CN102911263A - Soybean WRKY transcription factor GmWRKY40 and application thereof in plant drought resisting - Google Patents
Soybean WRKY transcription factor GmWRKY40 and application thereof in plant drought resisting Download PDFInfo
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- CN102911263A CN102911263A CN201210393181XA CN201210393181A CN102911263A CN 102911263 A CN102911263 A CN 102911263A CN 201210393181X A CN201210393181X A CN 201210393181XA CN 201210393181 A CN201210393181 A CN 201210393181A CN 102911263 A CN102911263 A CN 102911263A
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Abstract
The invention discloses a soybean WRKY transcription factor GmWRKY40 and an application thereof in cultivation of a plant drought-resistant variety and belongs to the technical field of plant genetic engineering. The soybean WEKY transcription factor comprises a protein which has the amino acid residue sequences shown as SEQ ID No:1 in the sequence table, or is formed by substitution, deletion or addition on one to ten amino acid residues in the amino acid residue sequence shown as SEQ ID No:1 in the sequence table or and has a transcription activation function and a plant drought resistance regulating function. A GmWRKY40 gene disclosed by the invention has significance to cultivation of a transgene crop in the drought-resistant variety and improvement of stress resistance of the crop.
Description
Technical field
The invention belongs to the plant gene engineering technology field, be specifically related to a kind of soybean WRKY class transcription factor GmWRKY40 and the application in the drought-resistant variety that cultivates plants thereof.
Background technology
WRKY class transcription factor is that a class contains the conservative amino acid whose zinc finger proteins such as WRKY, the wide participation of WRKY gene plant disease resistance response and grow.The WRKY transcription factor contains one or two very conservative WRKY zone, but generally very low in conservative overseas intergenic similarity.This structural domain comprises about 60 amino acids residues, and seven peptide WRKYGQK of N end guarded in ten minutes, and the C end has a C
2H
2Or C
2The zinc fingerprint of HC.
The system of defense of plant is a complicated system.Existing result of study shows, the crucial point of crossing in the signal conduction network that NPR1 is comprised of all kinds of known the signaling pathways of disease resistances participates in the regulation and control of all kinds disease resistance.The NPR1 gene promoter area contains the W-box element of series winding, the WRKY transcription factor (such as AtWKRY18 etc.) can combine with the W-box sequence of NPR1 gene promoter region, forward is regulated NPR1 and is expressed, thereby activates the downstream factor, strengthens the expression of PR gene.
Physics in the environment, chemistry and the environmental evolution such as biological are very large to the growth effect of plant, arid is a kind of a kind of common environmental factor that makes crop failure even total crop failure, soybean is a kind of important oil crops, it also is the main source of protein, understand fully its drought resistance mechanism, utilize its anti-drought gene to improve the drought resistance of crop, have important theory significance and realistic meaning.
Summary of the invention
The object of the present invention is to provide a kind of WRKY class transcription factor GmWRKY40 and encoding gene thereof relevant with drought resisting that derives from soybean.
The present invention also aims to provide GmWRKY40 transcription factor and the application of gene in the drought-resistant variety that cultivates plants thereof.
A kind of soybean WRKY class transcription factor is characterized in that, is the protein of one of following amino acid residue sequences:
1) amino acid residue sequence shown in the sequence table SEQ ID NO:1;
2) with the amino acid residue sequence shown in the sequence table SEQ ID NO:1 through replacement, disappearance or the interpolation of one to ten amino-acid residue and have the protein of the regulating plant drought resistance of transcriptional activation function.
Described sequence table SEQ ID NO:1's is the conserved domain of WRKY class transcription factor from the 135th to 188 amino acids residue sequence.
The gene of above-mentioned soybean WRKY class transcription factor is the gene of one of following nucleotide sequence:
1) nucleotide sequence shown in the sequence table SEQ ID NO:2;
2) polynucleotide of SEQ ID NO:1 protein sequence in the code sequence tabulation.
SEQ ID NO:2 in the sequence table is by 849 based compositions; Protein sequence shown in the SEQ ID NO:1 in the code sequence tabulation; Holding the 135th to 188 from 5 ' is the encoding sequence of WRKY structure.
The plant expression vector that contains above-mentioned soybean WRKY class transcription factor gene.
The Host Strains that contains above-mentioned soybean WRKY class transcription factor gene.
The primer of arbitrary GmWRKY40 gene fragment that increases.
The application of above-mentioned soybean WRKY class transcription factor gene in the plant of cultivating the drought resistance raising.
Described plant is Arabidopis thaliana or tobacco.
Utilize plant expression vector, agriculture bacillus mediated method can obtain the transfer-gen plant that arid resistivity is strengthened with the GmWRKY40 gene transfered plant.
