CN102260683A - Gene of coding rice transcription factor WRKY protein, expression vector and application thereof - Google Patents
Gene of coding rice transcription factor WRKY protein, expression vector and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biological engineering, in particular relates to a gene of coding rice transcription factor WRKY protein, expression vector and application thereof. The genes disclosed by the invention can be expressed in the tissues such as rice roots, stems, leaves, flowers and rice ears and the like; the gene expression abundance is the highest in roots and flowers and is lower in stems, leaves and rice ears. The genes disclosed by the invention are induced by jasmonic acid, ethylene and rhizoctonia solani, and the gene expression is inhibited by salicylic acid. The protein encoded by the genes disclosed by the invention has transcriptional activation activity; the strengthening of the resistance of T2-generation rice plants of over-expression transgenic line to rhizoctonia solani shows that the gene is a positive regulatory factor for activation of plant defense responses and can be applied to disease-resistant molecule breeding engineering of crops such as rice and the like.
Description
Technical field
The invention belongs to technical field of bioengineering, be specifically related to a kind of proteic gene of coding rice transcription factor WRKY and expression vector and application.
Background technology
Plant has formed complicated and exquisite disease-resistant defense mechanism in the evolution of long period of time process.The plant defense activation of a procedure relates to the sequential regulation and control that host gene is expressed mostly, and the regulation and control majority occurs in transcriptional level.Cis-acting elements specific combination in transcription factor and the gene promoter region, thereby the spatial and temporal expression of adjusting specific gene.Expression pattern analysis, mutant and transgenic experiments have proved that all transcription factor plays a crucial role in the regulation and control of plant defense.Therefore, separate and identify that the transcriptional regulatory component in the disease-resistant signal pathway is most important to the molecule mechanism of illustrating plant defense.
WRKY albumen be mainly be present in a class transcription factor in the plant (
And Somssich, 2004).The WRKY of higher plant constitutes the transcription factor superfamily, and 100 above members (Wu et al, 2005) are arranged in the paddy rice.Their common constitutional featuress are to contain 1-2 WRKY structural domain.This structural domain comprises about 60 amino-acid residues, and seven peptide WRKYGQK of N end guarded in ten minutes, were right after the zinc fingerprint of a C2H2 or C2HC thereafter.According to the number in the conservative territory of WRKY and the type of zinc fingerprint, WRKY albumen is divided into 3 groups (Eulgem et al, 2000).
The proteic major function of WRKY is the adjusting (Pandey and Somssich, 2009) of the disease-resistant defense response of involved in plant.Evidence shows that some paddy rice WRKY genes participate in the transmission and the adjusting of disease-resistant signal.OsWRKY03,71 acts on the disease-resistant signal pathway that SA relies on, and regulates the expression (Liu et al., 2005,2007) of downstream gene OsNPR1 and PR1b.OsWRKY13 is as the activator of SA signal pathway and the arrestin of JA signal pathway, and the mediation paddy rice is to the resistance (Qiu et al., 2007) of bacterial leaf-blight and rice blast; Further analysis revealed, OsWRKY13 is by activating gsh oxidation/restoring system and flavonoid biosynthetic pathway, redox state and stimulation plant protecting chemical with the monitoring cell are synthetic, thereby play a role (Qiu et al., 2008) in disease resistance response.Equally, the functional study of OsWRKY45 is also more deep, and the overexpression plant strengthens the resistance of rice blast, RNA interferes inhibition expression plant then the resistance of this disease to be weakened, this transcription factor acts on the disease-resistant signal pathway of SA mediation, but does not rely on NH1 (Shimono et al., 2007).Another one research group finds that the Arabidopis thaliana plant of overexpression OsWRKY45 all strengthens (Qiu and Yu, 2009) to pseudomonas syringae and resistance arid, high salt, points out it to play a role in the integration of biological and abiotic stress signal pathway.What is interesting is, derive from the OsWRKY45-2 of long-grained nonglutinous rice and the allelotrope OsWRKY45-1 of japonica rice 10 amino acid whose differences are only arranged, yet they show opposite adjusting effect in the mutual work of paddy rice and bacterial leaf spot bacterium, bacillary striped germ: the former is the positive regulon of disease resistance response, the latter is negative regulon, this may be because their mediate the cause (Tao et al., 2009) of different signal pathways.The experiment of Wang et al. (2007) shows, the rice plant of overexpression OsWRKY89 strengthens the resistance of rice blast fungus, the plant that suppresses to express then shows opposite phenotype, and this regulating effect realizes by changing blade surface wax metabolism and arranged distribution.Peng et al. (2008) report, OsWRKY62 plays the part of the role of a negative regulatory factor in the plant innate immunity, and the activation of defence gene is suppressed in the overexpression strain system, and the basic resistance of bacterial leaf-blight and the resistance of Xa21 mediation are all weakened to some extent.In addition, the regulatory function of OsWRKY53,31 and 23 in the plant disease-resistant reaction also obtained parsing in various degree (Chujo et al., 2007; Zhang et al., 2008; Jing et al., 2009).
