CN100366634C - Binding protein of response component of plant dehydration, and coding genes and application - Google Patents

Abstract

The present invention discloses a conjugated protein of a plant dewatering response element, a coded gene thereof and an application thereof. The conjugated protein is a protein with an amino acid residue sequence of SEQ ID No. 1 in a sequence list, or a protein for regulating and controlling plant stress resistance, which is used for substituting, deleting or adding one to ten amino acid residues of the amino acid residue sequence of SEQ ID No. 1 in the sequence list and has a transcriptional activation function. The coded gene of the conjugated protein is polynucleotide with a DNA sequence of SEQ ID No. 2 in a sequence list or a protein sequence of SEQ ID No. 1 in a coded sequence list, or a nucleotide sequence which can be hybridized with the DNA sequence limited by SEQ ID No. 2 in the sequence list under the high precision condition. The protein and the coded gene of the protein can be used for breeding plants with high stress resistance.

Description

Binding protein of response component of plant dehydration and encoding gene thereof and application

Technical field

The present invention relates in the plant a kind of with coerce relevant transcription factor and encoding gene thereof and application, particularly a kind of binding protein of response component of plant dehydration and encoding gene thereof and its application in cultivating the plant that resistance improves.

Background technology

Environment stresses such as arid, high salt and low temperature all can influence normal growth and the growth of plant.The understanding plant is replied and signal transduction mechanism adverse environmental factor, and the raising plant particularly resistance of crop is one of vital task of crop genetic research and breed improvement.

Under environment stress, can produce a series of responsing reactions in the plant materials, the variation that is accompanied by many Physiology and biochemistries and grows.Illustrate the reaction mechanism of plant, will provide scientific foundation for the molecular breeding of plant stress-resistance to adverse circumstance.Studies show that plant just can be made arid and comprises signal transduction, genetic expression and metabolism accommodation and continued growth and the growth in being adjusted in before water deficit causes plant injury.Gene and environment are two fundamental factor that coordinate plant growth is grown, explore envrionment conditions and how to regulate and control the genetic expression of plant, and envrionment conditions influence that the intravital physiological function of plant is changed become plant genetics and breeding scholar's ultimate challenge, and this is the hot issue of signal transduction (Signal transduction) Mechanism Study just.

Many genes of plant be proved to be subjected to that abiotic stress coerces induce directly performance function, the expression of the responsible regulation and control downstream gene that has and the conduction of adverse circumstance signal in resistance that these expression of gene products have.With coerce relevant gene product and can be divided into two big classes: the product of first kind genes encoding comprises that ionophorous protein, aquaporin, the osmoregulation factor (sucrose, proline(Pro) and trimethyl-glycine etc.) synthetic enzyme etc. participate in the gene product that plant stress is replied directly; The product of second genoid coding comprises the protein factor that participates in coercing relevant signal transmission and genetic expression adjusting, as protein kinase, transcription factor etc.Wherein, transcription factor plays an important role in the gene expression regulation that plant stress is replied.

Transcription factor is also referred to as trans-acting factor, is can be conjugated protein with the DNA of cis-acting elements generation specific effect in the eukaryotic gene promoter region, by between them and and other associated protein between interaction, activate or suppress and transcribe.The DNA land of transcription factor has determined it and cis-acting elements bonded specificity, and transcription regulatory region has determined it that genetic expression is risen to activate or restraining effect.In addition, himself activity also is subjected to appraising and deciding the influence of effects such as position and oligomerization.

At present known in plant with coerce relevant transcription factor and mainly contain: AP2 (APETALA2)/EREBP (element responsive to ethylene is conjugated protein, the ethylene responsive element bindingprotein) transcription factor family with AP2 structural domain, bZIP (basic region/leucine zipper motif transcription factors) the class transcription factor that contains alkalescence zone and leucine zipper, the WRKY transcription factor family that contains conservative WRKY aminoacid sequence, the MYC family and MYB family of containing alkaline helix-loop-helix (bHLH) and leucine zipper with tryptophane bunch (Trp cluster).These five transcription factor families, except that WRKY family not the water of involved in plant coerce the reaction, other four families all participate in regulating the environment stress reaction of plant to arid, high salt and low temperature etc.Wherein, AP2/EREBP class transcription factor extensively exists in higher plant, it is the peculiar class transcription factor of plant, in recent years, report is all arranged in Arabidopis thaliana, tobacco, corn, paddy rice and rape, and this shows AP2/EREBP class transcription factor ubiquity and have vital role in higher plant.

Yamaguchi-Shinozaki and Shinozaki are in the research to the promoter region of adverse circumstance response gene rd29A gene, found that an environment stress replys the functional element of taking advantage of a situation, be dehydration response element (DRE, dehydration-responsive element).After this, find that many adverse circumstance response gene promoter regions contain the DRE element.People such as Liu utilize yeast-one-hybrid system first, from the cDNA expression library of Arabidopis thaliana, be cloned into the transcription factor dehydration response element conjugated protein (DREB of two kinds of codings and DRE combination of elements, dehydration-responsive element binding protein) cDNA, difference called after DREB1A, DREB2A.They do not have significant homogeny on aminoacid sequence, but all contain one section very conservative DNA calmodulin binding domain CaM (EREBP/AP2 structural domain) of being made up of 58 amino acid.The protein three dimensional analysis shows that 3 beta sheets are contained in this zone, plays a crucial role to discerning all kinds of cis-acting elements.Wherein be arranged in the difference of two amino-acid residues of the 14th, 19 of second beta sheet, determine the specific combination of this class transcription factor and different cis-acting elements.DREB class transcription factor the 14th amino acids is Xie Ansuan (V14), and the 19th amino acids is L-glutamic acid (E19), and wherein the 19th amino acid is not conservative, and for example the 19th amino acids of the OsDREB1 transcription factor of paddy rice is exactly a Xie Ansuan.Aspect the decision DNA bonded specificity, the effect of V14 is obviously important than E19 in the DREB proteinoid; And ERF class transcription factor the 14th amino acids is a glycine, and the 19th is aspartic acid, thereby DREB specific combination DRE/CRT cis element, ERF specific combination GCC-box.The C-petiolarea of AP2/EREBP structural domain also comprises 1 core sequence of being made up of 18 amino-acid residues, and this sequence forms amphiphatic alpha-helix, and this alpha-helix may participate in the interaction between other transcription factor and DNA.

