CN103667315A - Salt-tolerant and drought-resistant gene TaDHN1 of wheat, recombinant plasmid and application - Google Patents

Abstract

The invention discloses a salt-tolerant and drought-resistant gene of wheat, namely the dehydrin gene TaDHN1 of wheat, as well as a plant expression vector containing the gene TaDHN1. The invention further discloses the application of the gene TaDHN1 to cultivation of salt-tolerant and drought-resistant plants. Experimental results show that the salt tolerance and drought resistance of the TaDHN1 gene transferred plants are remarkably improved.

Description

Wheat salt tolerance, anti-drought gene TaDHN1, recombinant plasmid and application

Technical field

The invention belongs to plant gene engineering technology field, relate in particular to wheat salt tolerance, anti-drought gene taDHN1,recombinant plasmid and application.

Background technology

In recent years, various abiotic stress have a strong impact on the output of crop, and wherein salt and drought stress are particularly outstanding on the impact of plant.By genetically engineered, cultivating resistance to contrary new variety is the drought-resistant abilities that effectively utilize saltings, improve plant, increases grain yield, the important means of Ensuring Food Safety.The research that utilizes at present genetic engineering technique to carry out plant salt tolerance, drought resisting aspect has obtained larger progress.Some experiments show, by gene transferred plant relevant to salt tolerant in plant itself and other biological, its allos is transcribed salt resistance ability that can render transgenic plant with translation product and improved.

Wheat is the staple food of the whole world 35% above population, one of Ye Shi Shandong Province staple food crop.But Triticum glycophyte, the extreme difference of growing in salinization soil, therefore clones resistant gene of salt, and cultivation Salt-tolerant Wheat new variety are extremely important.

Dehydrins (being called again Equations of The Second Kind embryonic development later stage Abundant protein) is the functional gene in the salt stress signal transduction pathway downstream of ABA dependence, and it is found in the embryonic development later stage the earliest, great expression in the dehydration in seed maturity later stage.Research afterwards shows, except involved in plant grows normally, dehydrin gene has mainly participated in the resistance of plant to high salt, arid, the various abiotic stress such as cold.

Summary of the invention

The object of this invention is to provide a kind of salt tolerant, anti-drought gene---wheat dehydrin gene taDHN1,recombinant plasmid and application thereof.We find in the wheat root of a probe that represents dehydrin gene after salt is processed significantly up-regulated expression in chip hybridization experiment in early stage, this research is on the basis of early-stage Study, cloned this dehydrin gene, functional study shows, crosses and expresses salt tolerant and the drought-resistant ability that this gene can improve plant.

Technical program of the present invention lies in: first according to the chip of expression spectrum data of wheat, select the dehydrin gene (probe) that is significantly subject to salt abduction delivering in Wheat Seedling Roots, then according to probe sequence design gene-specific primer, from the cDNA library of Wheat Seedling Roots, clone dehydrin gene taDHN1full-length cDNA.Order-checking obtains after sequence, and structure comprises taDHN1the stable over-express vector of gene complete ORF finally carries out functional verification in Arabidopis thaliana.

Wheat salt tolerance provided by the invention, anti-drought gene name are called taDHN1, the nucleotide sequence of described gene cDNA is as shown in SEQ ID No.1, and its aminoacid sequence is shown in SEQ ID No.2.

The present invention also provides containing above-mentioned wheat cdna taDHN1recombinant plasmid pCAMBIA-super1300/TaDHN1: by primer, to SEQ ID No.1 sequence both sides, introduce xbai and saci double enzyme site, then by xbai and sacgene segment shown in I double digestion plant expression vector pCAMBIA-super1300 and SEQ ID No.1, reclaims carrier large fragment and is connected acquisition with target gene fragment again,

Described primer sequence is: D1ORF5:5 '-TCTAGAATGGAGTACCAGGGGCAGCAC-3 ' ( xbai)

D1ORF3: 5’-GAGCTCTCAGTGCTGTCCGCCGGG-3’( SacI)。

Recombinant plasmid of the present invention is characterised in that in the expression cassette of foreign gene and contains taDHN1the complete encoder block of gene, i.e. total length ORF(open reading frame).

The invention provides a kind of wheat salt tolerance, anti-drought gene taDHN1application in cultivating salt tolerant and drought-resistant plant,

Salt tolerant of the present invention and drought-resistant plant are wheat, corn and paddy rice monocotyledons.

