CN101003808A - WRKY45 gene of paddy rice, preparation method and application - Google Patents
WRKY45 gene of paddy rice, preparation method and application Download PDFInfo
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Abstract
This invention discloses a method for preparing rice gene WRKY45 and its applications. This invention successfully separates gene WRKY45 from rice, determines its nucleotide sequence, and deduces its amino acid sequence. Gene functional analysis shows that rice gene WRKY45 can effectively improve the disease and drought resistance of Arabidopsis thaliana, rice, economic crops, horticultural plants, and pasture grass, thus has wide potential applications.
Description
Technical field
The present invention relates to Agricultural biotechnologies, more particularly, the present invention relates to utilize WRKY 45 gene of paddy rice to improve plant broad spectrum antidisease and drought-resistant ability based on the transgenic plant technology.
Background technology
Transcriptional regulator WRKY gene family is a plant specific super gene family, contains the aminoacid sequence WRKYGQK and the CX 4-5 CXnHXH/C (X is arbitrary amino acid) of high conservative at its protein N-terminal, belongs to zinc and refers to the type transcriptional regulator.WRKY albumen mass-energy is specifically in conjunction with the DNA distinguished sequence TTGACC (W box) of target gene promoters, thereby regulatory gene is expressed (EulgemT. etc., 2000, Trends Plant Sci., 5:199-206; Yu etc. 2001, The Plant Cell, 13:1527-1539).
The main biological function of present known WRKY gene family is the biological and biological environment stress reaction of regulation and control plant organic combination, grow and the signal transduction pathway of aspect such as metabolism adjusting, is in the regulation and control intersection point (cross-talk) of the collaborative integration of two of plants or many barss transduction pathway.Such as, Arabidopis thaliana WRKY6 gene product is induced the W box sequence of receptor-like kinase enzyme (SIRK) gene promoter region by being attached to aging specifically, thereby organic combination is regulated vegetable cell old and feeble and the foundation of plant disease-resistant proterties (Robatzek etc., 2002, Gene﹠amp; Dev., 16:1139-1149), and Arabidopis thaliana WRKY53 is regulation and control intersection point (Hinderhofer etc., 2001, Planta, 213 (3): 469-73) that old and feeble early stage generation of Arabidopsis leaf and disease-resistant proterties are built up.Arabidopis thaliana RRS1-R (WRKY16) gene is both as the special intrusion of resisting pathogenic bacteria Ralstonia solanacearum of typical disease-resistant gene; participate in disease-resistant signal conduction (the Deslandes L. etc. of plant again as typical WRKY gene; 2002; Proc Natl AcadSci USA; 99 (4): 2404-9); and the battlefield that plant protection is reacted is extended to nucleus, and (Lahaye T.2002; Trends in Plant Science; 7 (10): 425-427); the organic collaborative integration of signal transduction pathway of special disease resistance response that disease-resistant gene is brought out and broad-spectrum disease resistance reaction, expansion plant disease-resistant source bacterium spectrum.The foundation of two bars transduction pathway such as hair life of the collaborative integration of Arabidopis thaliana WRKY44 protein plant surface and the growth of seed exterior skin (Johnson etc., 2002, The Plant Cell, 14:1359-1375).Arabidopis thaliana WRKY70 protein activates the disease-resistant signal transduction pathway of plant of Whitfield's ointment mediation and suppresses the disease-resistant signal transduction pathway of plant that jasmonic mediates, be regulation and control point of crossing (the Li J. etc. of these two disease-resistant signal transduction pathways, 2004, The Plant Cell, 16:319-331).Collaborative foundation (the Pnueli L. etc. that integrate two bars transduction pathway such as leguminous plants drought resistance and seed dormancy of the WRKY gene of desert leguminous plants Retama raetam, 2002, Plant J., 31:319-330), and the LtWRKY21 protein that derives from shrubbery (Larreatridentata) participates in shrubbery drought resistance foundation (the Zou X. of ABA mediation as the incitant of plant hormone ABA signal transduction pathway, Deng, 2004, J.of Biological Chem., 279:55770-55779).The nearest research work of the inventor also confirms, the corresponding WRKY 45 gene of paddy rice of high expression level can improve the reaction level of transgenic plant to plant adverse circumstance associated hormone ABA significantly, can improve disease resistance and the drought-resistant level of transgenic plant again, be in the regulation and control intersection point that plant hormone ABA and disease-resistant signaling molecule Whitfield's ointment mediate two bars transduction pathway.