At present, in many plants, all find the transcription factor of this EREBP/AP2 of containing structural domain, and transmit relevant with signal such as disease-resistant, degeneration-resistant respectively.Liu Qiang etc. think that a dreb gene can be regulated and control a plurality of and plant arid, high salt and low temperature patience function associated expression of gene.Kasuga etc. studies confirm that, the DREB1A gene that imports to Arabidopis thaliana can promote the expression of gene rd29, rd17, kin1, cor6.6, Cor15a and the erd10 relevant with environment stress patience simultaneously, and the resistance of transfer-gen plant strengthens greatly.Equally, the low temperature tolerance ability of the transfer-gen plant of low temperature patience transcription factor CBF1 is significantly increased.Because the stress tolerance of plant is the complex character by the polygene regulation and control, rely on to import the comprehensive raising that the individual feature protein gene is difficult to realize stress resistance of plant.Therefore, utilize a key transcription factor to promote the expression of a plurality of functional genes, thereby strengthen the comprehensive resistance of plant, become the engineered research focus of plant stress-resistance.

The researchist finds that the earliest under environment stresses such as arid, high salt and low temperature, dormin (ABA) is a large amount of in the body accumulates, and many genes relevant with environment stress patience are to be subjected to the ABA inductive.Research ABA inducible gene expression regulation and control on transcriptional level find all to have a quite conservative sequence (PyACGTGGC) in the promoter region of functional gene, as the ABA response element (ABA responsive element, ABRE).Also cloned in succession with the trans-acting factor of ABRE specific combination.In addition, find that also the other cis-acting elements participates in the responsing reaction of ABA, (its core sequence is TGCCACCGG for Coupling element, CE) CE1 as the coupling element identified in barley HVA22 gene.

Meanwhile, research finds that also many expression of gene relevant with stress tolerance and ABA have nothing to do, and wherein, some gene is induced by arid and low temperature simultaneously, and some gene only has responsing reaction to arid or low temperature.Illustrate to have many barss pipeline in the plant materials, be responsible for the regulation and control of induction, transmission and the genetic expression of environment stress signal.Yamaguchi-Shinozaki etc. utilize the differential screening method from the Arabidopis thaliana that arid is handled, cloned a collection of drought-induced gene that is subjected to, wherein, the albumen that rd29 genes encoding and lea protein are closely similar, it all can produce responsing reaction to arid, high salt and low temperature stress, to its promotor discover two DRE cis-acting elements that do not rely on ABRE (dehydration responsive element, DRE).Its core sequence is TACCGACAT.The present gene relevant of separating clone with stress tolerance, as rd17, kin1, DRE element or DRE core sequence element have also all been found in the isogenic promoter region of cor6.6, in addition, also there is the CCGAC conserved sequence at low temperature response element CRT (C-repeat), LTRE (Low temperature responsive element), illustrates that the DRE element is prevalent in the promotor of environment stress response gene, and environment stress replied play an important role.

Comprehensive present result of study, the signal pipeline of plant under the environment stress condition has following six approach at least:

The signal pipeline that depends on ABA has three:

1) induced by arid, high salt, activate MYB, MYC class transcription factor gene, regulation and control have the target gene of MYBR or MYCR cis-acting elements.

2) induced by arid, high salt, activate ABF/AREB class transcription factor gene, regulation and control have the target gene of ABRE cis-acting elements.

3) induced by arid, high salt, activate CBF4, DREB1 class transcription factor gene, regulation and control have the target gene of DRE/CRT cis-acting elements.

The signal pipeline that does not rely on ABA has three:

1) induced by arid, high salt, activate DREB2 class transcription factor gene, regulation and control have the target gene of DRE/CRT cis-acting elements.

2) be subjected to low temperature induction, activate CBF1-3/DREB1A-C class transcription factor gene, regulation and control have the target gene of DRE/CRT cis-acting elements.

3) induced by arid, high salt or ethene, activate EREB class transcription factor gene, regulation and control have the target gene of DRE/CRT cis-acting elements.

At present, existing DREB transcription factor gene clone's report in staple crops such as paddy rice, corn, clone DREB transcription factor gene yet there are no report from soybean.

(DNA mobility shift assay EMSA), cries gel blocking (gelretardation) test again to the test of DNA mobility shifting, is the special gel electrophoresis technology of a kind of in vitro study DNA and protein interaction.Ultimate principle is: in gel electrophoresis, because effect of electric field, it is fast that the small molecule DNA fragment combines the speed that protein DNA fragment anode moves than it, therefore, but the short double chain DNA fragment of mark, it is mixed with protein, mixture is carried out gel electrophoresis, if target DNA combines with specific protein, the speed that its anode moves is blocked, gel is carried out radioactive automatic developing, combine with corresponding protein with regard to the susceptible of proof specific DNA.

Summary of the invention

The purpose of this invention is to provide a kind of binding protein of response component of plant dehydration and encoding gene thereof.

Binding protein of response component of plant dehydration provided by the present invention, name is called GmDREB3, derives from Glycine soybean (Glycine max (L.)), and its amino acid residue sequence is shown in the SEQ ID NO:1 in the sequence table.

