CN102277354B - Promoter of wheat dehydrin gene and application thereof - Google Patents

Abstract

The invention discloses a promoter TaDHNP of a wheat dehydrin gene and application thereof, aiming at providing a promoter capable of being induciblely expressed by abiotic stresses (salt/chilling) in a monocotyledonous plant, wherein, the nucleotide sequence of the promoter is shown as SEQ ID NO.1. The promoter induciblely expressed by abiotic stresses (salt/chilling), can be used for fusion with some salt resistant cold resistant genes to establish an inducible vector, thus having important significance in the breeding of plants, especially the breeding of salt resistant cold resistant genes of wheat.

Description

The promotor of wheat dehydrin gene and application thereof

Technical field

The invention belongs to the plant gene engineering technology field, relate to the promotor and the application thereof of a grow wheat dehydrin gene.

Background technology

In recent years, various abiotic stress have a strong impact on the output of crop.In various abiotic stress, salt stress and cold is coerced the reduction that usually causes crop yield owing to the dehydration that causes cell.Particularly along with industrial expansion, the soil salinization is more and more serious, has become the social concern that a whole world is paid close attention to.Therefore, cultivating anti-contrary new crop varieties through genetically engineered has become when the urgent task of previous ten minutes.In genetic engineering breeding, commonly used is the promotor of constitutive expression, for example cauliflower mosaic virus CaMV 35S promoter, Ubiquitin promotor etc.But the promotor of constitutive expression all can start transcribing of goal gene efficiently in the whole growth growth course; This efficiently expressing often is to be cost with a large amount of nutrient consumptions; Thereby directly influenced growing of plant, shown as that plant-growth retardation, plant are short and small, output reduction etc.And inducible promoter only under the stimulation of some physics or chemical signal or development of plants arrive particular phase, just start transcribing of foreign gene.It can not only make the expression product of goal gene in certain space-time accumulation; Increase regional expression amount; Improving the resistance of plant, can avoid simultaneously starting the negative impact to development of plants that the foreign gene overexpression causes by constitutive promoter, is the desirable promotor in the genetic engineering breeding.

The dehydration element is claimed embryonic development later stage Abundant protein again, great expression in the cell dehydration process that salt, drought, cold etc. causes, so the promotor of dehydrin gene is good candidate's inducible promoter in the breeding of adversity gene engineering.The promotor of dehydrin gene is cloned in several plants such as Arabidopis thaliana at present, but the clone of relevant wheat dehydrin gene promotor and application also do not appear in the newspapers.

Summary of the invention

The purpose of this invention is to provide a kind of can be in monocotyledons by the promotor of abiotic stress (salt, cold) abduction delivering TaDHNP

Technical program of the present invention lies at first selecting the dehydrin gene that in the wheat seedling root, significantly receives the salt abduction delivering according to the chip of expression spectrum data of wheat; Then according to gene order design gene-specific primer; Utilize the promotor of GenomeWalker method clone goal gene, in Arabidopis thaliana, carry out functional verification then.

Promotor provided by the present invention is from the wheat dehydrin gene TaDHNUpstream sequence, called after TaDHNPCan be one of following nucleotide sequence:

1) the SEQ ID No.1DNA sequence of sequence in the sequence table.

The nucleotide sequence of the dna sequence dna hybridization that 2) under the rigorous condition of height, can limit with sequence SEQ ID No.1 in the sequence table.The rigorous condition of above-mentioned height is can be in 0.1 * SSC (or SSPE), 0.1%SDS solution, hybridizes and wash film for 65 ℃.

The present invention also provides the plant expression vector that contains above-mentioned promotor pCAM1391Z/ TaDHNP

Promotor according to the invention TaDHNPApplication in cultivating salt tolerant, cold-resistant plant.The said plant of said plant is wheat or Arabidopis thaliana, preferably common wheat.

The application of the plant expression vector of the promotor of described wheat dehydrin gene in cultivating salt tolerant, cold-resistant plant, said plant is wheat or Arabidopis thaliana.

The downstream that any plant adverse circumstance genes involved are connected to inducible promoter according to the invention all can be by inducible transcription.And anyly can foreign gene be imported the carrier of expressing in the plant and can use.

Beneficial effect of the present invention: show that through transgenic experiments the promotor that the present invention cloned can be by salt and cold abduction delivering.This promotor can start any plant adverse circumstance Expression of Related Genes, can be widely used in to cultivate salt tolerant, cold-resistant new crop varieties.

