (about 24 ~ 28h) cultivate to OD value be 0.8 ~ 1.0 (about 10 ⁸cells/ml);

Harvested cell, washs once with 25ml sterilized water, the centrifugal 10min of 1500g under room temperature;

Suspension, in 1ml 100mM LiCl solution, is proceeded to 1.5ml centrifuge tube by re-suspended cell;

Whizzer maximum velocity centrifugation 15 seconds precipitation thalline, resuspended thalline is in 400ul 100mM LiCl solution;

By the packing of 50ul/ pipe, transform immediately;

Note: not by competence yeast ice bath.

The conversion of 1.6 yeast

(1) boil 1ml salmon sperm dna 5min, rapid ice bath is to prepare strand carrier DNA;

(2) by centrifugal for competence yeast, remaining LiCl solution is removed with Tips;

(3) each is transformed, adds in the following order:

(4) violent vortex mixing is until precipitation thalline is evenly distributed (about 1min) completely;

30min is hatched in (5) 30 DEG C of water-baths;

(6) 42 DEG C of water-bath heat-shocked 20 ~ 25min;

(7) 6000 ~ 8000rpm collected by centrifugation yeast thalline;

(8) resuspended yeast is in 1ml YPD substratum, and 30 DEG C of shaking tables are hatched;

After (9) 1 ~ 4h, get 25 ~ 100ul bacterium liquid paving selective medium dull and stereotyped, cultivate qualification in 2 ~ 3 days in 30 DEG C;

1.7 set up Mate & Plate library

(1) Y187 competent yeast cells is prepared;

(2) according to conversion operation 1.6 transformed yeast cell

20μl ds cDNA—2–5μg

6μl pGADT7-Rec(0.5μg/μl))

(3) yeast cell after conversion is resuspended in 15ml 0.9% (w/v) NaCl;

(4) 1/10,1/100 diluent of 100 μ l is plated on SD/ – Leu 100mm solid medium respectively, 30 DEG C, hatches 3d – 4d; Determine the storage capacity in library,

Independent cloning number=No.of cfu/ml on SD/ – Leu x re-suspension liquid volume (15ml)

This value indicates transformation efficiency;

(5) remaining suspension is plated on 150mm SD/ – Leu Selective agar medium

Every plate 150 μ l suspension, about uses 100 plates

Hatch 3d-4d for 30 DEG C;

(6) transformant is gathered in the crops:

(7) 4 DEG C, 3 – 4hr cool culture plate, add the frozen storing liquid of 5ml, use aseptic granulated glass sphere to be separated by the bacterium colony in plate.

(8) by all liquid collectings in aseptic single tube, calculate cell density with blood counting chamber.If cell density is less than <2x10 ⁷/ ml, the centrifugal volume reducing suspension.

1.8 yeast cDNA libraries carry out double cross

(1) with reference to the explanation of the Matchmaker Gold Yeast Two-Hybird System of Clontch, with the DNA in step 1.4 for template, utilize following primer SEQ ID No.7-8 to be reacted by the PCR of high-fidelity DNA polymerase and add top connection to GmCBL3, amplify the goal gene full length fragment of belt lacing;

P1 (upstream primer): 5'-CATGGAGGCCGAATTCATGGGAACTCAAGGATATGCAGCA-3'(SEQ ID No.7)

P2 (downstream primer): 5'-GCAGGTCGACGGATCCTTATGGGAATGCAGTAGGAGTATGA-3'(SEQ ID No.8);

(2) get 200 μ l PCR primer, utilize the gel of Tian Gen company to reclaim test kit, reclaim PCR primer;

(3) by the PCR primer of recovery and pGBKT7 carrier, double digestion is carried out with restriction enzyme BamHI and EcoRI of Takara company respectively;

(4) PCR primer of double digestion and pGBKT7 carrier are joined in new centrifuge tube according to the volume ratio of 5:1, then connect with the DNA ligation kit of Takara company, build pGBKT7-GmCBL3 bait protein recombinant vectors;

(5) get the reaction solution 1 μ l in (4), carry out intestinal bacteria conversion, and checking is positive;

(6) cultivate positive colony, extract and its plasmid of purifying;

(7) plasmid in (6) is added in the competent yeast described in step 1.5, carry out yeast conversion by the method for step 1.6, and obtain positive yeast bacterial strain;

(8) Dual culture is carried out by obtaining the Yeast libraries obtained in positive yeast bacterial strain and step 1.7, explanation with reference to the Matchmaker Gold Yeast Two-Hybird System of Clontch carries out double cross to above-mentioned yeast cDNA library, and the positive single bacterium colony of the interact protein obtaining GmCBL3.