Sequence 1 in the sequence table is made up of 313 amino-acid residues, is nuclear localization sequence from the 104th-105 amino acids residues of aminoterminal (N end), is conservative AP2/EREBP structural domain from the 120th-177 amino acids residue sequence of aminoterminal.

Above-mentioned binding protein of response component of plant dehydration encoding gene (GmDREB3), also belonging to protection scope of the present invention is one of following nucleotide sequence:

1) dna sequence dna shown in the SEQ ID NO:2 in the sequence table;

2) in the code sequence tabulation shown in SEQ ID NO:1 the polynucleotide of protein sequence.

Wherein, the SEQ ID № in the sequence table: 2 by 939 based compositions, and its encoding sequence is that coding has SEQ ID № in the sequence table: the protein of 1 amino acid residue sequence from 5 ' end the 1st to the 939th bit base; From 5 ' end the 358th to the 531st bit base is the encoding sequence of AP2/EREBP conserved domain, 58 amino acid of encoding, from 5 ' end the 397th to the 399th bit base is the encoding sequence of Xie Ansuan, is leucic encoding sequence from 5 ' end the 412nd to the 414th bit base.

Contain expression carrier of the present invention, clone and host bacterium and all belong to protection scope of the present invention.

Arbitrary segmental primer is to also within protection scope of the present invention among the amplification GmDREB3.

Northern blot experimental results show that the GmDREB3 gene is subjected to high salt abduction delivering, and be not subjected to arid, coldly coerce, the ABA abduction delivering; EMSA (gel retardation assasy) proves that GmDREB3 has binding specificity external with DRE; Yeast transcriptional activation system has proved interior binding specificity of the body of GmDREB3 and mobilizing function.GmDREB3 gene of the present invention will play an important role in cultivating resistance and resistance of reverse enhanced plant (particularly soybean) for the degeneration-resistant and anti-retrocorrelation expression of gene of artificial control provides the foundation.

Description of drawings

Figure 1A-Fig. 1 D is the situation that GmDREB3 is expressed by stress-inducing

Fig. 2 is the EMSA of GmDREB3

Embodiment

The clone of embodiment 1, GmDREB3 gene

Place 200mM NaCl solution to handle 12 hours the root system of rich No. 8 of iron (anti-saline and alkaline soybean varieties) soybean seedlings of growth about 14 days, adopt the Trizol method to extract the total RNA of soybean, (GIBCOBRL CAT.NO.18373-019) operates the full length sequence that obtains the GmDREB3 gene according to the step of its specification sheets to utilize 3 ' RACE System for RapidAmplification of cDNA Ends Kit.Wherein, the special primer of 3 ' RACE is: GSP1:5 ' GGTCGCTGAAATCAGACT 3 '; GSP2:5 ' GACTCCCAAAGAACCGCACGCGCCTCTG 3 '.The GmDREB3 gene has the nucleotide sequence of SEQ ID No:2 in the sequence table, and coding has the protein of the amino acid residue sequence of sequence 1 in the sequence table.Sequence 1 be nuclear localization sequence from the 104th-105 amino acids residue of aminoterminal, the encoding sequence of this nuclear localization sequence be sequence 2 from 5 ' end the 310th bit base-315 bit base; Sequence 1 be the AP2/EREBP structural domain from the 120th-177 amino acids residue of aminoterminal, from the 133rd of aminoterminal (in the AP2/EREBP conserved domain the 14th) is Xie Ansuan, is leucine from the 138th of aminoterminal (in the AP2/EREBP conserved domain the 19th).The encoding sequence of this AP2/EREBP structural domain be sequence 2 from 5 ' end the 358th bit base-531 bit base.GmDREB3 is a DREB albumen.

The sequence of GmDREB3 gene is compared on Genabnk, does not find and the soybean dreb gene of GmDREB3 dna homolog, proves that the GmDREB3 gene is a new gene.

Embodiment 2, Northern bolt detect GmDREB3 expression of gene characteristic

1, the preparation of vegetable material:

Get the soybean seedling about 14 days, carry out following processing:

(1) arid is handled: soybean seedling is taken out the moisture that blots on the root from soil, place on the exsiccant filter paper, arid is cultivated after 0.5 hour, 1 hour, 3 hours, 6 hours and 24 hours and is taken out material, uses liquid nitrogen flash freezer, and-80 ℃ of preservations are standby.

(2) salt marsh is handled: the root system of soybean seedling is placed 200mM NaCl solution, and illumination cultivation is taken out material after 0.5 hour, 1 hour, 3 hours, 6 hours and 24 hours, uses liquid nitrogen flash freezer, and-80 ℃ of preservations are standby.

(3) damage to plants caused by sudden drop in temperature processing: wheat seedling is placed 4 ℃ of incubators, and illumination cultivation takes out and uses liquid nitrogen flash freezer respectively after 0.5 hour, 1 hour, 3 hours, 17 hours and 72 hours, and-80 ℃ of preservations are standby.

(4) ABA handles: spray to soybean seedling in the ABA solution of 200 μ M, illumination cultivation takes out and uses liquid nitrogen flash freezer respectively after 0.5 hour, 1 hour, 3 hours, 17 hours and 72 hours, and-80 ℃ of preservations are standby.

(5) Dui Zhao processing: the soybean seedling of directly getting without any processing-80 is ℃ in contrast frozen.

Adopt the Trizol method to extract the total RNA of soybean of above-mentioned vegetable material respectively.