The application of recombinant plasmid pCAMBIA-super1300/TaDHN1 in cultivating salt tolerant, drought-resistant plant.

The invention provides to improve and contain gene taDHN1the method of plant salt tolerance and drought resistance is by gene taDHN1import host plant cell, tissue or plant individual, obtain having salt tolerant, the plant of drought resistance, described host plant is wheat, corn and paddy rice monocotyledons.

Beneficial effect of the present invention: the present invention clones first and obtained wheat dehydrin gene taDHN1, and the method mediating by agrobacterium tumefaciens proceeds to Arabidopis thaliana by this gene, through comparative analysis, proves, salt tolerant, the drought-resistant ability of transfer-gen plant obviously improve.

Accompanying drawing explanation

Fig. 1 is taDHN1the amplification of full length gene cDNA sequence,

Wherein M be λ DNA/ ( ecor I+ hind III) Marker; It is lower same,

Fig. 2 is after ABA and NaCl process taDHN1the RT-PCR of gene in Wheat Seedling Roots and leaf analyzes:

Fig. 2-a is taDHN1expression in the wheat seedling leaf of processing at ABA;

Fig. 2-b is taDHN1expression in the Wheat Seedling Roots of processing at ABA;

Fig. 2-c is taDHN1expression in the wheat seedling leaf of processing at NaCl;

Fig. 2-d is taDHN1expression in the Wheat Seedling Roots of processing at NaCl,

The plant expression vector pCAMBIA-super1300/TaDHN1's that Fig. 3 is structure xbari, saci double digestion the result,

Fig. 4 is the Agrobacterium bacterium liquid PCR detected result that turns expression vector pCAMBIA-super1300/TaDHN1,

Fig. 5 is the phenotypic evaluation of transgenic arabidopsis strain:

Fig. 5-A: wild-type Col-0 and transgenic arabidopsis strain be the long statistics of primary root root under different concns NaCl processes,

Wherein: WT: unconverted Col-0 wild-type Arabidopis thaliana; 8,20 for turning the different transgenic arabidopsis strains of pCAMBIA-super1300/TaDHN1; CK is contrast, on normal MS substratum, cultivates; ﹡: P ﹤ 0.05 ， ﹡ ﹡: P ﹤ 0.01,

Fig. 5-B: wild-type Col-0 and transgenic arabidopsis strain be the long statistics of primary root root under different mannitol concentrations are processed,

Fig. 5-C: the normal growth Arabidopis thaliana of 3 weeks and normal growth 3 weeks then arid are processed the Arabidopis thaliana growing state of 15 days.

Embodiment

embodiment 1

taDHN1the clone of gene cDNA sequence

1.1 taDHN1clone and the sequencing of full length gene cDNA sequence

1. primer sequence

According to the downstream primer of chip probe sequences Design gene specific, match with 5 of library ' end anchor primer, the full-length cDNA that the cDNA library of wheat young root of take is template amplification gene.

Primer sequence is: TaDHN1:5 '-GAGACGAACAGAGGTAGTACATG-3 ',

NT3：5’-ACTAAAGggaACAAAAGCTGG AG-3’。

2.PCR reaction system (50 μ L)

2×GC bufferⅠ 10μl

Template cDNA library 1ul

dNTPs（2.5mM each） 0.5μl

Primer1 (10μM) 1μl

Primer2(10μM) 1μl

LA Taq(TaKaRa) 0.5ul

DdH ₂o adds to final volume 50 μ l

3.PCR response procedures is

94 ℃ of denaturation 3min; 94 ℃ of sex change 45sec, 58 ℃ of renaturation 1min, 72 ℃ are extended 1min, circulate 35 times; 72 ℃ are extended 5min.

4.1% agarose gel electrophoresis

Pcr amplification product finds to have at 700bp place the band (Fig. 1) of an entry after detecting with 1% agarose gel electrophoresis.

5. the recovery of amplified fragments, with the linking of T carrier

To amplified band, adopt the agarose gel of Tiangen company to reclaim test kit, step by specification carries out.PCR product is connected with pGEM-T (Promega) carrier, and linked system is:

The PCR product 7 μ l that reclaim

10 * T4 ligase enzyme damping fluid, 1 μ l

PGEM-T carrier (50ng/ μ l) 1 μ l

T4DNA ligase enzyme (3U/ μ l) is 1 μ l (Takara)

DH2O to 10 μ l

In 16 ℃ of water-baths, connect and spend the night.