WRKY is involved in plant replying and some growth, metabolic process abiotic stress also.As participating in replying to abiotic stress such as low temperature, high salt, arid, oxidative stresses; Participate in the form structure of aging, epidermal hair and the control of seed size; Involved in sugar metabolism, the signal pathway that sesquiterpene is synthetic and dormin plant hormones such as (ABA) mediates.WRKY is also relevant to the resistance of UV-B with plant.
Paddy rice is the model plant of monocotyledons research, is the genome minimum, grass that the evolution degree is minimum, and has collinearity (colineariy) between other grass such as wheat, corn, jowar, sugarcane genome.Therefore, the research of rice genome structure and function has very big reference to the correlative study of other grass.Paddy rice is again important cash crop, it is the main food source of world's populations more than half, its genome complete sequence determination finish (International Rice Genome Sequencing Project, 2005) lay a good foundation for the research of paddy rice functional genomics, from rice genome, excavate useful gene, being used to improve output, making better products, improve resistance, is the research focus of present plant function genomics and agriculture high-tech sector.
Summary of the invention
First purpose of the present invention is to provide a kind of coding rice transcription factor WRKY proteic gene.
This gene is following (a) or (b) or (c) or (d) described nucleotide sequence:
(a) nucleotide sequence of SEQ ID No:1 in the sequence table;
(b) nucleotide sequence of the amino acid residue sequence of SEQ ID No:2 in the code sequence tabulation;
(c) with sequence table in the homology of nucleotide sequence 90% or more of SEQ ID No:1, and the proteinic nucleotide sequence of identical function of encoding;
(d) nucleotide sequence of the nucleotide sequence hybridization that under the rigorous condition of height, can limit with the SEQ ID No:1 in the sequence table;
The rigorous condition of described height is in the solution of 0.1 * SSPE or 0.1 * SSC, 0.1%SDS, hybridizes under 65 ℃ and washes film.