The residing surrounding environment of plant-growth is constantly changeable, often being subjected to the collaborative of a plurality of adverse circumstance factors coerces, a plurality of adverse circumstance factors are coerced adaptability set up comprehensive collaborative integration process beyond doubt, and coordinate closely mutually with phytomorph generation, growth and morphogenesis.Transcriptional regulator WRKY gene family is the crucial molecular biology function of performance among this comprehensively works in coordination with integration process, is that plant is coerced adaptability foundation to a plurality of adverse circumstance factors and phytomorph is built up the machine-processed at all place of coordinating integration.From present fundamental research achievement, transcriptional regulator WRKY gene can strengthen the adaptive faculty that plant is coerced the adverse circumstance factor effectively.Therefore, farm crop, gardening plant and the herbage new variety of utilizing the WRKY gene to cultivate degeneration-resistant border ability to significantly improve will become possibility.
But before the present invention, the function of WRKY45 gene is not also really disclosed; Still be not reported about WRKY45 gene raising plant broad spectrum antidisease and drought-resistant Research on ability; The function of this gene is not also illustrated.
Summary of the invention
At the deficiencies in the prior art, the present invention proposes following goal of the invention:
The first, the aminoacid sequence that a kind of WRKY 45 gene of paddy rice is provided and infers thus;
The second, disclose WRKY 45 gene of paddy rice molecular biology function and application thereof;
The 3rd, a kind of method of utilizing described WRKY 45 gene of paddy rice to cultivate farm crop, gardening plant and herbage new variety with broad-spectrum disease resistance proterties and drought-resistant ability is provided.
Purpose of the present invention is achieved by following technical proposals.
1. provide a kind of and isolate the WRKY45 gene from rice varieties Japan is fine, this gene has the described nucleotide sequence of SEQ ID No:1 in the sequence table; And provide the described aminoacid sequence of SEQ ID No:2 in the sequence table of having of inferring thus.
2. the WRKY 45 gene of paddy rice that utilizes the present invention to obtain has made up corresponding plant expression vector, transforms corresponding Agrobacterium.
3. utilize the foreign gene of plants such as reorganization agriculture bacillus mediated farm crop, gardening plant and herbage to transform, WRKY 45 gene of paddy rice is imported plants such as corresponding farm crop, gardening plant and herbage, obtain corresponding transgenic plant.
4. carry out disease resistance analysis and the drought-resistant capability evaluation of transgenic plant such as corresponding farm crop, gardening plant and herbage.
5. small area field experiment, disease resistance and the drought-resistant capability evaluation of transgenic plant such as corresponding farm crop, gardening plant and herbage have been carried out.
6. big area field experiment, disease resistance and the drought-resistant capability evaluation of transgenic plant such as corresponding farm crop, gardening plant and herbage have been carried out.The new variety that screening corresponding plant broad spectrum antidisease of acquisition and drought-resistant ability significantly improve, and apply.
The invention provides the preparation method of described WRKY45 gene, this method adopts the step of following order:
(1) will the grow Japanese fine paddy rice in 4 weeks carries out subzero treatment (4 ℃), adopts the amending method of Logemann etc. (1987), extracts total RNA.
Synthesizing of (2) first chains: adopt BD Clontech library construction test kit.The RNA sample that adds 1-3 μ l, the oligonucleotide of 1 μ l, 3 ' the PCR primer of 1 μ l, bathe 2min in 72 ℃ of temperature behind the mixing, place the 5 * first-strand buffer that adds 2 μ l behind the 2min on ice then, 1 μ lDTT, the ThermoScript II of 1 μ l dNTP Mix and 1 μ l is put on ice behind 42 ℃ of placement 1hr behind the mixing.
(3) amplification of cDNA: 2 μ l, first chain synthetic product and 80 μ l are taken off the ionic sterilized water, 10 μ l, 10 * Advantage, 2 PCR buffer, 2 μ l, 50 * dNTP Mix.2 μ l, 5 ' PCRprimer, 2 μ l, 3 ' PCR primer, with 2 μ l, 50 * Advantage Polymerarse Mix to 100 μ l, the centrifugal slightly PCR that carries out.Get 5 μ l PCR products and carry out the agarose gel detection.