2, change film

(1) processing of electrophoresis and commentaries on classics film apparatus: 0.1N NaOH/1mM EDTA soaks 1h, and is totally standby with flushing with clean water;

(2) prepare the denaturing formaldehyde gel: in an Erlenmeyer flask, add 10ml 10 * MOPs, water and 1g agarose that 73ml DEPC handles, heating and melting; Add 17ml formaldehyde when being cooled to 55 ℃ of left and right sides, mixing is poured in the glue groove of RNA special use;

(3) in the 0.5ml centrifuge tube that DEPC handles, add:

RNA+DEPC treating water 4 μ l (the total RNA of soybean is about 30 μ g)

Methane amide 12.5 μ l

Formaldehyde 4.0 μ l

10×MOPs 2.5μl

Bromjophenol blue (10 *) 2.5 μ l

(4) sample is at 65 ℃ of water bath processing 5min, and point sample begins electrophoresis then, and damping fluid is 1 * MOPs.When bromjophenol blue arrives gel 1/3-1/2 position, stop electrophoresis;

(5) use the distilled water flushing gel, change film with 20 * SSC.To be placed on the filter paper bridge after the gel upset, put one and the sizable nylon membrane (Hybond-N of gel ⁺, Amarsharm), catch up with except that bubble, on film, put 3 metafiltration paper, identical with the nylon membrane size, catch up with except that bubble, seal with film around the gel, put sizable thieving paper, press the weight of about 500g, change film 24h, change thieving paper therebetween.After the commentaries on classics film is finished, wash film 10min with 2 * SSC, blot with filter paper, preservative film is wrapped, and ultra violet lamp 3min deposits to hybridization for 4 ℃;

3, the preparation of probe

At 3 ' design special primer GmDREB3F and GmDREB3R (avoiding the AP2/EREBP zone) of GmDREB3 gene, be that template is carried out pcr amplification with the GmDREB3 gene of cloning, program and system are as follows:

Primer sequence:

GmDREB3F：5’CATCTAAGACACCACGGA?3’

GmDREB3R：5’CCAATCAATCTCCACAGA?3’

Reaction system (50 μ l):

Template (60ng/ul) 0.5 μ l

DNTP (every kind of 10mM) 1 μ l

Primer (each 25 μ M) 1 μ l

10 * damping fluid, 5 μ l

ddh ₂O 42.1μl

Taq(5U/μl) 0.4μl

Wherein, 10 * damping fluid: come from TaKaRa Taq test kit (TaKaRa company, Code No.:DR100A).Amplification condition (PTC-200): 94 ℃ of 2min of elder generation; Carry out 30 circulations by following condition then: 94 ℃ of 30sec, 58 ℃ of 45sec, 72 ℃ of 45sec; Last 72 ℃ of 5min.

Get amplified production 2ul electrophoresis detection in 1.2% sepharose, bromination second pyridine dyeing, the scanning of ultraviolet gel imaging instrument, there is a bright band position about 600bp.

4, the recovery of probe

Adopt Agarose Gel DNA Purification Kit Ver.2.0 (TaKaRa company, Code No.:DV807A) to reclaim the purifying probe.

5, probe mark

Probe mark is a random hexamer primer method, every 1-2 opens about 50-100ng of dna probe and the dna molecular amount mark that film adds step 4, add water to 14.5 μ l, 100 ℃ place ice bath 5min after boiling sex change 5min immediately, instantaneous centrifugal, add following ingredients then, final volume 25 μ l are more than 37 ℃ of reaction 5h.

5 * Oligo damping fluid, 5 μ l

Klenow?Fragment(5U/μl) 0.7μl

10 * damping fluid 2.5ul

BSA(10mg/ml) 1μl

α- ³²P-dCTP(10mCi/ml) 1.5μl

Attached: as to contain in 1ml 5 * Oligo damping fluid:

160μl H ₂O

250μl 1M?Tris-HCl

25μl 1M?MgCl ₂

5 μ l 1M mercaptoethanols

500μl 2M?HEPES

20μl 100mM?dATP

20μl 100mM?dGTP

20μl 100mM?dTTP

10 * damping fluid: come from Random Primer DNA Labeling Kit Ver 2.0 (TaKaRa company, Code No.:D6045)

6, prehybridization and hybridization

According to the quantity preparation prehybridization solution of the film that will hybridize, the composition of every 10ml prehybridization solution is composed as follows.

ddH ₂O 6.5ml

5×HSB 2ml

DenHardt’sIII 1ml

*ssDNA(10mg/ml) 0.5ml

* ssDNA needs sex change before salmon sperm dna (Sigma) adds: put 10min on ice immediately after boiling 10min.

Attached: 5 * HSB (pH6.8)Molecular weight (MW) g/200ml

NaCl(3M) 58.44 35.064

PIPES(0.1M) 302.4 6.048

EDTA(20mM) 372.24 1.488

Denhardt’sIII g/100ml

2％Gelatin 2

2％Ficoll-400 2

2％PVP-360 2

10％SDS 10

5％Na ₄P ₂O·10H ₂O 5

With the prehybridization solution mixing, put into nylon membrane after being preheated to clarification, 65 ℃ of prehybridization 5-6h (new film) in 65 ℃; After probe mark is intact, add isopyknic 0.4N NaOH solution, leave standstill 10min in room temperature behind the mixing and make the probe sex change, join then in the prehybridization solution, 65 ℃ of hybridization are spent the night;

7, wash-out

The film of having hybridized taken out from hybrid pipe put into the wash-out box, (excessive hybridization solution is removed in 2 * SSC/0.1%SDS) rinsings, washes twice, each 20min with washing lotion I in 45 ℃ then to add a small amount of washing lotion I; (0.2 * SSC/0.2%SDS) washes twice, each 15min in 45 ℃ to use washing lotion II again.After washing lotion I washes, detect hybridization signal and background, whether decision continues wash-out again.Blot washed film with filter paper, preservative film is wrapped (bubble is removed in attention);

8, radioautograph

In X-radiography magazine, Hybond membrane is placed on the intensifying screen, in the darkroom, the X-mating plate is placed on the Hybond membrane, put another intensifying screen again, compress X-radiography magazine ,-70 ℃ of refrigerators exposed 7-15 days.