Described SEQ ID No:1 nucleotide sequence, specific as follows:
AATACTGAAT?AGGCAGCAGC?AACAGCAGCA?GCAGCTAATT?AAGCATCAGA?CAGAAACTAG 60
AAAGAGCGAG?CACGCCACTA?TGGATATGAT?GGAGGAGGAG?GCTGCCAACG?CCGCCACTGC 120
TCAAGCTGCT?GCTGCTGGCG?ACCTCGCCGA?CGTCGTGGCC?CGTGCCAATG?CACGCGCATT 180
CCTCGTCTCC?ACCCCTCACC?ATCACCCCTC?ACCTCTGCAT?CCTCTTCCTC?CTCCTCCCAT 240
GCCTCAGGCG?CCTCACCAAT?ACTATCCCGC?CCCCCAGATC?ACCATCCCTT?ACCACCACCA 300
CCACCACGGA?GAGCTTCGGC?GCCCTACCAC?CATCGCTTAC?ACCGACGCAC?CTGTGCCGTT 360
CGAGACGGCG?GGGCCGCCAT?CCACGGTCGT?CGACTCATAC?CACCACCTCA?CACCCGGCGA 420
CGCCGGCTAC?GGGATGCCGC?GGCCGCTTGC?TCTCCAGATC?TCCCAGCACG?CCCTCTGTGG 480
CGGCGGCGAC?GTGGTGATGG?GCGGCGGAGG?CGCAGGCGCT?GCCGATGATG?GAGAAGAAGC 540
CATCAGGATC?TCGCCACTGA?CGCCGTCTGC?TCATCATCAA?ATGATGAAAA?GGAAGAATGA 600
GGTGAAGAAA?GTGGTGTGCA?TCCCGGCGCC?GCCAGCGACG?AGTAGCCGTG?GAGGTGGAGG 660
AGAGGTGATC?CCGTCTGATC?TATGGGCATG?GAGGAAGTAC?GGCCAGAAAC?CCATCAAAGG 720
CTCTCCTTAT?CCACGGGGTT?ACTACAGATG?CAGCAGCTCA?AAGGGATGTA?TGGCGAGGAA 780
GCAGGTGGAG?CGCAGCCGCA?GCGACCCCAA?CATGCTGGTG?ATCACCTACG?CGGCGGAGCA 840
CAACCACCCA?TGGCCGATGC?AACGTAATGT?GCTCGCCGGA?TATGCCCGTT?CTCATCACAG 900
TACTCATGCT?ACTGCCTCCA?GCAGCCGCCA?CAAGCAACAG?CAGCAGCAGC?AGACCAACCA 960
GCTGCAGCCT?GCACTGATCA?CAAGCTCATC?GTCTTCTTCC?TCCTCCCCAT?TCAATCTCTA 1020
CGCCGACGTC?GTGCTCGGTG?GCCAACAGGC?CAACATGATG?ATGACGACGG?AGGGCGCCGG 1080
CGCTGGACTT?GGCATCCAAC?CTTCAGCAGC?TGATGAGGTC?TTTGCAGAGC?TGGAGGAGTT 1140
GGAGCCTGAT?AATCCTACTA?TGATCAATGC?AAACATGCAG?GTGTACTCCA?CCACCAGCAG 1200
GCCAGGGGTA?AGCAGCTATG?ATCATCAGTG?GCACAAGTTC?TAATAATTAA?ACCATGCAGT 1260
ACCTATATAT?ATATATATCA?GAACTTCAAT?ATTCGAGAGA?GGAGATCAGA?GGAAATTGAA 1320
TTCTAGCACG?TACAATTAAT?ACATCATGAC?TATGCAAAAT?TTACATATAT?ATGATATGAT 1380
GGTCGCCTGT?GCGATTATTA?GTGACCAAAT?TAAAGATTGT?CAGAGATATA?GGCGTGTACT 1440
TACGCTTATA?TGTATCACGC?TGAGGTATAC?TGTATTTACA?AGAAGATAAA?TAAAATATCC 1500
ATATC 1505
Wherein, sequence table SEQ ID No:1 method by RT-PCR from the rice seedling leaf RNA of methyl jasmonate treatment is cloned, form by 1505 deoxynucleotides, comprise the opening code-reading frame of long 1164bp, infer the protein that coding is made up of 387 amino acid.From the 1st to the 79th at 5 ' end is 5 ' untranslated region sequence, and the 80th to 1240 deoxynucleotides are encoding sequence, and the 1241st to 1243 is terminator codon, and the 1244th to 1505 is 3 ' untranslated region sequence.