TaDHNPPromoter sequence is analyzed;

Wherein, the sequence that has a underscore is the cis-acting elements sequence; The cis-acting elements title is respectively: CGGGGG- GC-motif, CAGTTG- MBS, TTATTACGAA- WUN-motif, TGACG- CGTCA-motif, TACGTG- ABRE, ACGTGGC- ABRE, CACGTA- ABRE;

CACGGCCCGGGCTGGTAAAAACGAAGAGAGTATTGTGCATGGCGTTTCATACTTGCTTTTCATGGAAGTTCATACCAGGCTGTTGTCTCTCTCATTCATCTTGAATCTTGACAAATGCCATCCCGACAAGTTACAAGTCAGCTCAAATCTCGTCCGGAAACTTCAGCATAATGACAGTCCTGTTCTAGGCTCCTAGCTCAGATCACCGACCTCCTTTCCTCGATTACTTGTTCTCCTAACCAAAACTGAATGAAGTAACATGAGTCTTGCTTGTGGCTGGCCTACAACCGCACGCCCGCGACGGAGCTCCCATCGTGGTGACACCGCGCTGCGCACAAGCGACTAGCGCTGCCACAATCGAGGACGCCGCCGTGAAGACGAGCTCCGGACTATCACCAGCCGCCTGAGATCTCCCCATCAAAAGAGCAGCCGGCGAGGAGCACGAGCCCCGCCGCCGCCGGCTCCACGCATGCTTTGCATGTCAGAGGCCACCGGCGATGGCGAGGGGGTGGATGGGATGGGTGAGGCGAGGAGAGGCTGGTGTTTTTCGCCACCCGTGTCGCCCTGGGGAGGGCGCGACG CGGGGGCAGATGGGATCCACTAGTTCGAT CAGTTGAAGCCCTACGTATATTTTTTCCGATATGAAATATAAAATATTGGGAAGATATTTATTACTCAAATGCATAGAACAACA TTATTACGAAAGGAAGAAGAAAAAAAGGTGAACAACACCCCCAGCAAACGACAACAACAA TGACGACAAGCATCAATGATAGCACCCATAGGTAACAGTA TACGTGCTTTAACAATGATGCTTTTAGGGAGGTATAGGTGCTTCAACAATGATGCTTTCAGGAAGAGAACAACACATGAGCGCTGTCGTCGTTGTAACCAACCACTAAGGTTAGATCGTGAATTTTCACCTCGAAGAGGAGTTTGAGTAAACTTCATGCAATGTCTTCAACTAGAGAATGTCATATGAACAACAACATTCCCATGTTCAACCAATGAAGGTGGATCATAAGTTTTCACCCTGAGACTCAGCCCTTGCACTCAACTTCTGTCACCTAACCTACTTCGCCTTGTGTTGCTAGCATACCATCACCTGCCGACCCATTTTCCCATTTTTAATGAGCAGGGTTATGATTTTCAGTCGATGCATATTTTCATGGACGTGCGATGGACATCCTCGCGGGCATAAAAATGAATTTGATATTGCTCGATCATAATAACCCGAACGCGCCTACTTTTAATGATAGGATACCTTTGGTCCATATAGTTCCATTTTTGTTCCATGCCGACGCTTCGAGCTGTCAGTTTCCGAAGCAAACAAGACGACTTTTGGAAGTGTCCCTTGTGTGTCATCACCTCTTCGAGAACAAGCTCAG ACGTGGCAACCCGAAGGCGCCCAGTAGCTGCACACGTCCGACCTCTCTTTATGGGCTAGTCATCAGTCACCGGCAACCCGCTCCCGCGAGCA CACGTAGCGCCTCCGACGATCCTATCCCGTCGGCTATAAATTGTGCCTCGTGCTGGACCTGCCTACACAACACAGCCACGAGTAGCAGCCAAGTCGGGAAGACAACCAAGATCAACACCTGTGCAAGTCTGCAAG ATG。

Description of drawings

Dehydrin gene under Fig. 1 condition of salt stress TaDHNThe RT-PCR that melts in No. 3 seedling roots and the leaf on the wheat mountain analyzes;

Wherein, Actin is confidential reference items; Leaf: leaf; Root: root;

Fig. 2 melts No. 3 genomic dnas from the wheat mountain and increases TaDHNPThe electrophorogram of promotor;