1.9DNA order-checking

Picking contains the positive list bacterium colony of recombinant plasmid with containing Amp (Ampicillin Trihydrate, LB liquid medium 50mg/L) shakes bacterium and spends the night, then send Shenzhen Hua Da gene company limited to check order, obtain sequencing result: gene cDNA sequence as shown in sequence table SEQ ID No.1, total length 627bp.

Analyze through NCBIblast, the nucleotide homology 74.12% of above-mentioned cDNA sequence and soybean protein kinases Glycine max protein kinase (LOC100305401).What prove that experiment obtains is exactly the protein kinase of soybean, called after soybean GmCIPK2.

The thaliana flower of embodiment 2, agriculture bacillus mediated GmCIPK2 transforms

2.1 over-express vectors build

(1) the Gateway system of Invitrogen company is utilized, GmCIPK2 gene is carried out BP reaction forming to pDONR221 carrier (see Fig. 3), following primer SEQ ID No.9-10 is reacted by the PCR of high-fidelity DNA polymerase and adds top connection to GmCIPK2;

P3 (upstream primer): 5'-AAAAAAGCAGGCTTCATGGAGTACCCATACGACGT-3'(SEQ IDNo.9)

P4 (downstream primer): 5'-AGAAAGCTGGGTCGGTACGTCGTATGGGTACTCCAT-3'(SEQ IDNo.10);

(2) 25 μ l PCR primer are got, with TE Buffer (10mM Tris-HCl pH 7.5,1mM EDTA), the 30%PEG 8000/30mM MgCl of 1/2 volume ₂solution dilution 4 times, final concentration is 10%PEG and 10mM MgCl ₂, vortex mixes, the centrifugal 15min of 13,000rpm;

(3) gently move supernatant, by the resuspended precipitation of TE, make DNA final concentration be >10ng/ μ l;

(4) add following composition in 1.5ml centrifuge tube, and at room temperature mix to carry out BP reaction:

AttB-PCR product (>=10ng/ μ l, total content 15-150ng) 1-7 μ l

pDONR221vector(150ng/μl) 1μl

TE buffer,pH 8.0 to 8μl；

(5) take out BP Clonase II enzyme, after placing 2min on ice, vortex oscillation 2s, reapposes 2min on ice;

(6) (5) are repeated once, BP Clonase II enzyme of 2 μ l is added in the system of (4), mix gently, the dead slow speed centrifugal several seconds, reaction solution is made to be gathered at the bottom of centrifuge tube, 25 DEG C are reacted 1h or spend the night, and BP Clonase II enzyme places back in-20 DEG C or-80 DEG C of Refrigerator stores;

(7) 1 μ l Proteinase K is joined in reaction system, mix gently, hatch 10min with termination reaction for 37 DEG C;

(8) get 1 μ l reaction solution, carry out bacillus coli DH 5 alpha conversion, and checking is positive;

(9) escherichia coli cloning that is positive of incubated overnight, and extract its plasmid;

(10) purified positive colony plasmid (Entry clone) adds in 1.5ml centrifuge tube by following system, and at room temperature mixes to carry out LR reaction:

Entry clone(50-150ng) 1-7μl

Destination vector (150ng/ μ l, pK2GW7, Fig. 4) 1 μ l

TE buffer,pH 8.0 to 8μl；

(11) take out LR Clonase II enzyme, after placing 2min on ice, vortex oscillation 2s, reapposes 2min on ice.

(12) (11) are repeated once, LR Clonase II enzyme of 2 μ l is added in the system of (10), mix gently, the dead slow speed centrifugal several seconds, reaction solution is made to be gathered at the bottom of centrifuge tube, 25 DEG C are reacted 1h or spend the night, and LR Clonase II enzyme places back in-20 DEG C or-80 DEG C of Refrigerator stores;

(13) in reaction system, add 1 μ l Proteinase K termination reaction, slightly mix, hatch 10min for 37 DEG C;

(14) get 1 μ l reaction solution, carry out intestinal bacteria conversion, and checking is positive;

(15) cultivate positive colony, extract and its plasmid of purifying, finally transform plastids is proceeded in agrobacterium strains GV3101.2.2 thaliana flowers transform

(1) the flowering Arabidopsis plant of normal growth, at soil-grown under long-day conditions.If Arabidopis thaliana is bloomed, carrier is not also ready to, and can remove poppyhead, obtains more flower and transform after 4-6 days;

