CN102336826A - Transcription factor ERF related to soybean stress, coding gene thereof and application of coding gene - Google Patents

Abstract

The invention relates to a transcription factor ERF related to soybean stress, a coding gene thereof, and application of the coding gene, and belongs to the field of plant gene engineering. The amino acid sequence of the transcription factor ERF related to soybean stress is shown as a Sequence No.2, and the nucleotide sequence of the coding gene of the transcription factor is shown as a Sequence No.1. A soybean GmERF6 transcription factor gene related to stress induction is cloned, the expression mode and transcription regulator activity of the gene are researched, the drought resistance of wild arabidopsis is compared with that of a GmERF6 gene-transformed arabidopsis, a basis is provided for effectively applying the GmERF6 gene, and the GmERF6 gene has important significance for improving the stress resistance of plants, particularly breeding drought-resistant soybean varieties.

Description

A kind of soybean adverse circumstance associated transcription factor ERF and encoding sox and application

Technical field

The present invention relates to plant genetic engineering field, be specifically related to a kind of soybean adverse circumstance associated transcription factor ERF and encoding sox and application.

Background technology

Soybean is important cash crop, worldwide generally plants, and is not only the main source of human protein and lipid, also is important cattle food and industrial raw material simultaneously, and very big value is medically also being arranged.Yet biology and abiotic stress comprise that disease, salt damage, low temperature and arid etc. all produce totally unfavorable influence to the g and D of soybean, thereby cause soybean yields to reduce, and cause serious economy loss.Therefore, illustrate the response mechanism of soybean, identify that important gene that adverse circumstance is relevant and the resistance of utilizing them to improve soybean seem particularly important adverse circumstance.

Plant can conform through expression or synthetic a large amount of relevant stress protein of series of genes in the control agent.Wherein, transcription factor plays a significant role in the response coercing of plant owing to can regulate a plurality of expression of gene, becomes people's research focus gradually.The ERF transcription factor belongs to the ERF subfamily in the distinctive AP2/ERF transcription factor extended familys in the plant, from tobacco, separates obtaining at first.Their expressed proteins contain 1 conservative ERF that is made up of 58 or 59 amino acid jointly and combine the territory; This combines the territory to be made up of 3 βZhe Dies and 1 α spiral; Combination GCC that can be special and DRE/CRT element, and in the promotor of the pathogenesis-related proteins gene that these elements lay respectively at plant usually and arid, cold induction phase correlation gene.Thereby ERF just through regulate biology that these expression of gene participate in plant coerce with abiotic stress in go.The adverse circumstance response of plant is regulated by many barss approach; Wherein, Environment stress semiochemicalses such as ethene, Whitfield's ointment, jasmonic, dormin can be regulated the expression of ERF usually, and these four signaling molecules have been participated in different defence Expression of Related Genes through the ERF transcription factor.It is conjugated protein that tomato JERF3 is GCC box, can discern the DRE element again, in tomato, can be induced by ethene, jasmonic, low temperature, high salt and dormin, can improve the salt tolerance of transgene tobacco simultaneously.The Tsil of tobacco not only expresses in the acid-treated blade of ethene, NaCl, ABA and bigcatkin willow, can also be by arid and water logging abduction delivering.The overexpression of Tsil has improved the resistance of plant to high salt and cause of disease.The expression of JcERF in the wood peanut can be induced by high salt, arid, ethene and mechanical wounding, but is not induced by ABA, and overexpression JcERF has strengthened plant to salt and cold resistance in Arabidopis thaliana.

At present; From different plants such as Arabidopis thaliana, tobacco, tomato, capsicum, paddy rice, wheat, cotton, be cloned into a large amount of ERF genes; But in soybean, only reported the function of 3 ERF; They all participate in coercing in the response of soybean and go, the positive regulating and controlling effect of performance aspect the raising stress resistance of plant.All possibly there is a large amount of ERF transcription factors in monocotyledons and the dicotyledons; In view of ERF tackles the vital role of bringing into play in the adverse circumstance plant; Therefore, doing further to other member in the soybean ERF family, evaluation helps the improvement that soybean is coerced anti-(anti-) property with application.