Wherein, M:DNA molecular weight marker (down together);

The enzyme of Fig. 3 pCAM1391Z/TaDHNP promoter expression vector is cut checking;

The PCR that Fig. 4 transforms the transgenic arabidopsis of pCAM1391Z/TaDHNP carrier identifies;

Wherein, T1 is transgenic arabidopsis strain system;

Fig. 5 transforms the transgenic arabidopsis GUS coloration result of pCAM1391Z/TaDHNP carrier;

Wherein, A: the transgenic arabidopsis strain that transforms pCAM1391Z/TaDHNP ties up to the GUS coloration result under the collating condition; B, C, D: transform the GUS coloration result of transgenic arabidopsis strain system after 100mM NaCl handles 48 hours of pCAM1391Z/TaDHNP, C, D are B figure amplification effect; F: the transgenic arabidopsis that transforms pCAM1391Z/TaDHNP is through the GUS coloration result of 4 ℃ of processing after 48 hours.

Embodiment

Embodiment 1, wheat dehydrin gene TaDHNExpression analysis

1.1 material processing

No. 3 normal seed germination is melted on the wheat breed mountain, removes endosperm after the week, in the Hangload nutrient solution, is cultured to for two weeks, changes in the Hangload nutrient solution that contains 200mM NaCl and coerces processing.Got children tender blade and root system, and preserved in the liquid nitrogen in 0,0.5,3,12,24,48 hour after the processing.

1.2 the Trizol method is extracted wheat Total RNA

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

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

3. add the 0.2ml chloroform, thermal agitation mixing 15 seconds, room temperature was 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 mixing, precipitates 30min by-20 ℃;

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

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

8. repeat with RNA deposition of 75% washing with alcohol;

9. remove supernatant, RNA is deposited in to dry on the aseptic technique platform and showed slightly transparent to RNA in about 10-15 minute, and the RNase-free water that adds proper volume (30-50 μ l) fully dissolves (can be placed on-80 ℃ of prolonged preservation);

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

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

B) with quality and the size of 1%Agrose gel electrophoresis inspection 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.With EB dyeing, other gets the 1kb DNAMarker of 3 μ l as contrast behind the electrophoresis.

1.3 the first chain cDNA's is synthetic

Adopt PrimeScript ^TMRT-PCR Kit carries out.Reactions step is following:

1. in the 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 the PCR appearance, 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.

Above-mentioned sex change, annealing afterreaction liquid 10 μ l

5×PrimerScript ^TM?Buffer 4?μl

RNase?Inhibitor?(40U/μl)?0.5?μl

PrimScript ^TM?RTase 0.5?μl

Rnase?Free?dH ₂O 5?μl

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

42℃ 15-30?min

95℃ 5?min

4℃

1.4 PCR reaction and electrophoresis

1. be template with cDNA, carry out the PCR reaction.Primer is following

TaAct-S：?5’-?GTTCCAATCTATGAGGGATACACGC?-3’

TaAct-A:?5’-?GAACCTCCACTGAGAACAACATTACC?-3’

TaDHN-S:?5′-CAGCCAAGTCGGGAAGACAAC-3′

TaDHN-A:5′-TGGCCACCAGGGAGCTTCTC-3′

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

Rt cDNA template 1 μ l

Total?Volume 10μl

3. PCR program:

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

Confirm the cycle number of PCR according to the amplification situation of confidential reference items Actin, the add-on of adjustment cDNA template.

4. 1% agarose gel electrophoresis.The result sees Fig. 1.

Embodiment 2, wheat dehydrin gene promotor TaDHNPThe clone

2.1 the extraction of wheat cdna group DNA

1. adopt the CTAB method to extract wheat cdna group DNA.Step is:

(1) gets the about 0.5g of wheat root and in liquid nitrogen, grind, place the 1.5ml centrifuge tube, add 700 μ l and extract damping fluid (100 mmol/L Tris-HCl, 50 mmol/L EDTA-Na ₂, 100mmol/L NaCl, 1.5%CTAB, 2% mercaptoethanol), 65 ^oIncubation 30-60min puts upside down mixing 3-4 time therebetween in the C water-bath;

(2) take out centrifuge tube, in every pipe, add 700 μ l phenol: chloroform: primary isoamyl alcohol (25:24:1) solution, mixing 10min;

(3) the centrifugal 10min of 10000rpm;

(4) slowly extract supernatant to another 1.5ml centrifuge tube with rifle head (cutting off front end 1/3);

(5) add isopyknic chloroform: primary isoamyl alcohol (24:1) repeats above-mentioned extractive process.Slowly take out supernatant;

(6) isopropanol precipitating of 0.6 times of volume of adding, mixing leaves standstill for some time gently;

(7) go out the DNA deposition with 200 μ l rifle choicests;

(8) 70% alcohol flushing 2 ~ 3 times, DNA was drying at room temperature 10 minutes;

(9) add 500 μ l deionized waters, 4 ^oThe C dissolving.