(4) digestion of Proteinase K: the double-stranded cDNA that gets 2-3 μ g adds the Proteinase K of 2 μ l (20 μ g/ μ l) and takes out after preserving in 45 ℃ of temperature in-20 ℃ and bathe 20min, carry out phenol, chloroform extracting, get supernatant, add the 3M sodium acetate, glycogen (20 μ g/ μ l), 95% alcohol is centrifugal, removes supernatant, remove supernatant with 80% alcohol wash, precipitation is dissolved in water after the drying at room temperature.
(5) digestion of Sfi I: will carry out Sfi I enzyme through the double-stranded cDNA that protease K digesting is crossed and cut, 50 ℃ of temperature are bathed 2hr, add the blue dyestuff of 2 μ l, 1% dimethylbenzene nitrile mixing in centrifuge tube.
(6) clone of cDNA and mensuration: adopt the carrier that test kit provided, 16 ℃ of connections are spent the night, and transform back extraction plasmid and check order.
Compared with prior art, the present invention has following beneficial effect:
1. the present invention successfully isolates the WRKY45 gene first from rice varieties Japan is fine, and has determined its nucleotide sequence and the aminoacid sequence of inferring thus;
2. by gene function analysis, disclosed WRKY 45 gene of paddy rice and can improve disease resistance and the drought-resistant ability of plant significantly;
3. the gene function that is confirmed in model plant Arabidopis thaliana and rice plants also can be verified and production application in other important economic farm crop, gardening plant and forage grass effectively, is indicating wide application prospect.
Description of drawings
Fig. 1 is the screening of high expression level WRKY45 transfer-gen plant;
Fig. 2 compares the disease resistance that pseudomonas Pst.DC3000 infects;
Fig. 3 is the figure of the effect that significantly improves of the drought resisting level of transfer-gen plant;
Fig. 4 is the figure that drought environment is induced the effect that the transgenic plant pore closes rapidly;
Fig. 5 is the comparison diagram of transfer-gen plant and wild-type plant transpiration rate.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is done further clearly explanation, but they are not the qualification to protection domain of the present invention.Those of ordinary skill in the art can all belong to protection scope of the present invention from direct distortion of deriving of content disclosed by the invention or the technique means that is equal to.
The clone of embodiment 1--WRKY45 cDNA
The amending method of (1987) such as the extraction employing Logemann of the total RNA of rice seedling.The program of and library construction test kit (BD Clontech) synthetic according to cDNA makes up the cDNA library that low temperature (4 ℃) is induced 2h.Design corresponding primer according to the WRKY 45 gene of paddy rice nucleotide sequence, carry out the RT-PCR amplification, obtain part specific gene fragment.Subsequently, with the RT-PCR extension amplification outcome to pMD18-T carrier (Dalian TaKaRa company), and order-checking and predicted amino acid sequence.At last, as probe, Screening of Rice cDNA library obtains the WRKY45 full-length cDNA, specifically sees shown in the sequence table with the special paddy rice WRKY45 Gene Partial dna fragmentation that obtains.
The screening of embodiment 2--high expression level WRKY45 transfer-gen plant
With the WRKY45 full length cDNA clone that obtains in pOCA30.The plasmid that builds is transformed among the Agrobacterium LBA4404, adopts live body plant Agrobacterium flower-dipping method to transform, and the transgenic seed that obtains adopts and contains kantlex (50 μ g ml
-1) the MS substratum screen and obtain T
1Transfer-gen plant further utilizes northern blot analysis to confirm.Test-results as shown in Figure 1.
Grow the high expression level WRKY45 and the wild-type Arabidopis thaliana plant in 4 weeks, with the Arabidopsis leaf of Pseudomanassyringae pv.Tomato strain DC3000 injection, bacterial concentration is OD
600=0.001.Cultivating took a sample after 72 hours is coated with flat board, and the statistics colony number is analyzed.Test-results as shown in Figure 2.
Grow the high expression level WRKY45 and the wild-type Arabidopis thaliana plant in 4 weeks, saturated feedwater 1d, then arid 3 weeks continuously, test-results is as shown in Figure 3.
The mensuration of embodiment 6--blade transpiration rate
Be placed in the pan after getting growth time and the blade of similar wild-type of size and transfer-gen plant being cut, leaf back places on the experiment table up, room temp is 20 ℃, relative humidity is～50%, and weighing was carried out in dehydration respectively at 0,10,30,60,90,120 minute naturally.Test-results as shown in Figure 5.