Summary of the invention

The present invention provides a kind of soybean adverse circumstance associated transcription factor ERF and encoding sox and application.

The relevant ERF transcription factor of soybean adverse circumstance provided by the present invention derives from soybean varieties: Jilin 32, and by the Fu Jian researcher of academy of agricultural sciences, Jilin Province present, address: No. 1363 Jilin Academy of Agricultural Science, color space street, Changchun, Jilin Province; Called after GmERF6.

The aminoacid sequence of soybean adverse circumstance associated transcription factor ERF is Sequence NO.2.

The nucleotides sequence of the encoding sox of said soybean adverse circumstance associated transcription factor ERF is classified Sequence NO.1 as.

The application of described soybean adverse circumstance associated transcription factor ERF in the drought resistance that improves plant.

Sequence Sequence NO.1 is made up of 193 amino-acid residues 582 based compositions, Sequence NO.2, contains an ERF structural domain.。

Utilize any carrier that can guide foreign gene in plant, to express, the encoding sox importing vegetable cell with GmERF6 provided by the present invention can obtain the transfer-gen plant that arid resistance is improved.When using gene constructed plant expression vector of the present invention, can before its transcription initiation Nucleotide, can add any enhancing promotor or inducible promoter.Carry GmERF6 of the present invention expression vector can Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led, conventional biological method transformed plant cells or tissue such as agriculture bacillus mediated through using, and the plant transformed tissue cultivating is become plant.By host transformed both can be monocotyledons, also can be dicotyledons.

The invention has the beneficial effects as follows: the present invention has cloned an adverse circumstance and has induced relevant soybean GmERF6 transcription factor gene; Study expression pattern and the transcripting regulating activity of this gene in soybean; Wild-type and the drought resistance of changeing GmERF6 gene Arabidopis thaliana have been compared; For its effective application provides foundation, to improving the resistance of plant, it is significant particularly to cultivate the drought resisting soybean varieties.

Description of drawings

Fig. 1 is the pcr amplification figure as a result of GmERF6 gene of the present invention;

Fig. 2 is GmERF6 Recombinant Protein Expression figure of the present invention;

Fig. 3 is a GmERF6 gene adverse circumstance abduction delivering mode chart of the present invention;

Fig. 4 is the GmERF6 expression pattern figure of gene organization of the present invention;

Fig. 5 is reporter plasmid of the present invention, effect plasmid vector structural representation;

Fig. 6 is that activation analysis figure is regulated in GmERF6 genetic transcription of the present invention;

Fig. 7 be GmERF6 gene of the present invention at part T2 for the PCR qualification result figure in the transgenic arabidopsis;

Fig. 8 is that T3 of the present invention is for GmERF6 downstream gene semi-quantitative results figure in the transgenic arabidopsis;

Fig. 9 A is wild-type Arabidopis thaliana arid result figure of the present invention;

Fig. 9 B is that T3 of the present invention is for transgenic arabidopsis arid result figure;

Figure 10 is that wild-type Arabidopis thaliana of the present invention and T3 handle back percentage of water loss mensuration figure as a result for the transgenic arabidopsis arid.

Embodiment

The clone and the sequential analysis of embodiment 1:GmERF6 gene

The extraction of soybean RNA and cDNA's is synthetic: extract the total RNA of soybean Jilin 32 blades with RNAplant plus Reagent (available from TIANGEN), carry out reverse transcription with M-MLV reverse transcriptase (RNase H-) (available from TaKaRa), synthetic cDNA.

Primer design is with synthetic: carry out the Blast comparison among (20,30 and 50 days) immature embryo express spectra order-checking in 3 periods institute calling sequence (the big gene of China is accomplished) the input NCBI with soybean varieties Jilin 32, obtain one with plant in the higher unknown cDNA sequence of ERF transcription factor protein sequence homology.According to acquired unknown cDNA sequence, its nucleotide sequence such as Sequence NO.1 are said, design synthetic primer, F:5 '-CTTCCTACTCCTCCCTTTCAC-3 ', R:5 '-CGTAGTAGTGTTCCCAGATGC-3 '.With above-mentioned cDNA is template; Carry out pcr amplification according to following reaction system and condition: 25 μ L systems include 10 * PCR Buffer2.5 μ L, 2.5mM dNTP mix 2 μ L; Each 1 μ L of the primers F of 10 μ M and primer R; CDNA 1 μ L, EX Taq (available from TaKaRa) 0.3 μ L mends deionized water to 25 μ L.Reaction conditions: preparatory 94 ℃ of 8min of sex change; 94 ℃ of 40s, 60 ℃ of 40s, 72 ℃ of 1min, 30 circulations; 72 ℃ are extended 8min.Above-mentioned amplified fragments is reclaimed the back be connected with cloning vector pMD18-T (available from TaKaRa), send China big gene sequencing after the evaluation, the checking sequence is correct.