2. the purifying of wheat cdna group DNA

(1) in dissolved DNA, adds 1-5 μ lRNA enzyme (10mg/ml), 37 ^oC is incubated 1h;

(2) add isopyknic phenol: chloroform: primary isoamyl alcohol (25:24:1) solution, mixing;

(3) 4 ^oThe centrifugal 10min of C 10000rpm;

(4) get supernatant in another centrifuge tube;

(5) add the equal-volume chloroform: primary isoamyl alcohol (24:1) repeats above-mentioned extractive process;

(6) get supernatant and move on to a new 1.5ml centrifuge tube;

(7) the 3mol/L NaAc of adding 1/10 volume, the cold absolute ethyl alcohol of 2 times of volumes of adding, mixing gently behind the mixing;

(8) room temperature left standstill 10 minutes, chose the DNA deposition with toothpick;

(9) with 70% alcohol flushing 2 ~ 3 times, room temperature is placed to alcohol-free flavor;

(10) DNA adds 100 μ l deionized water dissolvings, measures DNA concentration with ultraviolet spectrophotometer, and passes through the quality that agarose gel electrophoresis detects DNA, and sample is-20 ^oC preserves.At last according to DNA concentration, with part dilution, 4 ^oC preserves for use.

2.2 wheat dehydrin gene promotor ( TaDHNP) the clone

The method of employing GenomeWalker (Clontech, CA, USA) clone's wheat dehydrin gene promotor TaDHNP, the detailed process by specification carries out.The primer sequence of gene specific is:

Dsp1:?5'-TTGGTCGCCTGGCCGTGCTGCTGCTGT-3'

Dsp2: 5'-GCACAGGTGTTGATCTTGGTTGTCTTC-3'

2.3 the agarose gel electrophoresis of pcr amplification product detects and reclaims

Pcr amplification product detects the band (Fig. 2) of finding to amplify a treaty 2kb with 1% agarose gel electrophoresis, adopts the agarose gel of Tiangen company to reclaim test kit to amplified band, and the step by specification carries out.

2.4 the PCR product is connected with pGEM-T (Promega) carrier

In the PCR pipe, add following component successively:

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

Spend the night in 16 ℃ of water-baths connections.

2.5 reclaim segmental clone and order-checking

1. the preparation of competent escherichia coli cell

(1) from-80 ℃ of refrigerators, takes out and be stored in the glycerine E.coliDH5a bacterial strain (is epoch biotech firms available from the sky) is placed on ice and slowly thaws.

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

(3) with flat board in 37 ℃ of constant temperature culture carton upside down overnight cultures.

(4) the single colony inoculation on the picking flat board is in the test tube that contains 5ml LB liquid nutrient medium, 37 ℃ shaking culture 14-16 hour.

(5) get in the triangular flask that 0.5ml bacterium liquid is inoculated in the 250ml that contains 50ml LB liquid nutrient medium, 2-3 hour (OD260=0.5) cultivated in 37 ℃ of vibrations (260rpm).

(6) the bacterium liquid of cultivating was put 1 hour on ice.

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

(8) solution A with the precooling of 25ml ice suspends thalline gently, places on ice 40-45 minute again.

(9) repeating step (8).

(10) solution B with the precooling of 2.5ml ice suspends thalline gently, then bacterium liquid branch is installed to (every pipe 100 μ l) in the 1.5ml centrifuge tube, and it is subsequent use to put into-80 ℃ of refrigerators.

2. connect the conversion of product

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

(2) in pipe, add 5 μ l ligation things on the Bechtop, shake up gently, put 30 minutes on ice.

(3) 42 ℃ of water-bath heat shocks 90 seconds were put rapidly 3-5 minute on ice.

(4) in centrifuge tube, adding 1ml LB liquid nutrient medium (not containing Amp), 37 ℃ of shaking culture 1 hour (260rpm) behind the mixing on the Bechtop.