Second purpose of the present invention is to provide a kind of rice transcription factor WRKY albumen of said gene coding, and this albumen is following (a) or (b) described amino acid residue sequence:
(a) the described amino acid residue sequence of SEQ ID No:2 in the sequence table;
(b) amino acid residue sequence in (a) is formed through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation have rice transcription factor WRKY protein function by (a) deutero-amino acid residue sequence;
The replacement of described one or several amino-acid residue and/or disappearance and/or interpolation are meant replacement and/or the disappearance and/or the interpolation of no more than ten amino-acid residues.
Described SEQ ID No:2 amino acid residue sequence, specific as follows:
Met?Asp?Met?Met?Glu Glu?Glu?Ala?Ala?Asn Ala?Ala?Thr?Ala?Gln Ala?Ala?Ala?Ala?Gly
5 10 15 20
Asp?Leu?Ala?Asp?Val Val?Ala?Arg?Ala?Asn Ala?Arg?Ala?Phe?Leu Val?Ser?Thr?Pro?His
25 30 35 40
His?His?Pro?Ser?Pro Leu?His?Pro?Leu?Pro Pro?Pro?Pro?Met?Pro Gln?Ala?Pro?His?Gln
45 50 55 60
Tyr?Tyr?Pro?Ala?Pro Gln?Ile?Thr?Ile?Pro Tyr?His?His?His?His His?Gly?Glu?Leu?Arg
65 70 75 80
Arg?Pro?Thr?Thr?Ile Ala?Tyr?Thr?Asp?Ala Pro?Val?Pro?Phe?Glu Thr?Ala?Gly?Pro?Pro
85 90 95 100
Ser?Thr?Val?Val?Asp Ser?Tyr?His?His?Leu Thr?Pro?Gly?Asp?Ala Gly?Tyr?Gly?Met?Pro
105 110 115 120
Arg?Pro?Leu?Ala?Leu Gln?Ile?Ser?Gln?His Ala?Leu?Cys?Gly?Gly Gly?Asp?Val?Val?Met
125 130 135 140
Gly?Gly?Gly?Gly?Ala Gly?Ala?Ala?Asp?Asp Gly?Glu?Glu?Ala?Ile Arg?Ile?Ser?Pro?Leu
145 150 155 160
Thr?Pro?Ser?Ala?His His?Gln?Met?Met?Lys Arg?Lys?Asn?Glu?Val Lys?Lys?Val?Val?Cys
165 170 175 180
Ile?Pro?Ala?Pro?Pro Ala?Thr?Ser?Ser?Arg Gly?Gly?Gly?Gly?Glu Val?Ile?Pro?Ser?Asp
185 190 195 200
Leu?Trp?Ala?Trp?Arg Lys?Tyr?Gly?Gln?Lys Pro?Ile?Lys?Gly?Ser Pro?Tyr?Pro?Arg?Gly
205 210 215 220
Tyr?Tyr?Arg?Cys?Ser Ser?Ser?Lys?Gly?Cys Met?Ala?Arg?Lys?Gln Val?Glu?Arg?Ser?Arg
225 230 235 240
Ser?Asp?Pro?Asn?Met Leu?Val?Ile?Thr?Tyr Ala?Ala?Glu?His?Asn His?Pro?Trp?Pro?Met
245 250 255 260
Gln?Arg?Asn?Val?Leu Ala?Gly?Tyr?Ala?Arg Ser?His?His?Ser?Thr His?Ala?Thr?Ala?Ser
265 270 275 280
Ser?Ser?Arg?His?Lys Gln?Gln?Gln?Gln?Gln Gln?Thr?Asn?Gln?Leu Gln?Pro?Ala?Leu?Ile
285 290 295 300
Thr?Ser?Ser?Ser?Ser Ser?Ser?Ser?Ser?Pro Phe?Asn?Leu?Tyr?Ala Asp?Val?Val?Leu?Gly
305 310 315 320
Gly?Gln?Gln?Ala?Asn Met?Met?Met?Thr?Thr Glu?Gly?Ala?Gly?Ala Gly?Leu?Gly?Ile?Gln
325 330 335 340
Pro?Ser?Ala?Ala?Asp Glu?Val?Phe?Ala?Glu Leu?Glu?Glu?Leu?Glu Pro?Asp?Asn?Pro?Thr
345 350 355 360
Met?Ile?Asn?Ala?Asn Met?Gln?Val?Tyr?Ser Thr?Thr?Ser?Arg?Pro Gly?Val?Ser?Ser?Tyr
365 370 375 380
Asp?His?Gln?Trp?His Lys?Phe
385 387
Wherein, sequence table SEQ ID No:2 is made up of 387 amino-acid residues, contains a WRKY structural domain (from N-terminal the 200th to 256 amino acids residue) and a C2H2 zinc fingerprint, classifies as the 3rd group of WRKY.