＜110〉Xishuangbanna Tropical Plant Garden, Chinese Academy of Sciences
＜120〉WRKY 45 gene of paddy rice and its production and application
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<141>2006-12-05
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＜213〉paddy rice (Oryza sativa)
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Met?Thr?Ser?Ser?Met
1
tcg?ccg?gcg?ccg?gcg?ccg?gcg?tac?gcg?cag?gtg?atg?gag?gac?atg?gag?166
Ser?Pro?Ala?Pro?Ala?Pro?Ala?Tyr?Ala?Gln?Val?Met?Glx?Asx?Met?Glx
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aag?ggg?aag?gag?ctg?gcg?gcg?cag?ctg?cag?ggg?ctc?ctc?cgc?gac?tcg?214
Lys?Gly?Lys?Glx?Leu?Ala?Ala?Gln?Leu?Gln?Gly?Leu?Lcu?Arg?Asx?Ser
25 30 35
ccg?gag?gcc?ggc?cgc?ttc?gtc?gac?cag?att?ctc?cac?acc?ttc?tcc?cgg?262
Pro?Glu?Ala?Gly?Arg?Phe?Val?Asx?Glx?Ile?Leu?His?Thr?Phe?Ser?Arg
40 45 50
gcg?atg?cgg?gcg?ctc?gac?aag?gcg?gcg?gtc?tcc?gcc?gcc?gga?gga?gaa?310
Ala?Met?Arg?Ala?Leu?Asx?Lys?Ala?Ala?Val?Ser?Ala?Ala?Gly?Gly?Glx
55 60 65
ggg?tcg?gag?gtg?cag?agc?gag?gtc?acc?tgc?ggg?ggc?ggg?gcc?agc?gcc?358
Gly?Ser?Glx?Val?Glx?Ser?Glx?Val?Thr?Cys?Gly?Gly?Gly?Ala?Ser?Ala
70 75 80
ggc?ggg?aag?agg?aaa?gcc?ccc?gcc?gcc?gac?cgg?aag?gcc?aac?tgc?cgc?406
Gly?Gly?Lys?Arg?Lys?Ala?Pro?Ala?Ala?Asx?Arg?Lys?Ala?Asx?Cys?Arg
85 90 95 100
agg?agg?acg?cag?caa?tcg?tcc?ggg?aat?tcg?gtg?gtc?gtc?aag?aac?ctc?454
Arg?Arg?Thr?Glx?Glx?Ser?Ser?Gly?Asx?Ser?Val?Val?Val?Lys?Asx?Leu
105 110 115
gac?gac?ggc?cag?gca?tgg?cgc?aag?tac?ggg?cag?aag?gag?atc?caa?aac?502
Asx?Asx?Gly?Glx?Ala?Trp?Arg?Lys?Tyr?Gly?Glx?Lys?Glu?Ile?Glx?Asx
120 125 130
tcc?aag?cac?cca?aag?gcc?tac?ttc?cgg?tgc?acg?cac?aag?tac?gac?cag?550
Ser?Lys?His?Pro?Lys?Ala?Tyr?Phe?Arg?Cys?Thr?His?Lys?Tyr?Asx?Glx
135 140 145
ctg?tgc?acg?gcg?cag?cgg?cag?gtg?cag?cgc?tgc?gac?gac?gac?ccg?gcg?598
Leu?Cys?Thr?Ala?Glx?Arg?Glx?Val?Glx?Arg?Cys?Asx?Asx?Asx?Pro?Ala
150 155 160
agc?tac?agg?gtc?acc?tac?atc?ggc?gag?cac?acc?tgc?cgg?gac?ccg?gcc?646
Ser?Tyr?Arg?Val?Thr?Tyr?Ile?Gly?Glu?His?Thr?Cys?Arg?Asx?Pro?Ala
165 170 175 180
acc?gcc?ccc?atc?atc?gcg?gcg?cac?gtc?atc?cac?cag?gtc?gcc?gcc?ggc?694
Thr?Ala?Pro?Ile?Ile?Ala?Ala?His?Val?Ile?His?Glx?Val?Ala?Ala?Gly
185 190 195
gac?aac?gac?gac?ggc?tgc?ggc?ggc?ctc?caa?gcg?ggg?tcc?cgc?ctc?atc?742
Asx?Asx?Asx?Asx?Gly?Cys?Gly?Gly?Leu?Glx?Ala?Gly?Ser?Arg?Leu?Ile
200 205 210
agc?ttc?gtc?gcc?gcg?ccg?gcg?gcg?cca?gta?gac?gct?gcc?gcg?gcg?ccg?790
Ser?Phe?Val?Ala?Ala?Pro?Ala?Ala?Pro?Val?Asx?Ala?Ala?Ala?Ala?Pro
215 220 225
acg?acc?agc?acg?atc?acc?acg?gtc?acc?gcg?ccg?ggc?ccg?ctg?ctg?cag?838