(2) contain in suitable antibiotic LB substratum by the correct Agrobacterium strain transfer of qualification to 5-10ml, 28 DEG C, 250rpm, cultivates 2 days;

(3) bacterium is transferred to 300ml containing in corresponding antibiotic LB substratum, 28 DEG C are cultivated OD600 is 1.5-2.0;

(4) 4000g, 15min room temperature collection bacterium, is resuspended to OD600=0.6 ~ 0.8 with 0.5% sucrose solution (matching while using) by cell;

(5) add Sliwet L-77 (tensio-active agent, Sigma), concentration 0.02 ~ 0.05% (vol/vol), mixing, can reduce Sliwet L-77 as far as possible;

(6) when transforming, mixed bacterium liquid is placed in suitable vessel, flower is immersed bacterium liquid 2-3 second.Also can with suction pipe by bacterium drop to taking;

(7) 16-24 hour is placed in the black plastic bag lucifuge moisturizing of the plant after conversion;

(8), after the lucifuge time terminates, plant is grown under normal operation, until seed maturity;

(9) the F1 generation seed received of above-mentioned transformed plant first dry 1 week, then broadcasts and is containing on suitable antibiotic flat board;

(10) seed disinfection: Arabidopis thaliana seed soaks 15min in 0.5% chlorine bleach liquor, then with aseptic washing 5 times;

(11) 4 DEG C of purifying 3 days, seedling, grow on plates 10 days, selects blade and the normal young plant of root growth is transplanted in soil;

(12) plant is grown under normal operation, until seed maturity, obtain F2 for seed, carry out functional verification test.

Embodiment 3, turn GmCIPK2 gene arabidopsis thaliana salt-tolerance checking

(1) extract the DNA sample of plant, and utilize following primer SEQ ID No.11-12 to be reacted by the PCR of Taq archaeal dna polymerase, verify and obtain and turn GmCIPK2 gene masculine plant;

P5 (upstream primer): 5'-ATGGAGTACCCATACGACGTACCAG-3'(SEQ ID No.11)

P6 (downstream primer): 5'-CTGGTACGTCGTATGGGTACTCCAT-3'(SEQ ID No.12);

(2) the disinfection respectively of two kinds of dissimilar Arabidopis thaliana seeds, these two kinds of seeds are: the Arabidopis thaliana transfer-gen plant of Arabidopis thaliana wild-type (negative control) and overexpression soybean GmCIPK2 gene;

(3) in NaCl concentration be 0.5% 1/2MS substratum on, cultivate through two kinds of Arabidopis thaliana seeds of disinfecting;

(4) normally cultivate after 10 days, find that the Arabidopis thaliana transfer-gen plant of overexpression GmCIPK2 gene can normal growth preferably, and Arabidopis thaliana WT lines can not normal growth (as shown in Figure 5).

Claims (5)

1. soybean salt-tolerance gene GmCIPK2, is characterized in that, the nucleotide sequence of its gene cDNA is as shown in SEQ ID No.1.

2. the plant expression vector pK2GW7-GmCIPK2 containing soybean salt-tolerance gene GmCIPK2 according to claim 1.

3. soybean salt-tolerance gene GmCIPK2 according to claim 1 is improving the purposes in plant salt endurance.

4. purposes as claimed in claim 3, it is characterized in that, described plant is Arabidopis thaliana.

5. improve a method for plant salt endurance, it is characterized in that, first utilize the yeast two-hybrid system of Clontech, construct the yeast cDNA library of soybean 371005; Then in soybean 371005 plant, be cloned into soybean salt-tolerance gene GmCBL3, constructed bait protein, obtained the interact protein GmCIPK2 of GmCBL3 albumen by the method for yeast two-hybrid; Then GmCIPK2 gene is carried out BP reaction forming to pDONR221 by sharp gateway system of crossing, cloned in a large number in bacillus coli DH 5 alpha by the method transformed, then carry out LR reaction gene GmCIPK2 is connected on expression vector pK2GW7, and express in bacillus coli DH 5 alpha; Then screening transgenic intestinal bacteria, and extract transform plastids, finally transform plastids is proceeded in Agrobacterium; Proceed to again in Arabidopis thaliana or soybean, obtain the plant turning GmCIPK2 gene that salt tolerance improves; The nucleotide sequence of the gene cDNA of described gene GmCIPK2, gene GmCBL3 is respectively as shown in SEQ IDNo.1-2.