6. reclaim clone and the order-checking of fragment

(1) preparation of competent escherichia coli cell

1. from-80 ℃ of refrigerators, take out and be stored in glycerine e.colidH5a bacterial strain (purchased from Tian Wei epoch biotech firm), is placed on ice and slowly thaws;

2. on Bechtop, with transfering loop, rule (LB solid medium does not contain Amp);

3. by flat board in 37 ℃ of constant temperature culture carton upside down overnight incubation;

4. the single colony inoculation on picking flat board is in containing in the test tube of 5 ml LB liquid nutrient mediums, 37 ℃ of shaking culture 14-16 hour;

5. get 0.5 ml bacterium liquid and be inoculated in the triangular flask containing the 250ml of 50 ml LB liquid nutrient mediums, 2-3 hour (OD260=0.5) of 37 ℃ of vibrations (260rpm) cultivation;

6. the bacterium liquid of cultivation is put 1 hour on ice;

7. 4 ℃ centrifugal 4 minutes (4000rpm), removes supernatant;

8. by the solution A of 25 ml ice precoolings, thalline is suspended gently, then place 40-45 minute on ice;

9. repeating step (8);

10. by the solution B of 2.5ml ice precooling, thalline is suspended gently, then bacterium liquid is divided and install to (every pipe 100 μ l) in 1.5ml centrifuge tube, put into-80 ℃ of refrigerators standby.

(2) connect the conversion of product

1. from-80 ℃ of refrigerators, take out 1 pipe (100 μ l) competent cell, put and slowly thaw on ice 30 minutes;

2. on Bechtop, in pipe, add 5 μ l ligation reactions, shake up gently, put 30 minutes on ice;

3. 42 ℃ of water-bath heat shocks are 90 seconds, put rapidly 3-5 minute on ice;

4. on Bechtop, in centrifuge tube, add 1ml LB liquid nutrient medium (not containing Amp), mix rear 37 ℃ of shaking culture 1 hour (260rpm);

5. centrifugal 6 minutes of 5000rpm under room temperature, discards 900 μ l supernatant liquors, will remain 100 μ l supernatant liquor Eddy diffusion thalline, adds the X-gal of 40 μ l and the IPTG of 4 μ l, mixes, and then with spreader, it is evenly coated onto on the flat board of preparation, places 30 minutes;

6. be inverted flat board and spend the night in 37 ℃ of constant incubators, when there is obvious single bacterium colony, take out;

7. put into 4 ℃ of refrigerator a few hours, make blue hickie color clearly demarcated;

8. with the toothpick picking hickie of sterilizing in the test tube of 10ml LB liquid nutrient medium (containing 60 μ g/mlAmp) is housed, 37 ℃ are shaken bacterium and spend the night.

(3) PCR of recombinant plasmid identifies and order-checking

There is T7 promotor this upstream of testing the cloning site of pGEM-T carrier used, and there is SP6 promotor in downstream, so can do primer pair Insert Fragment with these two promoter sequences, increases, and recombinant plasmid is identified;

1. PCR program: 94 ℃ of denaturation 10min; 94 ℃ of sex change 1min, 58 ℃ of renaturation 1min, 72 ℃ are extended 1min, circulate 35 times; 72 ℃ are extended 5min;

2. pcr amplification product detects by 1% agarose electrophoresis, and the Jun Yesong company of getting positive colony checks order.Sequencing result shows that institute's calling sequence comprises a complete ORF, long 402 bp.Total length ORF sequence is shown in SEQ ID No.1.

embodiment 2

under ABA, NaCl treatment condition taDHN1the expression analysis of gene

2.1 material processing

The normal seed germination of No. 3 is melted on wheat lines mountain, after 1 week, removes endosperm, and Hangload nutrient solution continues to cultivate 1 week.In salt stress liquid medium within, apply 200 mM NaCl, ABA processes and to apply 100 μ M ABA, and the tender blade of children and root system are got for extracting total RNA in 0,0.5,3,12,24,48 hour after processing respectively.