Northern blot analytical results shows that GmDREB3 genetic expression is unaffected under drought condition shown in Figure 1A-Fig. 1 D; The abduction delivering that coerced by high salt (200mM NaCl); Do not catch a cold coerce, ABA (200uM) stress-inducing GmDREB3 genetic expression.Illustrate that this gene is induced by high salt, be not subjected to arid, coldly coerce, ABA induces.

Embodiment 3, EMSA detect the external binding specificity of GmDREB3

1, GmDREB3 Prokaryotic Expression

The full length sequence of GmDREB3 gene is inserted among the multiple clone site EcoRI and XhoI of prokaryotic expression carrier pGEX-4T-1 (available from AmershamPharmacia Biotech company), because there is GST (Triptide) gene the multiple clone site front of pGEX-4T-1, GmDREB3 and GST albumen have just formed fusion rotein like this, with the expression vector transformation receptor e. coli bl21 that builds, cultivate after 6 hours for 37 ℃, in substratum, add IPTG to final concentration be 0.5mM abduction delivering 12h, collect thalline, behind the ultrasonic degradation in 4 ℃, centrifugal 20 minutes of 12000rpm, get supernatant and carry out the SDS-PAGE electrophoresis, the result detects the band of 64.43KD, shows that fusion rotein (GmDREB3-GST) expresses.

Use the step purifying GmDREB3-GST fusion rotein of MicroSpin GST Purification Module Kit (Amersham, product code:27-4570-03) by specification.

2, the mark of DRE probe

According to the GmDREB3 gene order, synthetic following DRE probe sequence: (base of band underscore is the core base of DRE in the following probe sequence)

DRE1： 5’＞ATT?TCA?TGG? CCG? ACC?TGC?TTT?CAT?GG C? CGA? CCT?GCT?T＜3’

DRE2： 5’＞AAG?CAG? GTC? GGC?CAT?GAA?AGC?AG G? TCG? GCC?ATG?AAA?T＜3’

DREm1(1)：5’＞ATT?TCA?TGG? AAG? ACC?TGC?TTT?CAT?GG A? AGA? CCT?GCT?T＜3’

DREm1(2)：5’＞AAG?CAG? GTC? TTC?CAT?GAA?AGC?AG G? TCT? TCC?ATG?AAA?T＜3’

DREm2(1)：5’＞ATT?TCA?TGG? CCT? CAC?TGC?TTT?CAT?GG C? CTC? ACT?GCT?T＜3’

DREm2(2)：5’＞AAG?CAG? TGA? GGC?CAT?GAA?AGC?AG T? GAG? GCC?ATG?AAA?T＜3’

DREm3(1)：5’＞ATT?TCA?TGG? TTT? TTC?TGC?TTT?CA T?GG T? TTT? TCT?GCT?T＜3’

DREm3(2)：5’＞AAG?CAG? AAA? AAC?CAT?GAA?AGC?AG A? AAA? ACC?ATG?AAA?T＜3’

Wherein, DRE1 and DRE2 are the DRE probe of wild-type, and DREm1, DREm2, DREm3 are the DRE probe of various sudden changes.

Use the T4 polynueleotide kinase (T4-Polynucleotide Kinase, T4-PNK) to the DRE probe carry out (γ- ³²P) dATP mark, the mark system is as follows:

H ₂O 18.6μL

10 * T4-PNK damping fluid (Promega company), 2.5 μ L

DRE probe (concentration is 1ug/ul) 1 μ L

T4-PNK (Promega company) (5U/ μ L) 1 μ L

(γ- ³²P) dATP (concentration is 10u Ci/ul) 2 μ L

37 ℃ of marks 30 minutes add 1 μ L 0.5M EDTA termination reaction, 65 ℃ of 10 minutes inactivators, and 4 ℃ are standby.

3, the GmDREB3-GST fusion rotein of the DRE probe of mark and purifying carries out association reaction

With GST albumen is contrast.

Combination anchor is as follows:

5 * binding buffer liquid 4uL

GmDREB3-GST fusion rotein of purifying (concentration is 0.5mg/ml) or GST albumen (concentration is 0.5mg/ml) 10uL

A kind of DRE probe 2uL of mark

50% glycerine 2uL

H ₂O 2uL

Wherein, 5 * binding buffer liquid composition is as follows:

12.5mM?HEPES-KOH(pH7.6)

250mM KCl

2.5mM DTT

2.5mM EDTA。

Association reaction carried out on ice 30 minutes, the SDS-PAGE glue 200V for preparing in the time of reaction carries out 1 hour prerunning, add 1 μ L sample-loading buffer (0.025% bromjophenol blue) behind the association reaction and carry out the SDS-PAGE electrophoresis, the 170V electrophoresis unloaded electrophoresis plate after 1-2 hour, glue is put in the water, the water on the glue is being blotted the binding characteristic that the DRE probe of X-ray sheet detection GmDREB3-GST fusion rotein and mark is pressed in the back.The result as shown in Figure 2, show wild-type DRE probe can with the GmDREB3-GST fusion rotein combination of purifying, and mutant DRE probe all can not with the combination of GmDREB3-GST fusion rotein, the result proves that the GmDREB3-GST fusion rotein and the DRE of purifying have binding specificity, and GmDREB is a DREB class transcription factor.Among Fig. 2, ml is DREm1 (1) and DREm1 (2) mutant probe, and m2 is DREm2 (1) and DREm2 (2) mutant probe, and m3 is DREm3 (1) and DREm3 (2) mutant probe, pro is wild-type DRE1 and DRE2 probe, and prob is not for adding proteic free probe (DRE1 and DRE2).