Beneficial effect of the present invention: GmWRKY40 gene pairs of the present invention is cultivated the transgenic crop of drought-resistant variety, and the resistance that improves farm crop is significant.
Description of drawings
The phylogenetic analysis of Fig. 1 soybean GmWRKY40 and other WRKY albumen;
Among the figure, GaWRKY1.pro represents Asiatic cotton WRKY1 albumen;
GhWRKY1.pro represents upland cotton WRKY1 albumen;
MtWRKY.pro represents clover WRKY albumen;
GmWRKY78.pro represents soybean WRKY 78 albumen;
AtWRKY8.pro, AtWRKY60.pro, AtWRKY40.pro represent Arabidopis thaliana
WRKY8, WRK60, WRKY40 albumen;
BrWRKY1.pro represents turnip WRKY1 albumen;
NtWRKY.pro, WIZZ.pro represent the WRKY albumen of tobacco;
PcWRKY4.pro represents the WRKY4 albumen of parsley;
VaWRKY4.pro represents the WRKY albumen of pigeon grape;
CaWRKY.pro represents the WRKY albumen of pimento;
LtWRKY21.pro represents the WRKY21 albumen of creosote bush (Larrea divaricata);
BnWRKY.pro represents the WRKY albumen of rape;
OsWRKY71.pro represents the WRKY71 albumen of paddy rice.
Turn the resistance of GmWRKY40 gene Arabidopis thaliana and not genetically modified Arabidopis thaliana (CK) under Fig. 2 drought condition;
Among the figure, A: plant strain growth situation B after drought is processed: the survival rate of plant after drought is processed.
Turn the resistance of GmWRKY40 genetic tobacco and not genetically modified tobacco (CK) under Fig. 3 drought condition;
Among the figure, A: plant strain growth situation B after drought is processed: the survival rate of plant after drought is processed.
Embodiment
The present invention will be further described below in conjunction with the drawings and specific embodiments.
Following examples do not limit the present invention, unspecified operation steps please refer to " molecular cloning experiment guide " third edition corresponding section (J. Pehanorm Brooker E.F. is Ritchie etc. not, Science Press) or consults the specification sheets of used kit among the embodiment.
(1) structure of soybean cDNA library and amplification
SuperScriptTM Plasmid System for cDNA Synthesis and Plasmid Cloning Kit specification sheets according to GibcoBRL company carries out.Get 5mg Soybean Root mRNA construction cDNA library, storage capacity is 5.2 * 10
6Cfu, carrier library are pPC86 (Trp+).
(2) screening of soybean cDNA library and sequential analysis
According to the synthetic W-box sequence of Arabidopsis thaliana NPR1 gene promoter region sequence design (W-box (+): 5 '-CTAGAGTTGACTTGACTTGGTTGACTTGACTTGGTTGACTTGACTTG-3 ' and W-box (-):
5 '-GATCCAAGTCAAGTCAACCAAGTCAAGTCAACCAAGTCAAGTCAACT-3 '), then (cut through Xba I and BamH I enzyme) being connected on pRS315His (Leu+) carrier after two chain annealing, the T4PNK phosphorylation, obtain bait carrier pRSWbox plasmid.
Preparation yWAM2 competent cell arrives yeast strain yWAM2 (Leu to the pRSWbox Plasmid Transformation
-, His
-, Trp
-) in, acquisition contains the yeast strain yWbox (His of pRSWbox
-, Trp
-).With the yWbox yeast that contains bait carrier the library is screened, transformant is coated onto His
-Select on the substratum, cultivated 3～5 days for 28 ℃.After yeast grows, carry yeast plasmid, Transformed E .Coli DH5 α extracts plasmid, restriction analysis, order-checking can obtain the clone's that sieves dna sequence dna, and sequence is analyzed.
The dna sequence dna that obtains is GmWRKY40 through comparison, and its nucleotide sequence is shown in sequence table SEQ ID NO:2, and its aminoacid sequence is shown in sequence table SEQ ID NO:1.The amino acid sequence analysis discovery, GmWRKY40 has conservative WRKY structural domain and nuclear localization signal, shows that GmWRKY40 belongs to WRKY class transcription factor.Amino acid sequence analysis also finds, with the GmWRKY40 sequence identity the highest be the sequence MtWRKY (ABE94581) that derives from Medicago truncatula, similarity is 51.0%.From phylogenetic analysis (Fig. 1), Gm WRKY40 and MtWRKY divide in a branch, show that the two has nearer sibship in evolution.
The binding specificity of embodiment 2GmWRKY40
The core sequence of W-box (+) (5 '-TTGACTTGAC-3 ') to be suddenlyd change, the sequence after the sudden change is 5 '-TcagCTcagC-3 ', called after m W-box; W-box and m W-box are building up on the pRS315His carrier, obtain pRSWbox and pRSmWbox plasmid.