The 3rd purpose of the present invention is to provide the above-mentioned coding rice transcription factor WRKY recombinant expression vector of proteic gene.
Gene of the present invention has expression in tissues such as rice root, stem, leaf, flower, fringe.Root, to express abundance in spending the highest, is stem, leaf secondly, and the abundance in the fringe is low.Gene of the present invention is induced by jasmonic, ethene and Rhizoctonia solani Kuhn, and Whitfield's ointment then suppresses its expression.
The 4th purpose of the present invention is to provide the above-mentioned coding rice transcription factor WRKY transgenic cell line of proteic gene.
The 5th purpose of the present invention is to provide the above-mentioned coding rice transcription factor WRKY engineering bacteria of proteic gene.
The 6th purpose of the present invention is to provide the application of the proteic gene of above-mentioned coding rice transcription factor WRKY in cultivating the resistance plant.More particularly, described resistance plant is a paddy rice.
Rice transcription factor WRKY albumen of the present invention has transcriptional activation activity.
The T2 of overexpression transgenic line of the present invention strengthens the resistance of banded sclerotial blight for rice plant, the defense response of paddy rice to disease that shown this gene mediated.
Description of drawings
Fig. 1 is the proteic transcriptional activation activity analysis chart group of embodiment of the invention genes encoding.
Fig. 2 is that embodiment of the invention gene is in HORMONE TREATMENT and the postvaccinal expression figure of sheath blight fungus group.
Fig. 3 be the overexpression vector of embodiment of the invention gene make up and T2 for the Northern analysis chart group of overexpression rice plant.
Fig. 4 is that the T2 of embodiment of the invention gene is for the anti-banded sclerotial blight analysis chart of overexpression rice plant.
Embodiment
The present invention is described in further detail below in conjunction with drawings and Examples.
Experimental technique among the following embodiment if no special instructions, is ordinary method.
Percentage composition among the following embodiment if no special instructions, is the quality percentage composition.
Restriction enzyme that relates in the experimentation and T
4Dna ligase is produced by precious biotechnology (Dalian) company limited, consults and uses specification sheets and uses.
One, the separation of the cDNA sequence of gene of the present invention:
Rice cropping kind show water seedling in 11 tri-leaf period is sprayed with 100 μ M methyl jasmonates, gets and handles back 1,6 and 12 hour blade extraction RNA, and balanced mix RNA sample after the reverse transcription (RT), carries out pcr amplification.Amplification condition is: pre-sex change is 3 minutes under 94 ℃ the temperature, again 94 ℃ following 30 seconds, 56 ℃ following 30 seconds, 72 ℃ following 1 minute 40 seconds, 30 circulations were extended 10 minutes down at 72 ℃ at last.Amplification obtains the cDNA fragment of 1505bp.Behind the PCR product purification, be connected on the pUCmT carrier sequence verification, i.e. SEQ ID № in the calling sequence table: 1.Primer is 5 '-AATACTGAATAGGCAGCAGCAACA-3 ' and 5 '-GCACAGGCGACCATCATATCATAT-3 '.