2.2 Trizol methods are extracted wheat Total RNA.

1. organization material is put into the mortar of Liquid nitrogen precooler, abundant grind into powder in liquid nitrogen;

2. treat that liquid nitrogen volatilization is dry, transfer to immediately in the centrifuge tube of 2ml, every 100mg material approximately adds the TRIzol extracting solution of the Invitrogen company of 1ml, and thermal agitation mixes sample, makes the abundant cracking of sample, and room temperature is placed 5 minutes;

3. add 0.2ml chloroform, thermal agitation mixes 15 seconds, and room temperature is placed 10 minutes;

4.4 ℃, centrifugal 15 minutes of 12000rpm;

5. with the careful sucking-off of pipettor upper strata water, add in the centrifuge tube of new 1.5ml, add the Virahol (1:1 volume) of 500 μ l, fully mix ,-20 ℃, precipitation 30min;

6.4 ℃, the centrifugal 10min of 12000rpm, careful abandoning supernatant;

75% washing with alcohol of 1ml for 7.RNA precipitation.4 ℃, the centrifugal 10min collecting precipitation of 8000rpm;

8. repeat by RNA precipitation of 75% washing with alcohol;

9. remove supernatant, RNA is deposited on aseptic operating platform and dries about 10-15 minute, and it is transparent that RNA shows slightly, and adds the RNase-free water of proper volume (30-50 μ l) fully to dissolve (can be placed on-80 ℃ long-term preserves);

10. ultraviolet spectrophotometer and 1%Agrose detected through gel electrophoresis RNA concentration and quality.

Note: a) use the output of UV spectrophotometer measuring RNA, the absorbancy at 260nm place, 1OD=40ug/ml.According to the light absorption value at 260nm and 280nm place, detect the purity of RNA, the OD of pure rna ₂₆₀/ OD ₂₈₀ratio should approach 2.0 (ratio is preferably between 1.9～2.1).

B) with 1%Agrose gel electrophoresis, examine quality and the size of side RNA.Draw the RNase-free water that 1ul RNA adds 3 μ l, add 65 ℃ of sex change of 1 μ l sample-loading buffer 5 minutes.After electrophoresis, with EB dyeing, separately get the 2kb DNAMarker of 3 μ l in contrast.

2.3 first chain cDNA's is synthetic

Adopt PrimeScript ^tMrT-PCR Kit carries out.Reactions steps is as follows:

1. in Microtube pipe, prepare following mixed solution.

dNTP Mixture (10 mM) 1μl

Oligo dT Primer (2.5μM) 1μl

Total RNA 4μl

RNase free H ₂O 4μl

2. on PCR instrument, carry out sex change, annealing reaction.

65℃ 5 min

4℃ 1 min

3. the centrifugal several seconds makes the mixed solution of RNA/ primer etc. be gathered in Microtube pipe bottom.

4. in above-mentioned Microtube pipe, prepare following inverse transcription reaction liquid

Reaction solution 10 μ l after above-mentioned sex change, annealing

5×PrimerScript ^TM Buffer 4 μl

RNase Inhibitor (40U/μl) 0.5 μl

PrimScript ^TMRTase 0.5 μl

Rnase Free dH ₂O 5 μl

5. on PCR instrument, by following condition, carry out reverse transcription reaction

42℃ 15-30 min

95℃ 5 min

4 ℃ of insulations

2.4 PCR reaction and electrophoresis

1. take cDNA as template, carry out PCR reaction.Primer is as follows

TaAct-S： 5’- GTTCCAATCTATGAGGGATACACGC -3’

TaAct-A: 5’- GAACCTCCACTGAGAACAACATTACC -3’

D1ORF5: 5’-TCTAGAATGGAGTACCAGGGGCAGCAC-3’（XbaI）

D1ORF3: 5’-GAGCTCTCAGTGCTGTCCGCCGGG-3’(SacI)

2.PCR system

ddH ₂O 4.7μl

10× buffer 2μl

Primer1（2μM） 1μl

Primer2（2μM） 1μl

dNTP（10mM each） 0.2μl

rTaq polymerase（5U/μl） 0.1μl

Reverse transcription cDNA template 1 μ l

Total Volume 10μl

3.PCR program

94℃ 5min；25～30 cycles，94℃ 20s，57℃ 60s，72℃ 45s；72℃ 5min。

According to the amplification situation of internal reference Actin, determine the cycle number of PCR, adjust the add-on of cDNA template.