(5) centrifugal 6 minutes of 5000rpm under the room temperature discards 900 μ l supernatants, will remain the 100 μ l supernatants thalline that suspends again, adds the X-gal of 40 μ l and the IPTG of 4 μ l, and mixing evenly is coated onto it on flat board of preparation with spreader then, places 30 minutes.

(6) be inverted and dull and stereotypedly in 37 ℃ of constant incubators, spend the night, take out when waiting to occur obvious single bacterium colony.

(7) put into 4 ℃ of refrigerator numbers hour, make blue hickie color clearly demarcated.

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

3. the PCR of recombinant plasmid identifies and order-checking

There is the T7 promotor at the upper reaches of the cloning site of the pGEM-T carrier that this experiment is used, and there is the SP6 promotor in downstream, increase to inserting fragment so can do primer with these two promoter sequences, recombinant plasmid are identified qualification program is following:

(1) gets the bacterium liquid 50 μ l that shaken in the 1.5ml centrifuge tube with pipettor on the Bechtop, in centrifuge tube, add 50 μ l ultrapure waters again.

(2) 100 ℃ are boiled sex change after 10 minutes, place on ice.

(3) pcr amplification TV 20 μ l: comprise plasmid template 10 μ l, 10 * buffer, 2 μ l (containing Mgcl2), dNTP 10mmol/L, each 20ng/ μ l of T7 and SP6 primer, 1U Taq enzyme.Reaction conditions is: 94 ℃, and 5 minutes; 30 circulations: 94 ℃, 1min; 56 ℃, 1min; 72 ℃, 2min; 72 ℃ are extended 5min.

Pcr amplification product detects with 1% agarose electrophoresis, and the bacterium liquid of getting positive colony send company to check order.Sequencing result shows the nucleotide sequence that the fragment of pcr amplification acquisition has sequence 1 in the sequence table.Sequence is carried out sequence alignment, and the result shows gene-specific primer used when 3 of this sequence ' end contains the amplification promotor, and 3 ' end and this laboratory clone's wheat dehydrin gene TaDHN5 ' terminal sequence identical, confirm that above-mentioned sequence fragment of cloning does TaDHNThe promoter sequence of gene.Utilize the plantCARE DB that the gained promoter sequence is carried out the cis-acting elements prediction, find that promoter region contains the cis-acting elements (Fig. 3) of response adverse circumstances such as ABRE, MBS.This is expressed by salt, stress-inducing such as cold with the TaDHNP promotor of being cloned be consistent.

Embodiment 3, wheat dehydrin gene promoter expression vector (PCAM1391Z/TaDHNP) Structure

According to promotor TaDHNPSequences Design contains respectively HindIII with EcoRThe promotor upstream and downstream primer of I, primer title and sequence be Dhy2p1:5 '-CCCAAGCTTCCCGGGCTGGTAAAAACGAAG-3 ' ( HindIII) Dhy2p2:5 '-CGGAATTCCTTGCAGACTTGCACAGGTGTTG-3 ' ( EcoRI).With the plasmid that contains promoter sequence is that template is carried out pcr amplification, and pcr amplification product is connected with pGEM-T easy carrier after electrophoresis, recovery, and transformed into escherichia coli extracts plasmid.Use HindIII with EcoRI respectively to the pCAM1391Z carrier with contain TaDHNPThe pGEM-T easy carrier of promotor carries out double digestion, reclaim respectively the big fragment of pCAM1391Z carrier with TaDHNPThe promotor small segment; Connect back transformed into escherichia coli DH5 α competent cell with T4 DNA ligase enzyme; Carry out enzyme behind the extraction recombinant plasmid and cut the evaluation recon; Enzyme is cut the purpose promotor band of product visible suitable size after electrophoresis detection and the carrier ribbon of incision, shows constructed plant expression vector correct (Fig. 4).

3.1 plasmid pCAM1391Z carrier and pGEM-T easy carrier HindIII with EcoRThe I double digestion

Alkaline lysis method of extracting pCAM1391Z empty carrier and pGEM-T easy plasmid are respectively got 10 μ g enzymes and are cut, and it is following that enzyme is cut system:

HindⅢ 1μl

EcoRI 1μl

PCAM1391Z carrier/pGEM-T easy plasmid 4 μ l

10×Buffer?M 1μl

ddH ₂O?To 20μl

Cut more than 3 hours in 37 ℃ of thermostat water bath enzymes.Be electrophoretic buffer with 1 * TAE behind the double digestion, enzyme cut product carry out 0.8% agarose gel electrophoresis.The goal gene band of about 1.6 kb among the big fragment of pCAM1391Z carrier and the pGEM-T reclaims this band under ultraviolet transilluminator, downcutting with clean blade.