Two, the proteic transcriptional activation analysis of genes encoding of the present invention:
Referring to Fig. 1, the A of Fig. 1 partly is the synoptic diagram group of the growing state of yeast transformant.The middle graph of A part is a subregion inoculation synoptic diagram: 1 expression pCL1 (over against shining) carrier, and 2 expression pGBKT7 (pBD, negative contrast) carrier, 3 expression pBD-WRKY carriers, 4 represent pBD-WRKY Δ C carriers.The structure and the implication of carrier stated " concrete experimentation " as follows.
The left figure of A part represents transformant all growths on the SD-Trp substratum of above-mentioned four kinds of carriers.
What the right figure of A part represented is that pCL1, pBD-WRKY and pBD-WRKY Δ C transformant are grown on selection substratum SD-Trp-Ade-His, and the pBD transformant is not grown.
The B of Fig. 1 partly is the relative reactivity chart of the alpha-galactosidase of transformant, and the alpha-galactosidase activity that contains the transformant of pCL1 (over against shining) is defined as 100%.Alpha-galactosidase activity is measured with test kit (available from Clotech company), is undertaken by supplier's specification sheets.
Concrete experimentation is as follows:
With primer 5 '-GGAATTC
CATATGTATGGATATGATGGAGGAGGAGG-3 ' (underscore is the NdeI site) and 5 '-CG
GGATCCThe whole coding region of GAACTTGTGCCACTGATGATCATAG-3 ' (underscore is the BamHI site) amplification gene of the present invention (from SEQ ID №: 1 79 to 1216) is with primer 5 '-GGAATTC
CATATGGCAACAGCAGCAGCAGCAGA-3 ' (underscore is the NdeI site) and 5 '-CG
GGATCCThe coding region of 102 amino-acid residues of disappearance C end of GAACTTGTGCCACTGATGATCATAG-3 ' (underscore is the BamHI site) amplification gene of the present invention (in SEQ ID No:1 934 to 1216), the PCR product is connected respectively to the GAL4DNA of pGBKT7 carrier in conjunction with on territory (BD), make up pBD-WRKY and pBD-WRKY Δ C carrier, respectively with pCL1 (coding total length GAL4) and empty carrier pGBKT7 (pBD) as positive and negative contrast, conversion AH109 yeast strains.Transformant selects on the substratum 30 ℃ to cultivate 3 days at SD substratum and SD-Trp-Ade-His, observe growing state, discovery pCL1, pBD-WRKY and pBD-WRKY Δ C transformant can be grown on above-mentioned selection substratum preferably, and the transformant that carries empty carrier pBD can not be grown, shown in the A part of Fig. 1, illustrate that the albumen of genes encoding of the present invention has the activity of transcriptional activation.The alpha-galactosidase activity of pBD-WRKY Δ C is 28.9% (shown in the B part of Fig. 1) of pBD-WRKY, illustrates that C-holds 102 amino-acid residues to be responsible for proteic most of transcriptional activation activity of genes encoding of the present invention.
Three, the abduction delivering analysis of gene of the present invention:
(1) paddy rice is cultivated and pathogenic bacteria inoculation, HORMONE TREATMENT:
Rice varieties show water 11 seeds soaked 1 day down at 37 ℃, and the back vernalization 1 day that shows money or valuables one carries unintentionally is sowed then in being added with the vermiculite of nutritive medium, cultivates in the greenhouse.28 ℃ of (daytime)/25 ℃ (night), photoperiod 14/10h, intensity of illumination 100 μ mol m
-2s
-1, relative humidity is 85%.Tri-leaf period, seedling was used for various processing, in the different time sampling, got blade and extracted RNA.
Sheath blight fungus GD118 obtains from national public welfare industry (agricultural) scientific research special project (nyhyzx3-16) rice sheath blight disease controlling cooperative groups.Inoculum preparation: the paddy kernel that will soak 2 days is packed in the culture dish, 121 ℃ of sterilizations 30 minutes, after the cooling, with the withered bacterium inoculated by hypha block of the line of 3 days cell ages in sterilized paddy kernel, in the dark down cultivation of 28 ℃ of high humidity environments 3 days.Two inoculums that carry mycelia are against rice strain base portion, about each one.Postvaccinal paddy rice is cultivated under room temperature, keeps humidity about 85%.Inoculating with the inoculum simulation of not carrying mycelia is contrast.Get blade respectively at 0,1,6,12, behind the 24h and extract total RNA.