4.1% agarose gel electrophoresis.The results are shown in Figure a in 2, b, c, d.

embodiment 3

the structure of 35S promoter plant expression vector

Plant expression vector pCAMBIA-super1300 is the binary vector that contains 35S promotor and NPT II gene, in its multiple clone site, contains restriction enzyme xbai and saci site.According to gene taDHN1cDNA sequence, design packet is containing the gene-specific primer of complete ORF, and in the introducing of primer 5 ' end xbai, saci double enzyme site, primer sequence is:

D1ORF5: 5’-TCTAGAATGGAGTACCAGGGGCAGCAC-3’（ XbaI）

D1ORF3: 5’-GAGCTCTCAGTGCTGTCCGCCGGG-3’( SacI)

With this to primer amplification taDHN1cDNA sequence.Then use respectively restriction enzyme xbai and saci double digestion carrier pCAMBIA-super1300 and goal gene segment.The carrier of complete degestion after electrophoretic separation, reclaims through glue on 1% sepharose, then links with the cDNA fragment of double digestion, builds and obtains plant expression vector pCAMBIA-super1300/TaDHN1.

3.1 plasmid pCAMBIA-super1300 empty carriers and goal gene segment xbai and saci double digestion

It is as follows that enzyme is cut system:

XbaI lμl

SacI 1μl

PCAMBIA-super1300 carrier

(or goal gene fragment) 5 μ l

10×Buffer M 1μl

ddH ₂O To 20μl

In 37 ℃ of thermostat water bath enzymes, cut more than 3 hours.

3.2 enzymes are cut electrophoresis and the recovery of product

After double digestion completes, take 1 * TAE as electrophoretic buffer, enzyme is cut to product and carry out 0.8% agarose gel electrophoresis.Large fragment and the goal gene fragment of carrier cut pCAMBIA-super1300 with clean blade under ultraviolet transilluminator in, sepharose reclaims test kit and reclaims object band.

3.3 connect

The pCAMBIA-super1300 carrier segments of cutting through enzyme and goal gene fragment are carried out 16 ℃ of connections with the ratio of mol ratio 1:4 and are spent the night.

3.4 transform

Connect product heat shock method and transform bacillus coli DH 5 alpha competent cell, transformed bacteria containing on the LB solid plate of Kan 50 μ g/ml 37 ℃ cultivate about 16 hours.

The evaluation of 3.5 recons

Plasmid enzyme restriction is identified.Extract positive colony plasmid, plasmid is carried out xbai and saci double digestion, enzyme is cut system with 3.1.Enzyme is cut product after 0.8% agarose gel electrophoresis, goal gene band and the carrier strap of the suitable size of having cut detected, and proves vector construction correct (Fig. 3).

embodiment 4

the competent preparation of Agrobacterium and conversion

The competent preparation of 4.1 Agrobacterium GV3101

1. the single bacterium colony of picking agrobacterium tumefaciens from YEP dull and stereotyped (containing 50 μ g/ml Rifampins), is inoculated in containing 50 μ g/ml

In the YEP liquid nutrient medium of Rifampin, 200rpm/min, 28 ℃ of overnight incubation;

2. get 2ml incubated overnight liquid be inoculated in 50ml containing in identical antibiotic YEP liquid nutrient medium at the same terms

Under be cultured to OD ₆₀₀reach 0.5;

3. bacterium liquid ice bath 30min, 4 ℃, the centrifugal 10min of 5000rpm, collects thalline;

4. thalline is resuspended in the NaCl of 10ml 0.15mol/L of ice bath to centrifugal collection thalline;

5. be suspended in again in the CaCl2 solution of 1ml 20mmol/L ice precooling, with 200 μ l/ pipes, bacterium liquid be divided in to 1.5ml

In Eppendorf pipe, put quick-frozen 1min in liquid nitrogen ,-70 ℃ save backup.

4.2 freeze-thaw methods transform agrobacterium tumefaciens GV3101

1. at room temperature melt Agrobacterium competent cell, add 1 μ g expression vector plasmid DNA, mix rear ice bath

30min；

2. put liquid nitrogen flash freezer 1min, move to rapidly 37 ℃ of insulation 3min;

3. the YEP 800 μ l that add antibiotic-free, 3hr is cultivated in 28 ℃ of concussions;

The centrifugal 30s of 4.7000rpm collects thalline, is applied on the YEP flat board containing 50 μ g/ml Rifampins, 50 μ g/ml Kan, is inverted the dark 2-3 days of cultivation for 28 ℃.