Obtain including the sequence of GmERF6 full length gene OFR through pcr amplification, the protein of being made up of 193 amino-acid residues of encoding comprises a conservative ERF and combines territory, two nuclear localization signals and an EAR straining element.

The expression of embodiment 2:GmERF6 gene in protokaryon

According to GmERF6 gene order design synthetic primer, and above that, introduce Sac I and Hind III restriction enzyme site respectively, F:5 '-GAGCTCATGGCCCCAAGAGACAGC-3 ' in the downstream primer; R:5 '-TTCGAATCAGGCAACCTCCGGGAG-3 '.With the pMD18-T-GmERF6 plasmid is template, carries out pcr amplification.With amplified production behind sepharose DNA purifying and recovering test kit (available from TIANGEN) purifying; With Sac I and Hind III (various restriction enzymes are all available from TaKaRa) double digestion; After reclaiming purifying, be connected with same pET28a (available from Novagen) carrier with Sac I and Hind III double digestion.Connect product and transform dust Xi Shi intestinal bacteria (E.coli) DH5 α (available from Biovector), after the empirical tests, change among the host bacterium Rosetta (DE3) (available from Novagen) and express.

The prokaryotic expression of recombinant protein: get respectively and positive be connected to 5mL with the empty carrier bacterial strain and add in the LB substratum (peptone 10g/L, yeast powder 5g/L, NaCl 10g/L) of 50mg/L kantlex and 15mg/L paraxin 37 ℃ of jolting overnight cultures.The ratio of getting in 1: 100 is transferred in the LB substratum of additional 50mg/L kantlex of 10mL and 15mg/L paraxin, and 37 ℃ of concussions are cultured to bacterium liquid OD ₆₀₀During for 0.4-0.6, adding final concentration is the IPTG of 0.3mmol/L, after 3h is cultivated in 37 ℃ of concussions, gets 1mL bacterium liquid, and the centrifugal 1min of 12000rpm collects thalline.

The SDS-PAGE of expression product analyzes: carry out according to SDS-PAGE electrophoresis ordinary method.

After IPTG induced, GmERF6 recombinant protein (the molecular weight size is about 30kDa) can be stablized expression (Fig. 2) effectively in Rosetta (DE3) bacterial strain.

The expression pattern of embodiment 3:GmERF6 gene under adverse circumstance is induced

The adverse circumstance in soybean Jilin 32 is handled: with Hoagland nutrient solution (Ca (NO ₃) ₂4H ₂O 0.62g/L, KNO ₃0.34g/L, KH ₂PO ₄0.06g/L, NH ₄NO ₃0.053g/L, MgSO ₄7H ₂O 0.493g/L, MgCl ₂0.67mg/L, H ₃BO ₃0.38mg/L, ZnSO ₄7H ₂O 0.29mg/L, CuSO ₄5H ₂O 0.015625mg/L, FeSO ₄7H ₂O0.02785g/L, EDTA-Na ₂0.0373g/L, PH 5.7-5.8), 28 ℃, 16h illumination/8h dark condition cultivation soybean water planting seedling, the water planting seedling of getting for four leaf phases carries out following processing:

High salt (NaCl) is handled: soybean seedling is placed the Hoagland nutrient solution that contains 200mM NaCl, and sampling respectively places liquid nitrogen rapidly behind processing 0h, 1h, 2h, 5h, 10h and the 24h, and-80 ℃ of preservations are subsequent use;