Thr?Thr?Ser?Thr?Ile?Thr?Thr?Val?Thr?Ala?Pro?Gly?Pro?Leu?Leu?Glx
230 235 240
ccg?ctc?aag?gtg?gag?ggc?ggc?gtc?ggc?tcg?tcc?gac?cag?gag?gag?gtg?886
Pro?Leu?Lys?Val?Glu?Gly?Gly?Val?Gly?Ser?Ser?Asx?Glx?Glu?Glu?Val
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ctg?agc?agc?ctc?acg?ccc?ggc?agc?tcc?gcg?gcg?cgc?ggc?ggc?ggc?ggc?934
Leu?Ser?Ser?Leu?Thr?Pro?Gly?Ser?Ser?Ala?Ala?Arg?Gly?Gly?Gly?Gly
265 270 275
ggc?ggc?gga?gtc?gcg?ggt?ccc?ttc?ggg?ccg?gac?cag?ggc?gat?gtc?acg?982
Gly?Gly?Gly?Val?Ala?Gly?Pro?Phe?Gly?Pro?Asx?Glx?Gly?Asx?Val?Thr
280 285 290
tcc?tcc?ctg?cac?tgg?agc?tac?gac?gcc?gtc?gcc?ggc?atg?gag?ttc?ttc?1030
Ser?Ser?Leu?His?Trp?Ser?Tyr?Asx?Ala?Val?Ala?Gly?Met?Glu?Phe?Phe
295 300 305
aag?aac?gac?gag?gtt?gtc?ttc?gat?ctg?gac?gac?att?atg?ggt?ttg?agc?1078
Lys?Asx?Asx?Glu?Val?Val?Phe?Asx?Leu?Asx?Asx?Ile?Met?Gly?Leu?Ser
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ttt?tgatcaccga?agaatcatgg?atggacacgg?gccgggtaaa?acgatcgaaa?1131
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gaagatggat?tccacgcgtg?tgtacagaaa?taattagcgg?cagcgcggat?cttaatttgg?1191
aacttgcaaa?gatactccta?attagcctgg?ctagattagt?ttgtaaattc?cttgttgatg?1251
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Met?Thr?Ser?Ser?Met?Ser?Pro?Ala?Pro?Ala?Pro?Ala?Tyr?Ala?Gln?Val
1 5 10 15
Met?Glx?Asx?Met?Glx?Lys?Gly?Lys?Glx?Leu?Ala?Ala?Gln?Leu?Gln?Gly
20 25 30
Leu?Leu?Arg?Asx?Ser?Pro?Glu?Ala?Gly?Arg?Phe?Val?Asx?Glx?Ile?Leu
35 40 45
His?Thr?Phe?Ser?Arg?Ala?Met?Arg?Ala?Leu?Asx?Lys?Ala?Ala?Val?Ser
50 55 60
Ala?Ala?Gly?Gly?Glx?Gly?Ser?Glx?Val?Glx?Ser?Glx?Val?Thr?Cys?Gly
65 70 75
Gly?Gly?Ala?Ser?Ala?Gly?Gly?Lys?Arg?Lys?Ala?Pro?Ala?Ala?Asx?Arg
80 85 90 95
Lys?Ala?Asx?Cys?Arg?Arg?Arg?Thr?Glx?Glx?Ser?Ser?Gly?Asx?Ser?Val
100 105 110
Val?Val?Lys?Asx?Leu?Asx?Asx?Gly?Glx?Ala?Trp?Arg?Lys?Tyr?Gly?Glx
115 120 125
Lys?Glu?Ile?Glx?Asx?Ser?Lys?His?Pro?Lys?Ala?Tyr?Phe?Arg?Cys?Thr
130 135 140
His?Lys?Tyr?Asx?Glx?Leu?Cys?Thr?Ala?Glx?Arg?Glx?Val?Glx?Arg?Cys
145 150 155
Asx?Asx?Asx?Pro?Ala?Ser?Tyr?Arg?Val?Thr?Tyr?Ile?Gly?Glu?His?Thr
160 165 170 175
Cys?Arg?Asx?Pro?Ala?Thr?Ala?Pro?Ile?Ile?Ala?Ala?His?Val?Ile?His
180 185 190
Glx?Val?Ala?Ala?Gly?Asx?Asx?Asx?Asx?Gly?Cys?Gly?Gly?Leu?Glx?Ala
195 200 205
Gly?Ser?Arg?Leu?Ile?Ser?Phe?Val?Ala?Ala?Pro?Ala?Ala?Pro?Val?Asx
210 215 220
Ala?Ala?Ala?Ala?Pro?Thr?Thr?Ser?Thr?Ile?Thr?Thr?Val?Thr?Ala?Pro
225 230 235
Gly?Pro?Leu?Leu?Glx?Pro?Leu?Lys?Val?Glu?Gly?Gly?Val?Gly?Ser?Ser
240 245 250 255
Asx?Glx?Glu?Glu?Val?Leu?Ser?Ser?Leu?Thr?Pro?Gly?Ser?Ser?Ala?Ala
260 265 270
Arg?Gly?Gly?Gly?Gly?Gly?Gly?Gly?Val?Ala?Gly?Pro?Phe?Gly?Pro?Asx