3.2 enzyme is cut the connection of product

The pCAM1391Z carrier vector fragment of cutting through enzyme and pGEM-T easy double digestion reclaim fragment (about 1.6kb) to carry out 16 ℃ with the ratio of mol ratio 1:4 and is connected and spends the night.

3.3 connect the conversion of product

Connect product heat shock method transformed into escherichia coli DH5 α competent cell, transformed bacteria on the LB solid plate that contains Kan 50 μ g/ml 37 ℃ cultivated about 16 hours.

3.4 the evaluation of recon

Plasmid enzyme restriction is identified

Extract the positive colony plasmid, plasmid is carried out HindIII with EcoRIt is the same that I double digestion, enzyme are cut system.The promotor purpose band that the detection of 0.8% agarose gel electrophoresis is cut open and the carrier segments of incision, two stripe size are correct, show vector construction correct (Fig. 4).

Embodiment 4, the competent preparation of Agrobacterium and conversion

4.1 the competent preparation of Agrobacterium GV3101

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

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

(2) getting 2ml incubated overnight liquid is inoculated in 50ml and contains in the 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 among the NaCl of 10ml 0.15mol/L of ice bath centrifugal collection thalline.

(5) resuspending is divided in 1.5ml with 200 μ l/ pipe with bacterium liquid in the CaCl2 solution of 1ml 20mmol/L ice precooling

In the Eppendorf pipe, put quick-frozen 1min in the liquid nitrogen ,-70 ℃ of preservations are subsequent use.

4.2 freeze-thaw method transforms agrobacterium tumefaciens GV3101

(1) at room temperature melts the Agrobacterium competent cell, add 1 μ g expression vector DNA, ice bath behind the mixing

30min。

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

(3) the YEP 800 μ l of adding antibiotic-free, 3hr are cultivated in 28 ℃ of concussions.

(4) the centrifugal 30s of 7000rpm collects thalline, is applied on the YEP flat board that contains 50 μ g/ml Rifampins, 50 μ g/ml Kan, is inverted dark the cultivation 2-3 days for 28 ℃.

4.3 thalline PCR identifies and electrophoresis

Thalline PCR method and program and electrophoresis extend time 2min with 1.4 for PCR72 ℃ according to the expanding fragment length adjustment.

The evaluation of the functional verification of embodiment 5, promotor-Arabidopis thaliana conversion and transgenic line

5.1 Arabidopis thaliana plantation

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

5.2 the preparation of Agrobacterium

Respectively the inoculation GV3101 Agrobacterium that contains the pCAM1391Z/TaDHNP carrier in the 10mLYEB substratum (100 mg/L Rifampins, 25 mg/L celebrating big with 100 mg/L cards that) in, cultivate shaken overnight for 28 ℃.So containing the back changes and to be inoculated in 500 ml and to contain enlarged culturing in the identical antibiotic YEB substratum.The centrifugal 15 min deposition of 5 000 rpm thalline.With Agrobacterium be resuspended in 200 ml transform in the damping fluid (the MS macroelement, 3% sucrose, 0.03%Silwet-77), OD600 ≈ 0.8-1.

5.3 Arabidopis thaliana transforms

Pour 200 ml bacterium liquid in the tray into.The Arabidopis thaliana back-off that pruning is good also immerses in suspension bacteria liquids all inflorescences,

Stir to be stained with gently and spend 30 sec-1 min.Take out flowerpot and be sidelong in pallet, wrap up to preserve moisture with freshness protection package.Pallet is put the dark place cultivate 24 h.Take out Nursery and upright the placement then, recover illumination, continue to cultivate plant to ripe.

5.4 the screening of positive plant

After T0 sterilized with 7.5% chlorine bleach liquor (comprising 7.5% Youxiaolin and 0.01% Triton-X 100) for seed, program request was selected on the culture plate (30 mg/L Totomycin) at MS.In 4 ℃ of following vernalization 2-3 days.Move in the culturing room and cultivate.About about 10 days, select hygromycin resistance plant (it is right to grow true leaf 1-2, and root is stretched in the substratum) and be transplanted in the Nursery.Cultivation is until seed maturity.T1 obtains T2 for plant for seed with the quadrat method screening.