Arid (Drought) is handled: soybean seedling is placed the Hoagland nutrient solution that contains 20%PEG8000, and sampling respectively places liquid nitrogen rapidly behind processing 0h, 1h, 2h, 5h, 10h and the 24h, and-80 ℃ of preservations are subsequent use;

Low temperature (Cold) is handled: soybean seedling is placed 4 ℃ of incubators, and sampling respectively places liquid nitrogen rapidly behind processing 0h, 1h, 2h, 5h, 10h and the 24h, and-80 ℃ of preservations are subsequent use;

Mechanical wounding (Wound) processing: the scalper with sterilization causes number place wound to the soybean seedling blade, and sampling respectively places liquid nitrogen rapidly behind processing 0h, 1h, 2h, 5h, 10h and the 24h, and-80 ℃ of preservations are subsequent use;

Dormin (ABA) is handled: spray soybean seedling with the Hoagland nutrient solution that contains 200 μ M ABA, sampling respectively places liquid nitrogen rapidly behind processing 0h, 1h, 2h, 5h, 10h and the 24h, and-80 ℃ of preservations are subsequent use;

Ethene (ETH) is handled: soybean seedling is placed plastic tank with cover, is placed with the 500mL beaker in the bucket, in 200mL zero(ppm) water, 2mL 40% ethrel, 1gNaHCO are housed ₃, using rubber belt sealing, sampling respectively places liquid nitrogen rapidly behind processing 0h, 1h, 2h, 5h, 10h and the 24h, and-80 ℃ of preservations are subsequent use;

Whitfield's ointment (SA) is handled: spray soybean seedling with the Hoagland nutrient solution that contains 2mM SA, sampling respectively places liquid nitrogen rapidly behind processing 0h, 1h, 2h, 5h, 10h and the 24h, and-80 ℃ of preservations are subsequent use;

Methyl jasmonate (MeJA) is handled: spray soybean seedling with the Hoagland nutrient solution that contains 100 μ M MeJA, sampling respectively places liquid nitrogen rapidly behind processing 0h, 1h, 2h, 5h, 10h and the 24h, and-80 ℃ of preservations are subsequent use;

The extraction of total RNA and the compound method of cDNA are with embodiment 1.According to the cDNA sequences Design real-time fluorescence quantitative PCR primer of GmERF6 (F:5 '-CAACAACATTCGCAGTCCCA-3 '; R:5 '-AGTCGTTACGGCGGAAATC-3 ').With soybean constitutive expression gene β-Tubuin be internal reference (F:5 '-GGAAGGCTTTCTTGCATTGGTA-3 '; R:5 '-AGTGGCATCCTGGTACTGC-3 ').The cDNA that utilizes ABIPRISM7500 real-time quantitative PCR appearance to handle sampling spot with each adverse circumstance of soybean is that template is carried out the real-timeRT-PCR analysis.Reaction system contains 2 * SYBR Premix Ex Taq (available from TaKaRa), 10 μ L, ROX ReferenceDye II 0.14 μ L, cDNA 2 μ L, Primer F 0.4 μ L, Primer R 0.4 μ L, moisturizing to TV 20 μ L.Response procedures is 95 ℃ of 30s; 95 ℃ of 5s, 58 ℃ of 34s, 72 ℃ of 30s, 40 circulations.Adopt 2 ^{-Δ Δ CT}The method analytical data is confirmed the relative expression quantity of gene.Each sampling spot is established 3 technology and is repeated, and test is established 3 secondary pollutants altogether and learned repetition.

Adverse circumstance handle GmERF6 down because of relative expression quantity;

The result shows that all there is up-regulated expression trend in GmERF6 under handling in various adverse circumstances.

The expression pattern of embodiment 4:GmERF6 gene in soyabean tissue

Root, stem, leaf, flower and the total RNA and the reverse transcription of 20 days embryos of extracting soybean Jilin 32 respectively become cDNA, and method is with embodiment 1.CDNA with above-mentioned, stem, leaf, flower and 20 days embryos is that masterplate carries out real-time fluorescence quantitative PCR, and method is with embodiment 3.

GmERF6 is because of the relative expression quantity in the assorted soybean different tissues:

The result shows that GmERF6 is the highest because of expression amount in the root of soybean, and expression amount is followed successively by: root＞20 day embryo＞leaf＞flower＞stem.