Binding specificity and activation characteristic in the body of embodiment 4, GmDREB3

One, GmDREB3 is gene constructed to expression vector YEP-GAP

1, obtains the complete sequence of GmDREB3 gene coding amino acid part

Sequences Design primer according to the GmDREB3 gene of having cloned, the primer end adds EcoRI and XhoI restriction enzyme site respectively, rich No. 8 genomic dna of handling with salt among the embodiment 1 of iron is a template, and pcr amplification obtains the complete sequence of coded amino acid part, and program and system are as follows:

Primer sequence:

GmDREB3FF：5’TATCCCGGGACCCCAGACCAAATTGTTCAG?3’

GmDREB3FR：5’GCAGAGCTCGCGGCCGCTTGCAGAAGCAGCCAAAGACA?3’

Reaction system (50 μ l):

Template (60ng/ul) 0.5 μ l

DNTP (every kind of 10mM) 1 μ l

Primer (every kind 25 μ M) 1 μ l

10 * damping fluid, 5 μ l

ddh ₂O 42.1μl

Taq(5U/μl) 0.4μl

Wherein, 10 * damping fluid: come from TaKaRa Taq test kit (TaKaRa company, Code No.:DR100A).

Amplification condition (PTC-200): 94 ℃ of 2min of elder generation; Carry out 30 circulations by following condition then: 94 ℃ of 30sec, 58 ℃ of 45sec, 72 ℃ of 70sec; Last 72 ℃ of 5min.

Get amplified production 2ul electrophoresis detection in 1.2% sepharose, bromination second pyridine dyeing, the scanning of ultraviolet gel imaging instrument, there is a bright band position of result about 939bp.

Adopt Agarose Gel DNA Purification Kit Ver.2.0 (TaKaRa company, Code No.:DV807A) to reclaim purified pcr product.

2, GmDREB3 is gene constructed to expression vector YEP-GAP

PCR product and expression vector YEP-GAP (Liu Q, Kasuga M, Sakuma Y with purifying in the step 1, Abe H, Miura S, Yamaguchi-Shinozaki K, Shinozaki K., 1998), with EcoRI (Takara) and XhoI (Takara) respectively enzyme cut 4-6hr.Adopt Agarose Gel DNA Purification KitVer.2.0 (TaKaRa company, Code No.:DV807A) to reclaim purifying enzyme and cut product.The enzyme of purifying is cut the PCR product cut carrier YEP-GAP with enzyme and be connected 4-8hr, get 0.5 μ l and connect product, electric shock transforms the JM109 bacterial strain, 37 ℃ of incubated overnight, the picking positive colony, whether the sequencing analysis sequence is correct, obtains containing the recombinant expression vector YEP-GAP-GmDREB3 of GmDREB3 gene.

Two, the checking of binding specificity and activation characteristic in the body of GmDREB3

1, the structure of yeast reporter

Fragment 5 '-GAATTC-DRE-DRE-DRE-DRE-GTCGAC-3 ' (core sequence of DRE: TACCGACAT) be building up to the Pmin of pHis-1 carrier (MATCHMAKER One-Hybrid System, Clontech company) respectively that will contain 4 DRE elements _HIS3Promotor and pLacZi carrier (MATCHMAKER One-Hybrid System, Clontech company) P _CYCIThe promotor upstream obtains recombinant vectors pHis-1-DRE and pLacZi-DRE respectively, respectively pHis-1-DRE and pLacZi-DRE carrier is cut into wire with Xho I and Nco I restriction endonuclease.Earlier wire pHis-1-DRE carrier is transformed in the yeast cell (YM4271 strain system, MATCHMAKER One-Hybrid System, Clontech company), acquisition can be at SD/His ^-The yeast transformant of normal growth on the substratum (Yeast transformant).Be host cell then, continue to transform the pLacZi-DRE carriers that contain 4 repetition DRE elements with this yeast transformant.The SD/His that lacks Histidine and uridylic so at the same time ^-/ Ura ^-On the substratum, select to obtain to contain the normal dual yeast reporter of pHis-1-DRE and pLacZi-DRE; The core sequence CCGAC of 4 DRE elements is mutated into TTTTT (MDRE), i.e. 5 '-GAATTC-MDRE-MDRE-MDRE-MDRE-GTCGAC-3 ', by normal dual yeast reporter construction process, make up a dual yeast reporter of mutant that contains 4 MDRE boxes again.

2, PEG/LiAc method transformed yeast and interpretation of result

(1) inoculation yeast bacterial strain (YM4271 strain system, MATCHMAKER One-Hybrid System, Clontech company) in 1ml YPD liquid nutrient medium, concuss 2 minutes, disperse behind the agglomerate suspension to be gone in the triangular flask that contains 50ml YPD liquid nutrient medium, 30 ℃/250rpm shakes and spends the night, and surveys OD600=1.7-1.8 and (counts about 4 * 10 ⁷Individual/mL);

(2) get 30ml step (1) overnight culture and receive in the fresh YPD substratum of 300ml, 30 ℃/250rpm cultivates, and about 3 hours to OD600=0.5 ± 0.1, the centrifugal 5min of room temperature 1000g collects thalline, abandons supernatant, suspend with 1/2 volume, 1 * TE, 1000g/5min is centrifugal;

(3) supernatant is abandoned in suction, suspends with the freshly prepared 1 * TE/LiAc solution of 1.5ml, and the vibration mixing is standby;

(4) taking out 0.1ml yeast competence transforms, add following solution successively: 0.1 μ g expression vector YEP-GAP-GmDREB3,0.1mg ssDNA (salmon sperm dna, Sigma), 0.6mlPEG/LiAc vibrated 30 ℃/200rpm shaking culture 30 minutes at a high speed 1 minute;

(5) add 70ul DMSO (sigma#D8779), be inverted mixing gently, 42 ℃ of heat shocks 30 minutes, vibration gently therebetween, ice bath 2 minutes, the centrifugal 5min of room temperature 1000g;

(6) supernatant is abandoned in suction, adds 0.5ml 1 * TE buffer suspension cell;

(7) dip in transfering loop and get suspension, containing 0 respectively, the SD/His of 15mmol/L 3-AT ^-/ Ura ^-/ Trp ^-Setting-out is cultivated on the selective medium.