275 280 285
Glx?Gly?Asx?Val?Thr?Ser?Ser?Leu?His?Trp?Ser?Tyr?Asx?Ala?Val?Ala
290 295 300
Gly?Met?Glu?Phe?Phe?Lys?Asx?Asx?Glu?Val?Val?Phe?Asx?Leu?Asx?Asx
305 310 315
Ile?Met?Gly?Leu?Ser?Phe
320 325
Claims (5)
1. WRKY45 gene of separating from paddy rice, it is characterized in that: this gene has SEQ ID № in the sequence table: 1 described nucleotide sequence.
2. WRKY45 gene according to claim 1 is characterized in that: this gene has SEQ ID № in the sequence table: 2 described aminoacid sequences.
3. the preparation method of the described WRKY45 gene of claim 1 is characterized in that adopting the step of following order:
(1) will the grow Japanese fine paddy rice in 4 weeks carries out subzero treatment (4 ℃), adopts the amending method of Logemann etc. (1987), extracts total RNA;
Synthesizing of (2) first chains: adopt BD Clontech library construction test kit.The RNA sample that adds 1-3 μ l, the oligonucleotide of 1 μ l, 3 ' the PCR primer of 1 μ l, bathe 2min in 72 ℃ of temperature behind the mixing, place the 5 * first-strand buffer.1 μ lDTT that adds 2 μ l behind the 2min on ice then, the ThermoScript II of 1 μ l dNTP Mix and 1 μ l is put on ice behind 42 ℃ of placement 1hr behind the mixing;
(3) amplification of cDNA: 2 μ l, first chain synthetic product and 80 μ l are taken off the ionic sterilized water, 10 μ l, 10 * Advantage, 2 PCR buffer, 2 μ l, 50 * dNTP Mix.2 μ l, 5 ' PCRprimer, 2 μ l, 3 ' PCR primer, with 2 μ l, 50 * Advantage Polymerarse Mix to 100 μ l, the centrifugal slightly PCR that carries out gets 5 μ l PCR products and carries out the agarose gel detection.
(4) digestion of Proteinase K: the double-stranded cDNA that gets 2-3 μ g adds the Proteinase K of 2 μ l (20 μ g/ μ l) and takes out after preserving in 45 ℃ of temperature in-20 ℃ and bathe 20min, carry out phenol, chloroform extracting, get supernatant, add the 3M sodium acetate, glycogen (20 μ g/ μ l), 95% alcohol is centrifugal, removes supernatant, remove supernatant with 80% alcohol wash, precipitation is dissolved in water after the drying at room temperature;
(5) digestion of SfiI: will carry out the SfiI enzyme through the double-stranded cDNA that protease K digesting is crossed and cut, 50 ℃ of temperature are bathed 2hr, add the blue dyestuff of 2 μ l, 1% dimethylbenzene nitrile mixing in centrifuge tube;
(6) clone of cDNA and mensuration: adopt the carrier that test kit provided, 16 ℃ of connections are spent the night, and transform back extraction plasmid and check order.