Activation analysis is regulated in embodiment 5:GmERF6 genetic transcription

The construction process of plant expression vector is identical with embodiment 2 methods.CaMV35S promotor with AtPDF1.2 promotor (comprising GCC-box) replacement pCAMBIA1301 (available from CAMBIA) makes up the reporter plasmid expression vector; Gus gene with GmERF6 and GmERF7 (another kind of ERF transcription factor in the soybean) replacement pCAMBIA1301 makes up the effect plasmid expression vector, and its structural representation is as shown in Figure 5.Order-checking transforms Agrobacterium EHA105 (available from Biovector) after identifying correctly.

With reference to the method for Hu Xinwen etc., agrobacterium-mediated transformation soybean transformation callus.The soybean callus tissue is cut into the fritter of mung bean size, be immersed in (the resuspended back of thalline OD in the resuspended liquid of Agrobacterium EHA105 of the resuspended liquid of Agrobacterium EHA105 and reporter plasmid, pCAMBIA1301-CmERF6 and three kinds of carriers of pCAMBIA1301-CmERF7 effect plasmid of the resuspended liquid of Agrobacterium EHA105, reporter plasmid and two kinds of carriers of pCAMBIA1301-CmERF7 effect plasmid of the resuspended liquid of Agrobacterium EHA105, reporter plasmid and the two kinds of carriers of pCAMBIA1301-CmERF6 effect plasmid that contain the reporter plasmid carrier respectively ₆₀₀Be about 0.2), vacuum pressure 0.09-0.1MPa infected 15 minutes.In the dark photoperiod of 16h illumination/8h, under 22 ℃ of conditions, cultivate 1d altogether.

GUS fluorometric assay: carry out with reference to " plant genetic engineering principle and technological second edition " and the method for Jefferson.Get and do not infect and infect the about 100mg of soybean callus, put into mortar respectively, use the liquid nitrogen grinding powdered, add 600uL and extract damping fluid, the supernatant after centrifugal is the gus protein crude extract.The sample crude extract is divided into two portions, and a part adopts the Bradford method to measure gus protein content; Another part is used for fluorescent quantitation and detects, and adds the GUS reaction substrate 4-MUG of 2mmol/L in the crude extract, behind 37 ℃ of insulation 15min, adds 0.2mmol/L Na ₂CO ₃The reaction terminating liquid termination reaction at excitation wavelength 365nm, under the emission wavelength 455nm, is measured fluorescent value.GUS is active to obtain relative reactivity with the fluorescent value that obtains divided by protein concentration and time.

The fluorometric assay result is as shown in Figure 6, and GmERF6 not only can suppress the expression of downstream gene, and the activation that can also suppress other transcription factors is active.

The expression of embodiment 6:GmERF6 gene in Arabidopis thaliana

The construction process of plant expression vector is identical with embodiment 2 methods, GmERF6 is subcloned on the MCS of plant expression vector pBI121 (available from Clontech), and order-checking transforms Agrobacterium EHA105 after identifying correctly.

It is environmental with pBI121-GmERF6 arabidopsis thaliana transformation Columbia that colored method is stained with in utilization.Concrete grammar is following:

Arabidopis thaliana transforms:

1 places the 1.5mL centrifuge tube with wild-type Arabidopis thaliana seed, the Youxiaolin soaking disinfection 3min with 10%, and soak with sterilized water 4-5 back of aseptic water washing, and 4 ℃, vernalization 4-6d;

2 are laid on the MS substratum (regulating pH value to 5.7-5.8 with KOH) with the Arabidopis thaliana seed after the vernalization, culture condition: 16h (24 ℃) illumination/8h (22 ℃) dark, and humidity 80% is cultivated 7-10d;