Use respectively pRSmWbox and pRSWbox Plasmid Transformation yeast yWAM2 (Leu
-, His
-, Trp
-), obtain ymWbox yeast (His
-, Trp
-) and yWbox yeast (His
-, Trp
-), GmWRKY40 is transformed respectively yWbox and ymWbox yeast, be coated on His-and select on the substratum, the result shows that GmWRKY40 is transformed in the yWbox yeast, it is at His
-Select can grow on the substratum, and be transformed in the ymWbox yeast, it is at His
-Select can not to grow on the substratum, illustrate the expression product of GmWRKY40 gene in the yeast cell body that be structured on the pPC86 carrier can with the specific combination of W-box, activated reporter gene His
+Expression, therefore can be at His
-Select to grow on the substratum, and when W-box sported mW-box, the expression product of GmWRKY40 in the yeast cell body can not with the specific combination of mW-box, do not activate reporter gene His
+Expression, therefore can not be at His
-Select to grow on the substratum.Therefore, screen the GmWRKY40 that obtains and in the yeast body, have W-box cis element binding specificity.
Transformation of Arabidopsis thaliana and the Transformation of tobacco of embodiment 3GmWRKY40 gene
(1) vector construction and Agrobacterium-mediated Transformation
GmWRKY40 is cut with Sal I enzyme, and the Klenow enzyme fills, and cuts with Sac I enzyme again, reclaims the purpose fragment; Carrier pPZP212-R cuts with Xba I enzyme, and the Klenow enzyme fills, and cuts with Sac I enzyme again, reclaims the purpose fragment; Connect, Transformed E .coli.JM109, the upgrading grain, enzyme is cut evaluation, chooses required clone, and order-checking obtains pPZP212-40.
Preparation Agrobacterium LBA4404 competence, method is with reference to MicroPulser
TMELectroporation Apparatus Operating Instructions and AppLications Guide (BIO-RAD company), get 20 μ L LBA4404 competent cells, add 1 μ L DNA (0.5 μ g), change electric shock cup electric shock over to and transform (1.8KV).Added 1mL YEB liquid nutrient medium renewal cultivation 3 hours (28 ℃, 200rpm).Get respectively 20 μ L, 200 μ L and be coated with YEB flat board (Kan 50mg/L, Rif 50mg/L).Two clones of picking carry the Agrobacterium plasmid with alkaline process, and PCR detects.
(2) transformation of Arabidopsis thaliana
The Arabidopis thaliana seed is 4 ℃ of vernalization treatment of carrying out 2-3 days, and every basin is sowed 6～8 seeds (Nutrition Soil and vermiculite were by 2: 1); Be put in and cultivate (22 ℃, illumination 16h) in the greenhouse; After Arabidopis thaliana is extracted just mossy out, cut off just mossy, treat that it extracts more time mossy out, and minority can be used for transforming when beginning to bear pods.
Choose the single colony inoculation of Agrobacterium at 3mLYEB (Kan 50mg/L, Rif 50mg/L), 28 ℃, 250rpm cultivated 30 hours; Enter among the fresh YEB (Kan 50mg/L, Rif 50mg/L) of 200mL by switching in 1: 400,28 ℃, 250rpm cultivated approximately 14 hours, surveyed OD
600≈ 1.5; 7500rpm, 4 ℃, the centrifugal collection thalline of 10min; Resuspended thalline is in the penetrating fluid (10% sucrose, SILWET 0.02%) of diploid long-pending (400mL).
The bud of Arabidopis thaliana is immersed in the penetrating fluid 1 minute; Transform complete after, plastics bag on the Flowerpot casing, horizontal positioned makes it after growing 24～48 hours under the low light intensity, can normally cultivate.
The seed of results is put into the 1.5mL centrifuge tube, add the ethanol (containing 0.05%Tween 20) of 1mL 75%, on shaking table, shake 10 minutes (300rpm), centrifugal, remove supernatant; The ethanol that adds 1mL 95% is washed once, and is centrifugal, removes supernatant; Repeat once; The dehydrated alcohol that adds 0.3mL in super clean bench moves on on the aseptic filter paper, dries up; The seed that dries up is spread on the 1/2MS dull and stereotyped (Kan 50mg/L); 4 ℃ of vernalization are after 2 days, and 22 ℃, the 16h illumination cultivation; With positive plant (T
1Generation) is transplanted in the basin and cultivates, and collect seed and carry out T
2The generation screening.
(3) Transformation of tobacco
With the above-mentioned agrobacterium strains that contains plasmid pPZP212-40, adopt leaf dish method transformation of tobacco NC89, screen at the substratum that contains the 150mg/L kantlex, obtain having the transgenic tobacco plant of resistance.After plant to be planted is taken root, transplant to basin and cultivate, collect seed.