4.3 thalline PCR evaluations, qualification result is as Fig. 4.

Thalline PCR the primer is with embodiment 3.Method and program are with 2.4.

embodiment 5

transgenosis functional verification---transformation of Arabidopsis thaliana, screening and phenotype analytical

The plantation of 5.1 Arabidopis thalianas

By 7.5% chlorine bleach liquor (comprising 7.5% clorox and 0.01% Triton-X 100) sterilization 15 minutes for wild-type Arabidopis thaliana seed, then use rinsed with sterile water 5-6 time, point is sowed on MS flat board, in 4 ° of C vernalization 2-3 days.Then be transplanted to (Nutrition Soil mixes by equal proportion with vermiculite) in nutrition pot, 23 ° of C cultivate, 16/8 h photoperiod, light intensity 30-40 μ molm-2 s-1; After plant to be planted is bloomed, cut off its major branch top, promote side shoot development.In 4-6 after beta pruning days, carry out Agrobacterium-mediated Transformation.

5.2 transformation of Arabidopsis thaliana

200 ml bacterium liquid are poured in tray.By the Arabidopis thaliana back-off of having pruned and all inflorescences are immersed in suspension bacteria liquids, stir to be stained with gently and spend 30 sec-1 min.Take out flowerpot and be sidelong in pallet, with freshness protection package, wrap up with moisturizing.Pallet is put to dark place and cultivate 24 h.Then take out nutrition pot upright placement, recover illumination, continue to cultivate plant to ripe.

The screening of 5.3 positive plants: T0 is for seed with after 7.5% chlorine bleach liquor (comprising 7.5% clorox and 0.01% Triton-X100) sterilization, and program request is selected on culture plate (30 mg/L Totomycin) at MS.Vernalization 2-3 days at 4 ℃.Move in culturing room and cultivate.About about 10 days, select hygromycin resistance plant (grow true leaf 1-2 couple, root is stretched in substratum) and be transplanted in nutrition pot.Cultivate until seed maturity.Same method screening T1 obtains T2 for plant for seed.And at T2, select resistance in for plant than for the single of 3:1 inserts independent strain, and obtain the T3 that isozygotys and for strain, carry out Molecular Detection and the phenotypic evaluation of transgenic arabidopsis.

The PCR of 5.4 transgenic arabidopsis identifies

1. the extraction of arabidopsis thaliana genomic dna

(1) get the fresh blade of about 100mg, put into 1.5ml centrifuge tube, liquid nitrogen flash freezer, grinds in mill, adds 600 μ l to be preheated to 2 * CTAB Extraction buffer of 65 ℃, mixes to be placed in 65 ℃ of water-baths and to place 90min;

(2) mixture adds isopyknic phenol/chloroform/primary isoamyl alcohol after being chilled to room temperature, mix, and 4 ℃, the centrifugal 10min of 12000rpm;

(3) get supernatant, add isopyknic chloroform/primary isoamyl alcohol, mix, 4 ℃, the centrifugal 10min of 12000rpm;

(4) get supernatant, add the 3mol/L NaAc(pH5.3 of 1/10 volume) and the Virahol of 0.7 times of volume, carefully mixing, room temperature is placed 15min, precipitation DNA;

(5) with a glass hook, DNA hook is gone out, and be transferred to one and be equipped with in the clean Eppendorf pipe of 800 μ l 70% ethanol, room temperature is placed washing precipitation in several minutes, the centrifugal 5min of 6000g;

(6) remove most supernatant, dry air number minute, is dissolved in appropriate TE damping fluid as far as possible.

2.PCR amplification

The arabidopsis thaliana genomic dna extracting above of take is template, with gene-specific primer (with embodiment 3), carries out pcr amplification.

PCR system is with 2.4.PCR response procedures is: 94 ℃ of denaturation 5min; 94 ℃ of sex change 45sec, 58 ℃ of renaturation 45sec, 72 ℃ are extended 1min, circulate 35 times; 72 ℃ are extended 7min.Pcr amplification product detects the object band that amplifies a 400bp left and right in transgenic arabidopsis plant after agarose gel electrophoresis, in turning the plant of empty carrier, has no amplified band.