3 mixed land for growing field crops soil, turfy soil, vermiculite and to place Nursery by 1: 1: 1, irrigate PNS nutritive medium (KNO ₃0.2525g/L, MgSO ₄7H ₂O 0.2465g/L, Ca (NO ₃) ₂7H ₂O 0.236g/L, FeSO ₄7H ₂O 6.9mg/L, EDTA-Na ₂2H ₂O 9.3mg/L, H ₃BO ₃4.34mg/L, MnCl ₂4H ₂O 2.772mg/L, CuSO ₄5H ₂O0.125mg/L, ZnSO ₄7H ₂O 0.2875mg/L, Na ₂MoO ₄2H ₂O 0.0726mg/L, NaCl 0.0585mg/L, CoCl ₂6H ₂O 0.00238mg/L, KH ₂PO ₄0.163g/L, K ₂HPO ₄0.0114g/L, regulate pH value to 5.7-5.8 with KOH), in Nursery, preservative film 4-6d puts into incubator on the cover with the Arabidopis thaliana kind in the substratum, and culture condition is the same;

About 4 about 4 weeks, Arabidopis thaliana begins to bloom, and cuts off the flower of having opened, waits to infect;

5 picking pBI121-GmERF6 mono-clonals are in the YEP (peptone 10g/L, yeast powder 10g/L, NaCl 5g/L) of additional 50 μ g/mL Rifampins of 5mL and 50 μ g/mL kantlex, and 28 ℃, 120rpm cultivates 24h;

6 pour this 5mL YEP among the YEP of additional 50 μ g/mL Rifampins of 500mL and 50 μ g/mL kantlex into, and 28 ℃, 120rpm cultivates about 7-8h, to OD ₆₀₀=1.0-1.2;

75000rpm, centrifugal 15min under the room temperature removes supernatant, thalline is resuspended in conversion medium liquid MS cultivates and concentrate (1 * molysite, 1 * organic, 2% sucrose is regulated pH value to 5.7-5.8 with KOH for 1/2 macroelement, 1 * trace element), to OD ₆₀₀About=0.8, infect forward direction and wherein add several tweens;

8 with in the Arabidopis thaliana top immersion conversion medium, vacuum infestation 10min;

9 Arabidopis thalianas after will infecting are secretly cultivated 10-24h, take out plant, water sufficient nutritive medium, cultivate under the normal condition;

10 individual plants results seed, promptly T0 is for seed.

Genetically modified screening and evaluation

1 is laid on the MS substratum enterprising row filter that contain 50mg/L kantlex for seed after by method sterilization among the embodiment 6 with T0; With the resistant plant kind normal cultured in Nursery that filters out; Get its blade, extract the total DNA of blade according to the method in UniversalGenomic DNA Extraction Kit Ver.3.0 (available from the TaKaRa) specification sheets;

2 with after 50 times of the DNA dilutions of extracting, and getting 1 μ L is template, carries out conventional PCR checking with the primer among the embodiment 1.Wherein with the negative contrast of unconverted wild Arabidopis thaliana, the positive contrast of plasmid pBI121-GmERF6;

3 gather in the crops the seed of the plant of being positive, and promptly T1 is for seed;

4 with T1 for seed continue as stated above the screening, again results seed be the T2 seed.

Fig. 7 be GmERF6 at part T2 for the PCR qualification result in the transgenic arabidopsis, show that the GmERF6 gene successfully is incorporated in the arabidopsis gene group, and can be in Arabidopis thaliana genetic stability.

Embodiment 7:T3 is for GmERF6 downstream gene expression component analysis in the transgenic arabidopsis

Extract the wild-type Arabidopis thaliana according to the method for embodiment 1 and become cDNA for plant leaf RNA and reverse transcription, be used as the template of sxemiquantitative RT-PCR after diluting 5 times with transgenic arabidopsis T3.Do internal reference with the homogenization of sample cDNA template concentrations with Arabidopis thaliana AtActin2 gene: the amplified production of getting equal volume carries out agarose gel electrophoresis; Brightness through band; The adjustment that the amount of adding template in the PCR reaction system is carried out; Resulting amplified band brightness reaches consistent up in identical cycle number the time, and uses the reverse transcription product of this volume to carry out subsequent P CR reaction as template, thereby judges the expression amount of downstream goal gene.

Reaction system is following:

Moisturizing to 25 μ L, pcr amplification program: 94 ℃ of 8min; 94 ℃ of 40sec, 51-57 ℃ of 30s, 72 ℃ of 1min, 25-30 circulation; 72 ℃, 10min.