5.5 the PCR of transgenic arabidopsis identifies

The extraction of arabidopsis thaliana genomic dna is with 2.1

PCR system, program and deposition condition are with 4.3.Detected result is illustrated in the foreign gene band (Fig. 5) that can see suitable size in the transgenic arabidopsis strain system.Show that exogenous promoter has changed the Arabidopis thaliana plant over to.

6.GUS dyeing

(1) draws materials: collect the whole strain seedling of T2 generation in growth 1-2 week, put into the 90% acetone test tube that precooling is housed rapidly.Room temperature 30 min that decolour.

(2) with material change over to precooling the dyeing damping fluid that does not contain X-gluc (0.2% Triton X-100,2 mmol/L yellow prussiate of potash, the 2mmol/L Tripotassium iron hexacyanide, 50 μ mol/L sodium phosphate buffers, pH7.2) in the washing.

(3) dyeing: remove washing dyeing damping fluid, add the dyeing damping fluid (0.2% Triton X-100, the 2 mmol/L yellow prussiate of potash that contain X-gluc; The 2 mmol/L Tripotassium iron hexacyanides; 2 mmol/L X-gluc, 50 μ mol/L sodium phosphate buffers, pH7.2); And sample is put into vacuum filtration 15-20 min on ice, all be immersed in the solution until all material.

(4) material transfer to 37 ℃ dyeing is spent the night or dyeed two days.

(5) dehydration: when waiting tissue blueness to occur, remove staining fluid, with 20%, 35%, 50% ethanol gradient dehydration, per steps 30 min.

(6) fixing: that material is placed among the FAA (50% ethanol, 10% glacial acetic acid, 5% formaldehyde) in fixing 30 min of room temperature.

(7) remove FAA, add 70% ethanol.

(8) directly examine under a microscope material and photograph.Coloration result shows: under the normal cultured condition, the TaDHNP promotor can not start exogenous gene expression, and transgenic arabidopsis strain system does not see has GUS dyeing (Fig. 5 A); Handle and 4 ℃ of processing 48h at 100 mM NaCl, the TaDHNP promotor is induced the startup exogenous gene expression, and GUS is coloured to the positive (Fig. 5 B, C, D, E).

Sequence table

?