Drought resisting experiment: transfer-gen plant and transfer-gen plant not placed under the normal growth conditions of Arabidopis thaliana do not feed water cultured continuously 15～20 days; Then feedwater is cultivated.
In the drought resisting experiment, do not feed water in continuous 15 days, begin the to dewater flavescence, 17 days blades of adjoining tree blade occurs withered; The blade overwhelming majority of transfer-gen plant is still acted normally, and strain 1 and strain 2 are all survived, and strain 3 and strain 4 survival rates are respectively 81.3% and 85.4% (Fig. 2).As seen the drought resistance of transgenic arabidopsis plant is improved.
Embodiment 5 turns the Physiological Analysis of GmWRKY40 genetic tobacco
Drought resisting experiment: transfer-gen plant and transfer-gen plant not placed stop feedwater under the normal growth conditions of tobacco, until most of wild-type plant dehydration rehydration when dead, rehydration was added up survival rate after 3 days.
In the drought resisting experiment, do not feed water in continuous 24 days, most of wild-type plant dehydration is dead, carries out rehydration, adds up survival rate after 3 days.Wherein the wild-type contrast has 15.6% plant survival; Transgenic line 3,4 is all survived, and strain 1 and strain 2 survival rates are respectively 86.7% and 80% (Fig. 3).As seen the drought resistance of transgenic tobacco plant is improved.
Claims (8)
1. a soybean WRKY class transcription factor is characterized in that, is the protein of one of following amino acid residue sequences:
1) amino acid residue sequence shown in the sequence table SEQ ID NO:1;
2) with the amino acid residue sequence shown in the sequence table SEQ ID NO:1 through replacement, disappearance or the interpolation of one to ten amino-acid residue and have the protein of the regulating plant drought resistance of transcriptional activation function.
2. soybean WRKY class transcription factor according to claim 1 is characterized in that, described sequence table SEQ ID NO:1's is the conserved domain of WRKY class transcription factor from the 135th to 188 amino acids residue sequence.
3. the gene of claim 1 or the 2 described soybean WRKY class transcription factors of encoding.
4. gene according to claim 3 is characterized in that, is the gene of one of following nucleotide sequence:
1) nucleotide sequence shown in the sequence table SEQ ID NO:2;
2) polynucleotide of SEQ ID NO:1 protein sequence in the code sequence tabulation.
5. the plant expression vector that contains claim 3 or 4 described soybean WRKY class transcription factor genes.
6. the Host Strains that contains claim 3 or 4 described soybean WRKY class transcription factor genes.
7. claim 3 or the 4 described soybean WRKY class transcription factor genes application in the plant of cultivating the drought resistance raising.
8. the application of soybean WRKY class transcription factor gene according to claim 8 in the plant of cultivating the drought resistance raising is characterized in that described plant is Arabidopis thaliana or tobacco.
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|---|---|---|---|
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104046634A (en) * | 2014-06-19 | 2014-09-17 | 天津农学院 | Triticum aestivum WRKY transcription factor gene and application thereof in high-temperature stress response of Arabidopsis thaliana |
| CN116769819A (en) * | 2023-05-11 | 2023-09-19 | 广东省农业科学院果树研究所 | Application of citrus CsWRKY40 gene in repelling diaphorina citri |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1814620A (en) * | 2005-02-01 | 2006-08-09 | 中国科学院遗传与发育生物学研究所 | Soybean WRKY transcription factor GmWRKY6 and its coding gene and use |
-
2012
- 2012-10-17 CN CN201210393181XA patent/CN102911263A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1814620A (en) * | 2005-02-01 | 2006-08-09 | 中国科学院遗传与发育生物学研究所 | Soybean WRKY transcription factor GmWRKY6 and its coding gene and use |
Non-Patent Citations (2)
| Title |
|---|
| LIGANG CHEN ET AL: "The role of WRKY transcription factors in plant abiotic stresses", 《BIOCHIMICA ET BIOPHYSICA ACTA》 * |
| NCBI: "ACCESSION NO：XM_003545361", 《GENBANK》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104046634A (en) * | 2014-06-19 | 2014-09-17 | 天津农学院 | Triticum aestivum WRKY transcription factor gene and application thereof in high-temperature stress response of Arabidopsis thaliana |
| CN116769819A (en) * | 2023-05-11 | 2023-09-19 | 广东省农业科学院果树研究所 | Application of citrus CsWRKY40 gene in repelling diaphorina citri |
| CN116769819B (en) * | 2023-05-11 | 2024-01-02 | 广东省农业科学院果树研究所 | Application of citrus CsWRKY40 gene in repelling diaphorina citri |
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Application publication date: 20130206 |