The primer of using in the experiment:

GmERF6：5′-CTTCCTACTCCTCCCTTTCAC-3′，5′-CGTAGTAGTGTTCCCAGATGC-3′；

AtActin2：5′-CCTTGAAGTATCCTATTGAGC-3′，5′-GGTCTTTGAGGTTTCCATCT-3′；

AtKin1：5′-CATCATCACTAACCAAAACACAC-3′，5′-GATACACTCTTTCCCGCCT-3′；

AtSOS1：5′-CATCCTCACAATGGCTCTAA-3′，5′-CTCCTTCCTTTTCACTTTCA-3′；

AtPR3：5′-CACTTACAACGCCTTTATCACC-3′，5′-AGTCAACTCCTATTGCTCTACCG-3′；

AtRD22：5′-CCAAACACTCCCATTCCC-3′，5′-TGCCTCCGTAACCATCCT-3′；

AtPDF1.2：5′-AGAAGCCAAGTGGGACAT-3′，5′-CGATTTAGCACCAAAGATT-3′；

AtPR4：5′-CTTTTATCATACACAGTGGCTACG-3′，5′-CATCCAAATCCAAGCCTCC-3′；

AtERF7：5′-TCATCAGCGAGGAGACAAG-3′，5′-CGAAACAGGAAAAGCGA-3′；

AtERF4：5′-AGGTGGGATGGAGAAGAGA-3′，5′-GAAAGCCAATAGAAGGAGC-3′；

AtNIMIN1：5′-ATCTAACGGCGGAGAAAGG-3′，5′-CACAACGCTAACAAATGAAAC-3′。

RT-PCR result is as shown in Figure 8 in the downstream gene sxemiquantitative; T3 is for AtKin1 in the transgenic arabidopsis (TT), and AtSOS1, the expression amount of AtPR3 and AtRD22 contrast wild-type (WT) all obviously to be reduced; And the expression amount of AtPDF1.2 and AtPR4 obviously raises; We infer that this mainly is because GmERF6 has suppressed the relevant expression of transcribing inhibition of some adverse circumstances in the transgenic arabidopsis, thereby have removed the inhibition to some adverse circumstance related gene expression, cause the rising of its expression amount.In order to verify this inference, we have analyzed transcribing in the Arabidopis thaliana and have suppressed subbase because of AtERF4, the expression amount of AtERF7 and AtNIMIN1, and the result shows AtERF4, the expression of AtERF7 and AtNIMIN1 has been suppressed really, has proved above inference.

Embodiment 8:T3 is for transgenic arabidopsis arid Treatment Analysis

, treat to stop to water 18 days when Arabidopis thaliana seedling normal growth one in Nursery is all for transgenic arabidopsis according to the method for embodiment 6 plantation wild-type Arabidopis thaliana and T3, note wild-type Arabidopis thaliana and T3 phenotype respectively for transgenic arabidopsis with digital camera.

The result is as shown in Figure 9, and the wild-type Arabidopis thaliana is serious wilts and with slight etiolation, hypoevolutism; Transgenic arabidopsis is grown normal by comparison and is begun to bloom.

Embodiment 9:T3 handles back percentage of water loss analysis for the transgenic arabidopsis arid

According to the method for embodiment 6 plantation wild-type Arabidopis thaliana and T3 for transgenic arabidopsis.Wild-type Arabidopis thaliana and T3 are stripped down for the lotus throne blade of transgenic arabidopsis, weigh rapidly and be put on the exsiccant filter paper, weigh and record in time point shown in Figure 10.

The result is shown in figure 10, and the percentage of water loss of transgenic arabidopsis (TT) will be starkly lower than wild-type Arabidopis thaliana (WT).

Sequence table

Claims (3)

1. soybean adverse circumstance associated transcription factor ERF, it is characterized in that: its aminoacid sequence is Sequence N0.2.

2. soybean adverse circumstance associated transcription factor ERF as claimed in claim 1, it is characterized in that: the nucleotides sequence of its encoding sox is classified Sequence N0.1 as.

3. the application of soybean adverse circumstance associated transcription factor ERF as claimed in claim 1 in the drought resistance that improves plant.

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