< 110>University Of Ji'nan, Shandong University

< 120>promotor of wheat dehydrin gene and application thereof

<141>2010-6-1

<160>?1

<210>?1

<211>1615

<212>DNA

< 213>wheat

< 221>promotor of wheat dehydrin gene

<222>（1）…（1615）

<400>1

1 CACGGCCCGG?GCTGGTAAAA?ACGAAGAGAG?TATTGTGCAT?GGCGTTTCAT?ACTTGCTTTT

61 CATGGAAGTT?CATACCAGGC?TGTTGTCTCT?CTCATTCATC?TTGAATCTTG?ACAAATGCCA

121 TCCCGACAAG?TTACAAGTCA?GCTCAAATCT?CGTCCGGAAA?CTTCAGCATA?ATGACAGTCC

181 TGTTCTAGGC?TCCTAGCTCA?GATCACCGAC?CTCCTTTCCT?CGATTACTTG?TTCTCCTAAC

241 CAAAACTGAA?TGAAGTAACA?TGAGTCTTGC?TTGTGGCTGG?CCTACAACCG?CACGCCCGCG

301 ACGGAGCTCC?CATCGTGGTG?ACACCGCGCT?GCGCACAAGC?GACTAGCGCT?GCCACAATCG

361 AGGACGCCGC?CGTGAAGACG?AGCTCCGGAC?TATCACCAGC?CGCCTGAGAT?CTCCCCATCA

421 AAAGAGCAGC?CGGCGAGGAG?CACGAGCCCC?GCCGCCGCCG?GCTCCACGCA?TGCTTTGCAT

481 GTCAGAGGCC?ACCGGCGATG?GCGAGGGGGT?GGATGGGATG?GGTGAGGCGA?GGAGAGGCTG

541 GTGTTTTTCG?CCACCCGTGT?CGCCCTGGGG?AGGGCGCGAC?GCGGGGGCAG?ATGGGATCCA

601 CTAGTTCGAT?CAGTTGAAGC?CCTACGTATA?TTTTTTCCGA?TATGAAATAT?AAAATATTGG

661 GAAGATATTT?ATTACTCAAA?TGCATAGAAC?AACATTATTA?CGAAAGGAAG?AAGAAAAAAA

721 GGTGAACAAC?ACCCCCAGCA?AACGACAACA?ACAATGACGA?CAAGCATCAA?TGATAGCACC

781 CATAGGTAAC?AGTATACGTG?CTTTAACAAT?GATGCTTTTA?GGGAGGTATA?GGTGCTTCAA

841 CAATGATGCT?TTCAGGAAGA?GAACAACACA?TGAGCGCTGT?CGTCGTTGTA?ACCAACCACT

901 AAGGTTAGAT?CGTGAATTTT?CACCTCGAAG?AGGAGTTTGA?GTAAACTTCA?TGCAATGTCT

961 TCAACTAGAG?AATGTCATAT?GAACAACAAC?ATTCCCATGT?TCAACCAATG?AAGGTGGATC

1021?ATAAGTTTTC?ACCCTGAGAC?TCAGCCCTTG?CACTCAACTT?CTGTCACCTA?ACCTACTTCG

1081?CCTTGTGTTG?CTAGCATACC?ATCACCTGCC?GACCCATTTT?CCCATTTTTA?ATGAGCAGGG

1141?TTATGATTTT?CAGTCGATGC?ATATTTTCAT?GGACGTGCGA?TGGACATCCT?CGCGGGCATA

1201?AAAATGAATT?TGATATTGCT?CGATCATAAT?AACCCGAACG?CGCCTACTTT?TAATGATAGG

1261?ATACCTTTGG?TCCATATAGT?TCCATTTTTG?TTCCATGCCG?ACGCTTCGAG?CTGTCAGTTT

1321?CCGAAGCAAA?CAAGACGACT?TTTGGAAGTG?TCCCTTGTGT?GTCATCACCT?CTTCGAGAAC

1381?AAGCTCAGAC?GTGGCAACCC?GAAGGCGCCC?AGTAGCTGCA?CACGTCCGAC?CTCTCTTTAT

1441?GGGCTAGTCA?TCAGTCACCG?GCAACCCGCT?CCCGCGAGCA?CACGTAGCGC?CTCCGACGAT

1501?CCTATCCCGT?CGGCTATAAA?TTGTGCCTCG?TGCTGGACCT?GCCTACACAA?CACAGCCACG

1561?AGTAGCAGCC?AAGTCGGGAA?GACAACCAAG?ATCAACACCT?GTGCAAGTCT?GCAAG

Claims (4)

1. the promotor of a grow wheat dehydrin gene TaDHNP, it is characterized in that: the nucleotide sequence of said promotor is shown in SEQ ID No.1.

2. contain the plant expression vector of the promotor of the said wheat dehydrin gene of claim 1, it is characterized in that, according to promotor TaDHNPSequences Design contains respectively HindIII with EcoRThe promotor upstream and downstream primer of I, primer title and sequence are Dhy2p1:5 '-CCCAAGCTTCCCGGGCTGGTAAAAACGAAG-3 '; Dhy2p2:5 '-CGGAATTCCTTGCAGACTTGCACAGGTGTTG-3 ' is that template is carried out pcr amplification with the plasmid that contains promoter sequence, and pcr amplification product is connected with pGEM-T easy carrier after electrophoresis, recovery, and transformed into escherichia coli extracts plasmid; Use HindIII with EcoRI respectively to the pCAM1391Z carrier with contain TaDHNPThe pGEM-T easy carrier of promotor carries out double digestion, reclaim respectively the big fragment of pCAM1391Z carrier with TaDHNPThe promotor small segment; Connect back transformed into escherichia coli DH5 α competent cell with T4 DNA ligase enzyme; Carry out enzyme behind the extraction recombinant plasmid and cut the evaluation recon; Enzyme is cut the purpose promotor band of product visible suitable size after electrophoresis detection and the carrier ribbon of incision, shows that constructed plant expression vector is correct.

3. the application of the promotor of wheat dehydrin gene as claimed in claim 1 in cultivating salt tolerant, cold-resistant plant, it is characterized in that: said plant is wheat or Arabidopis thaliana.

4. the application of plant expression vector in cultivating salt tolerant, cold-resistant plant that contains the promotor of wheat dehydrin gene as claimed in claim 2, it is characterized in that: said plant is wheat or Arabidopis thaliana.

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