The phenotypic evaluation of 5.5 transgenic arabidopsis

1. the plantation of Arabidopis thaliana

T3 sterilizes 15 minutes with 7.5% chlorine bleach liquor (comprising 7.5% clorox and 0.01% Triton-X 100) for the isozygoty seed of Arabidopis thaliana strain of single copy, then use rinsed with sterile water 5-6 time, point is sowed on MS flat board, in 4 ° of C vernalization 2-3 days, then be transplanted to (Nutrition Soil mixes by equal proportion with vermiculite) in nutrition pot, 23 ° of C cultivate, 16/8 h photoperiod, light intensity 30-40 μ molm-2 s-1.

2.NaCl and drought stress are processed

NaCl processes: by careful the transplanting in the MS culture dish that contains 50 mM, 100 mM NaCl of Arabidopsis thaliana Seedlings (contrast and transgenic line) of sprouting 2 days, vertically cultivate and within one week, observe phenotype.Result shows on normal MS substratum, Col-0 wild-type Arabidopis thaliana and turning taDHN1the Arabidopis thaliana phenotypic difference of gene is little, and on the MS substratum that contains 50 mM or 100 mM NaCl, turns taDHN1the root system of the Arabidopis thaliana plant of gene is obviously longer than wild-type Col-0, and T test shows that the long difference of the two root has reached significance level (Fig. 5-A).

Osmotic stress is processed: by careful the transplanting in the MS culture dish that contains 50 mM, 100 mM, 150 mM N.F,USP MANNITOL of Arabidopsis thaliana Seedlings (contrast and transgenic line) of sprouting 2 days, vertically cultivate and within one week, observe phenotype.Result shows on normal MS substratum, Col-0 wild-type Arabidopis thaliana and turning taDHN1the Arabidopis thaliana phenotypic difference of gene is little, and on the MS substratum that contains 50 mM 100 mM or 150 mM N.F,USP MANNITOL, turns taDHN1the root system of the Arabidopis thaliana plant of gene is obviously longer than wild-type, and T test shows that the long difference of the two root has reached significance level (Fig. 5-B).

Arid is processed: the Arabidopsis thaliana Seedlings (contrast and transgenic line) of sprouting a week is transplanted in compost, cultivated and after 20 days, start to control the processing of water (not watering) arid.Arid is processed after 15 days and is observed phenotype.Result shows that arid processes after 15 days, wild-type Arabidopis thaliana plant wither, the leaf of some strains become withered (Fig. 5-C); And turned taDHN1the Arabidopis thaliana plant growing way of foreign gene is vigorous, can normally bear pod (Fig. 5 C TaDHN1-8 and TaDHN1-20 strain).