(8) the normal dual yeast reporter of half dull and stereotyped culturing step 1 structure, the dual yeast reporter of mutant that second half culturing step 1 makes up is so that do check analysis.

(9) be placed upside down in incubator, cultivated 3-4 days for 30 ℃.Found that SD/His at 0mmol/L 3-AT ^-/ Ura ^-/ Trp ^-Culture medium flat plate on the yeast reporter of normal yeast reporter and sudden change growth is all arranged, but the diameter of the yeast reporter of sudden change is obviously little; And at the SD/His of 15mmol/L 3-AT ^-/ Ura ^-/ Trp ^-Culture medium flat plate on normal yeast reporter can normal growth, but the yeast reporter of sudden change is not restrained not growth.

3, galactosidase activity detects

(1) from the SD/His of 0mmol/L 3-AT ^-/ Ura ^-/ Trp ^-Culture medium flat plate on the yeast reporter bacterium colony of the normal yeast reporter of picking and sudden change respectively.Go in the YPD liquid nutrient medium,, wait to grow to the logarithmic growth later stage, get 1.5ml bacterium liquid, the centrifugal 30s of 3000rpm in 30 ℃ of shaking culture;

(2) abandon supernatant, liquid in the control main places liquid nitrogen quick-frozen 10min with centrifuge tube, taking-up is melted it naturally, adds 50ul Z/X-gal solution, 30 ℃ of incubations, found that normal yeast reporter becomes blue in 6-8h, and the yeast reporter of sudden change changes not in 12h, still is white.Illustrate that transcription factor GmDREB3 can combine with the DRE cis-acting elements, and have mobilizing function, activated the Pmin promotor and (comprised Pmin _HIS3Promotor and P _CYCIPromotor), impel reporter gene to express.Thereby interior binding specificity of the body that has proved GmDREB3 and mobilizing function.

Three, medicament preparation:

(1) YPD liquid nutrient medium

Microbial culture yeast extract (Bacto-Yeast Extract) 10g/L

Microbial culture tryptone (Bacto-Peptone) 20g/L

Regulate pH to 5.8,60 ℃ of glucose of adding 40% are later on reduced in 121 ℃/15min sterilization, and making its final concentration is 20g/L.

(2) SD/His ^-/ Ura ^-/ Trp ^-Selective medium

Do not contain amino acid whose yeast nitrogen (Yeast nitrogen base) 6.7g/L

Auxotroph mixture (drop-out media without His/Ura/Trp) 100ml

Agar powder (Bacteriological agar) 20g/L

Regulate pH to 5.8,121 ℃/15min sterilization adds/goes into 40%Glucose after reducing to 60 ℃, and making its final concentration is 20g/L.

(3) auxotroph mixture (Drop-out mix): (10X)

L-Isoleucine (Isoleucine) 300mg/L

L-Valine (Xie Ansuan) 1500mg/L

L-Adenine (VITAMIN B4) 200mg/L

L-Arginine (arginine) 200mg/L

L-Histidine Hcl monohydrate (Histidine) 200mg/L

L-Leucine (leucine) 1000mg/L

L-Lysine Hcl (Methionin) 300mg/L

L-Methionine (methionine(Met)) 200mg/L

L-Phenylalanine (phenylalanine) 500mg/L

L-Threonine (Threonine) 2000mg/L

L-Tyrosine (tyrosine) 300mg/L

(4)1×PEG/LiAc：

50％(w/v)PEG3350 8ml

10×TE?buffer 1ml

10×LiAc 1ml

(5)10×TE?Buffer：

100mM?Tris-Hcl

10mM?EDTA，pH＝7.5

121 ℃ of autoclavings, room temperature preservation.

(6)1×TE/LiAc：

10×TE?buffer 1ml

10×LiAc 1ml

ddH ₂O 8ml

(7)Z?Buffer：

Na ₂HPO ₄·7H ₂O 16.1g/L

NaH ₂PO ₄·H ₂O 5.5g/L

KCl 0.75g/L

MgSO ₄·7H ₂O 0.246g/L

Regulate pH to 7.0,121 ℃/15min sterilization, 4 ℃ of preservations.

(8) X-gal storage liquid (X-gal Stock Solution):

Use N, N-dimethyl-formamide (DMF) dissolves X-gal, and making its final concentration is 20mg/ml ,-20 ℃ of storages.

(9) contain the Z buffer damping fluid 100ml (Z buffer with X-gal) of X-gal, note matching while using:

Z?buffer 98ml

Beta-mercaptoethanol (the 0.27ml of β-mercaptoethanol)

X-gal storage liquid (X-gal stock solution) 1.67ml

Sequence table

<210>1

<211>312

<212>PRT

＜213〉Glycine soybean (Glycine max (L.))