4. the application of the described WRKY45 gene of claim 1 in cultivating the disease resistance plant variety.
5. the application of the described WRKY45 gene of claim 1 in cultivating the drought-resistant plant kind
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| WO2011103714A1 (en) * | 2010-02-24 | 2011-09-01 | Institute Of Genetics And Developmental Biology, Chinese Academy Of Sciences | Genes conferring stress tolerance in plants and uses thereof |
| CN102344487A (en) * | 2011-10-18 | 2012-02-08 | 中国科学院植物研究所 | Plant resistance-related protein ATWRKY46 as well as encoding gene and application thereof |
| CN102358904A (en) * | 2011-10-30 | 2012-02-22 | 中国科学院西双版纳热带植物园 | Arabidopsis thaliana WRKY57 gene and preparation method and application in improving crop drought resistance thereof |
| CN102653556A (en) * | 2011-03-04 | 2012-09-05 | 中国科学院遗传与发育生物学研究所 | Plant adverse resistance related transcription factor GmWRKY78 as well as encoding gene and application thereof |
| CN109136260A (en) * | 2018-10-11 | 2019-01-04 | 中国科学院微生物研究所 | The application of WRKY20 protein and its encoding gene in regulation stress resistance of plant |
| WO2019203633A3 (en) * | 2018-04-18 | 2019-11-28 | Kimagri Corporation Sdn Bhd | Recombinant gene |
| CN110663709A (en) * | 2019-11-15 | 2020-01-10 | 中南民族大学 | Preparation method and application of paris polyphylla extractive solution |
| CN113214371A (en) * | 2021-05-17 | 2021-08-06 | 西南大学 | Loquat drought-resistant related EjWRKY17 gene and encoding protein and application thereof |
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2006
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Cited By (14)
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| WO2011103714A1 (en) * | 2010-02-24 | 2011-09-01 | Institute Of Genetics And Developmental Biology, Chinese Academy Of Sciences | Genes conferring stress tolerance in plants and uses thereof |
| CN102653556B (en) * | 2011-03-04 | 2013-10-09 | 中国科学院遗传与发育生物学研究所 | Plant adverse resistance related transcription factor GmWRKY78 as well as encoding gene and application thereof |
| CN102653556A (en) * | 2011-03-04 | 2012-09-05 | 中国科学院遗传与发育生物学研究所 | Plant adverse resistance related transcription factor GmWRKY78 as well as encoding gene and application thereof |
| CN102344487B (en) * | 2011-10-18 | 2013-05-29 | 中国科学院植物研究所 | Plant resistance-related protein ATWRKY46 as well as encoding gene and application thereof |
| CN102344487A (en) * | 2011-10-18 | 2012-02-08 | 中国科学院植物研究所 | Plant resistance-related protein ATWRKY46 as well as encoding gene and application thereof |
| CN102358904A (en) * | 2011-10-30 | 2012-02-22 | 中国科学院西双版纳热带植物园 | Arabidopsis thaliana WRKY57 gene and preparation method and application in improving crop drought resistance thereof |
| CN102358904B (en) * | 2011-10-30 | 2013-03-20 | 中国科学院西双版纳热带植物园 | Arabidopsis thaliana WRKY57 gene and preparation method and application in improving crop drought resistance thereof |
| WO2019203633A3 (en) * | 2018-04-18 | 2019-11-28 | Kimagri Corporation Sdn Bhd | Recombinant gene |
| CN112313338A (en) * | 2018-04-18 | 2021-02-02 | 金农有限公司 | Recombinant gene |
| CN109136260A (en) * | 2018-10-11 | 2019-01-04 | 中国科学院微生物研究所 | The application of WRKY20 protein and its encoding gene in regulation stress resistance of plant |
| CN109136260B (en) * | 2018-10-11 | 2022-03-15 | 中国科学院微生物研究所 | WRKY20 protein and application of encoding gene thereof in regulation and control of plant stress resistance |
| CN110663709A (en) * | 2019-11-15 | 2020-01-10 | 中南民族大学 | Preparation method and application of paris polyphylla extractive solution |
| CN113214371A (en) * | 2021-05-17 | 2021-08-06 | 西南大学 | Loquat drought-resistant related EjWRKY17 gene and encoding protein and application thereof |
| CN113214371B (en) * | 2021-05-17 | 2022-04-05 | 西南大学 | Loquat drought-resistant related EjWRKY17 gene and encoding protein and application thereof |
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