sequence table

SQ ID No.1

<110> University Of Ji'nan

<120> wheat salt tolerance, anti-drought gene TaDHN1 and application thereof

<141>2013-10-22

<160> 1

<210> 1

<211>402

<212>cDNA

<213> wheat

<221> wheat salt tolerance, anti-drought gene TaDHN1 and application thereof

<222>（1）…（402）

<400>1

1 ATGGAGTACC AGGGGCAGCA CGGCCACGCC ACCGACAAGG TGGAGGAGTA CGGCCAGCCT

61 GTGGCCGGGC ACGGCGGTTT CACCGGCAGG CCCACGGGGA CGCACGGCGC GCAGCTCCAG

121 GCGACGAGGG ACGACCACAA GACCGACGGC GTCCTTCGCC GCTCCGGCAG CTCCAGCTCC

181 AGCTCGTCCG AGGACGACGG CGTGGGCGGC AGGAGGAAGA AGGGGATGAA GGAGAAGATC

241 AAGGAGAAGC TCCCCGGCGG AGCCCACAAG GACGCCACCG CCGGGCAGCA GCACACGGCG

301 GTGGCGGGCG AGTACGCGGG CACGCACGGC ACCGAGGCCA CCGGCGAGAA GAAGGGCGTC

361 ATGGACAAGA TCAAGGAGAA GCTTCCCGGC GGACAGCACT GA

SQ ID No.2

<110> University Of Ji'nan

<120> wheat salt tolerance, anti-drought gene TaDHN1 and application thereof

<141>2013-10-22

<160> 1

<210> 1

<211>133

<212>AA

<213> wheat

<221> wheat salt tolerance, anti-drought gene TaDHN1 and application thereof

<222>（1）…（133）

<400>1

1 MEYQGQHGHA TDKVEEYGQP

21 VAGHGGFTGR PTGTHGAQLQ

41 ATRDDHKTDG VLRRSGSSSS

61 SSSEDDGVGG RRKKGMKEKI

81 KEKLPGGAHK DATAGQQHTA

101 VAGEYAGTHG TEATGEKKGV

121 MDKIKEKLPG GQH*

sequence table

SQ ID No.1

<110> University Of Ji'nan

<120> wheat salt tolerance, anti-drought gene TaDHN1 and application thereof

<141>2013-10-22

<160> 1

<210> 1

<211>402

<212>cDNA

<213> wheat

<221> wheat salt tolerance, anti-drought gene TaDHN1 and application thereof

<222>（1）…（402）

<400>1

1 ATGGAGTACC AGGGGCAGCA CGGCCACGCC ACCGACAAGG TGGAGGAGTA CGGCCAGCCT

61 GTGGCCGGGC ACGGCGGTTT CACCGGCAGG CCCACGGGGA CGCACGGCGC GCAGCTCCAG

121 GCGACGAGGG ACGACCACAA GACCGACGGC GTCCTTCGCC GCTCCGGCAG CTCCAGCTCC

181 AGCTCGTCCG AGGACGACGG CGTGGGCGGC AGGAGGAAGA AGGGGATGAA GGAGAAGATC

241 AAGGAGAAGC TCCCCGGCGG AGCCCACAAG GACGCCACCG CCGGGCAGCA GCACACGGCG

301 GTGGCGGGCG AGTACGCGGG CACGCACGGC ACCGAGGCCA CCGGCGAGAA GAAGGGCGTC

361 ATGGACAAGA TCAAGGAGAA GCTTCCCGGC GGACAGCACT GA

SQ ID No.2

<110> University Of Ji'nan

<120> wheat salt tolerance, anti-drought gene TaDHN1 and application thereof

<141>2013-10-22

<160> 1

<210> 1

<211>133

<212>AA

<213> wheat

<221> wheat salt tolerance, anti-drought gene TaDHN1 and application thereof

<222>（1）…（133）

<400>1

1 MEYQGQHGHA TDKVEEYGQP

21 VAGHGGFTGR PTGTHGAQLQ

41 ATRDDHKTDG VLRRSGSSSS

61 SSSEDDGVGG RRKKGMKEKI

81 KEKLPGGAHK DATAGQQHTA

101 VAGEYAGTHG TEATGEKKGV

121 MDKIKEKLPG GQH*

Claims (8)

1. a wheat salt tolerance, anti-drought gene taDHN1, it is characterized in that: the nucleotide sequence of described gene cDNA is as shown in SEQ ID No.1.

2. a kind of wheat salt tolerance as claimed in claim 1, anti-drought gene taDHN1, it is characterized in that: its aminoacid sequence is shown in SEQ ID No.2.

3. recombinant plasmid pCAMBIA-super1300/TaDHN1: introduce to SEQ ID No.1 sequence both sides by primer xbai and saci double enzyme site, then by xbai and sacgene segment shown in I double digestion plant expression vector pCAMBIA-super1300 and SEQ ID No.1, reclaims carrier large fragment and is connected acquisition with target gene fragment again,

Described primer sequence is: D1ORF5:5 '-TCTAGAATGGAGTACCAGGGGCAGCAC-3 ' ( xbai)

D1ORF3: 5’-GAGCTCTCAGTGCTGTCCGCCGGG-3’( SacI)。

4. recombinant plasmid according to claim 3, is characterized in that: described recombinant plasmid is characterised in that in the expression cassette of foreign gene and contains taDHN1the complete encoder block of gene, i.e. total length ORF(open reading frame).

5. a wheat salt tolerance, anti-drought gene taDHN1application in cultivating salt tolerant and drought-resistant plant.

6. application according to claim 5, is characterized in that: described salt tolerant and drought-resistant plant are the monocotyledonss such as wheat, corn and paddy rice.

7. the application of recombinant plasmid pCAMBIA-super1300/TaDHN1 in cultivating salt tolerant, drought-resistant plant.

8. improve and contain gene described in claim 1 taDHN1the method of plant salt tolerance and drought resistance is by gene described in claim 1 taDHN1import host plant cell, tissue or plant individual, obtain having salt tolerant, the plant of drought resistance, described host plant is the monocotyledonss such as wheat, corn and paddy rice.

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