<400>1

Met?Gly?Thr?Ala?Ile?Asp?Met?Tyr?Asn?Ser?Ser?Ash?Ile?Val?Ala?Asp

1 5 10 15

Phe?Leu?Asp?Pro?Tyr?Ser?Glu?Glu?Leu?Met?Lys?Ala?Leu?Lys?Pro?Phe

20 25 30

Met?Lys?Ser?Asp?Tyr?Phe?Ser?Ala?Ser?Ser?Ser?Ser?Ser?Leu?Glu?Ser

35 40 45

Gln?Pro?Cys?Ser?Phe?Ser?Ser?Asn?Ser?Leu?Pro?Thr?Ser?Tyr?Pro?Ser

50 55 60

Ser?Asn?Gln?Ile?Lys?Leu?Asn?Gln?Leu?Thr?Pro?Asp?Gln?Ile?Val?Gln

65 70 75 80

Ile?Gln?Ala?Gln?Ile?His?Ile?Gln?Gln?Gln?Gln?Gln?His?Val?Thr?Gln

85 90 95

Thr?Gln?Thr?His?Leu?Gly?Pro?Lys?Arg?Val?Pro?Met?Lys?His?Ala?Gly

100 105 110

Thr?Ala?Ala?Lys?Pro?Thr?Lys?Leu?Tyr?Arg?Gly?Val?Arg?Gln?Arg?His

115 120 125

Trp?Gly?Lys?Trp?Val?Ala?Glu?Ile?Arg?Leu?Pro?Lys?Asn?Arg?Thr?Arg

130 135 140

Leu?Trp?Leu?Gly?Thr?Phe?Asp?Thr?Ala?Glu?Glu?Ala?Ala?Leu?Ala?Tyr

145 150 155 160

Asp?Asn?Ala?Ala?Phe?Lys?Leu?Arg?Gly?Glu?Phe?Ala?Arg?Leu?Asn?Phe

165 170 175

Pro?His?Leu?Arg?His?His?Gly?Ala?Phe?Val?Phe?Gly?Glu?Phe?Gly?Asp

180 185 190

Tyr?Lys?Pro?Leu?Pro?Ser?Ser?Val?Asp?Ser?Lys?Leu?Gln?Ala?Ile?Cys

195 200 205

Glu?Ser?Leu?Ala?Lys?Gln?Glu?Glu?Lys?Pro?Cys?Cys?Ser?Val?Glu?Asp

210 215 220

Val?Lys?Pro?Val?Ile?His?Ala?Ala?Glu?Leu?Ala?Glu?Val?Glu?Ser?Asp

225 230 235 240

Val?Ala?Lys?Ser?Asn?Ala?Glu?Tyr?Val?Tyr?Pro?Glu?Phe?Glu?Asp?Phe

245 250 255

Lys?Val?Glu?His?Glu?Asn?Pro?Met?Phe?Ser?Gly?Glu?Ser?Ser?Ser?Pro

260 265 270

Glu?Ser?Ser?Val?Thr?Phe?Leu?Asp?Phe?Ser?Asp?Phe?Ser?Asp?Ser?Asn

275 280 285

Asn?Gln?Trp?Asp?Glu?Met?Glu?Asn?Phe?Gly?Leu?Glu?Lys?Phe?Pro?Ser

290 295 300

Val?Glu?Ile?Asp?Trp?Glu?Ala?Ile

305 310

<210>2

<211>939

<212>DNA

＜213〉Glycine soybean (Glycine max (L.))

<400>2

atgggaactg?ctatagacat?gtacaacagc?agcaacatcg?tagcggattt?cctagatccg 60

tatagtgaag?agctgatgaa?agcacttaag?ccttttatga?aaagtgatta?tttctctgcc 120

tcttcttctt?cttcactcga?atcacagcct?tgttcttttt?catctaattc?tctccccact 180

tcgtatccct?cttccaacca?aatcaagctc?aaccaactca?ccccagacca?aattgttcag 240

attcaggccc?aaatccacat?tcagcagcag?cagcagcacg?tgacccaaac?ccaaacccac 300

ctgggcccaa?aacgcgtccc?catgaagcac?gctggcacgg?ccgcgaaacc?cacgaagctc 360

taccgcgggg?tgcggcaacg?gcattggggc?aagtgggtcg?ctgaaatcag?actcccaaag 420

aaccgcacgc?gcctctggct?aggaacattc?gacaccgcag?aggaagcagc?attagcgtac 480

gacaacgcag?cgtttaagct?cagaggcgag?ttcgcgcgtc?tcaattttcc?tcatctaaga 540

caccacggag?ccttcgtttt?cggcgagttc?ggagattaca?agcctctacc?ttcttccgtg 600

gattccaaac?tgcaagctat?ttgcgaaagc?ttagcgaaac?aagaggaaaa?gccgtgttgc 660

tccgtcgaag?acgtgaagcc?cgtgatacac?gctgctgagc?tggcagaggt?cgagtctgac 720

gtggcaaaat?cgaacgctga?atatgtttat?cccgagttcg?aggattttaa?ggtcgagcac 780

gagaacccaa?tgttttctgg?tgaatcttct?tcgcctgaat?ccagtgttac?tttcttggat 840

ttctcggact?tctcggattc?taataatcag?tgggatgaaa?tggagaattt?tgggttggag 900

aagttccctt?ctgtggagat?tgattgggaa?gctatatga 939

Claims (9)

1. binding protein of response component of plant dehydration, its amino acid residue sequence is shown in SEQ ID NO:1.

2. protein according to claim 1 is characterized in that: SEQ ID № in the described sequence table: 1 be the AP2/EREBP structural domain from aminoterminal 120-177 position.

3. the gene of coding claim 1 or 2 described binding protein of response component of plant dehydration.

4. gene according to claim 3 is characterized in that: the nucleotide sequence of described gene is selected from down one of group:

1) dna sequence dna shown in SEQID NO:2 in the sequence table;

2) in the code sequence tabulation shown in SEQ ID NO:1 the polynucleotide of protein sequence.

5. the plant expression vector that contains claim 3 or 4 described binding protein of response component of plant dehydration genes.

6. the clone that contains claim 3 or 4 described binding protein of response component of plant dehydration genes.

7. the host bacterium that contains claim 3 or 4 described binding protein of response component of plant dehydration genes.

8. claim 1 or the 2 described binding protein of response component of plant dehydration application in the plant of cultivating the salt resistance raising.

9. claim 3 or the 4 described binding protein of response component of plant dehydration genes application in the plant of cultivating the salt resistance raising.

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