HORMONE TREATMENT: with 100 μ mol L
-1Methyl jasmonate (MeJA), 2mmol L
-1Whitfield's ointment (SA) and 1mmol L
-1Ethrel (ET) sprays the rice seedling in tri-leaf period, respectively at 0,1,6,12,24h gets blade and extract total RNA.Wherein methyl jasmonate is dissolved in small amount of methanol earlier, uses ddH again
2O is molten, and the methyl alcohol final concentration is 0.1% (volumn concentration).
(2) RNA extraction, Northern hybridization and real-time fluorescence RT-PCR analysis
Adopt the PureYield of Promega company
TMRNA Midiprep System extracts total RNA and extracts from the leaf of 3 leaf phase paddy rice (elegant water 11), undertaken by working instructions.The amplimer of the specific probe of gene of the present invention is: 5 '-CAATGCACGCGCATTCCTCG-3 ' and 5 '-CGTCAGTGGCGAGATCCTGA-3 '.This probe is used for the Northern hybridization analysis.The total RNA of 10 μ g electrophoresis classification in 1.2% denaturing formaldehyde agarose gel is transferred to Hybond-N then
+On the nylon membrane, with [α-
32P]-gene-specific probe of mark is 65 ℃ of hybridization down.Under 65 ℃, add 0.1%SDS with 2 * SSC and washed 5 minutes, under same temperature, add 0.1%SDS and washed again 5 minutes with 1 * SSC.Mould phosphorus screen (Amersham Pharmacia, UK).
With the total RNA of 2 μ g is that template is carried out reverse transcription, adopts Invitrogen Corporation's Super Scriptreverse transcriptase II, presses the working instructions operation.Real-time fluorescence RT-PCR adopts ARI PRISM7000 Sequence Detection System (Applied Biosystems), is undertaken by following condition: 94 ℃ of pre-sex change 2 minutes; 94 ℃ of sex change 5 seconds, 54 ℃ of annealing 10 seconds; 72 ℃ were extended 40 circulations 20 seconds.Paddy rice Actin1 (AK071586) is as interior mark.The Auele Specific Primer of amplification gene of the present invention is: 5 '-AACAGCAGCAGCAGCAGA-3 ' and 5 '-GTCGGCGTAGAGATTGAATGG-3 '.The amplimer of Actin1 is: 5 '-GGCACCACACCTTCTACAATGAG-3 ' and 5 '-ACACCATCACCAGAGTC AAGCA-3 '.
The result as shown in Figure 2, the A of Fig. 2 partly analyzes for the Northern that gene of the present invention is expressed under sheath blight fungus, methyl jasmonate and ethrel are handled, the B of Fig. 2 partly for Whitfield's ointment handle under the real-time fluorescence quantitative RT-PCR analysis of genetic expression of the present invention.As seen, gene of the present invention is expressed by sheath blight fungus inoculation, jasmonic and ethephon-induction, and is not subjected to Induced by Salicylic Acid, illustrates that this gene may participate in the disease-resistant defence signal pathway of jasmonic/ethene mediation.
Four, the disease-resistant analysis of the overexpression transfer-gen plant of gene of the present invention:
Referring to Fig. 3, A is partly for the overexpression vector synoptic diagram of gene of the present invention, WRKY refers to the complete encoding sequence (in SEQ ID No:1 the 80th to 1240) of gene of the present invention, LB and RB refer to the left and right border of pCambia1301 carrier respectively, T7 terminator refers to the T7 terminator, and Ubi refers to the promotor of corn ubiquitin gene.B partly is the Northern analysis chart of gene of the present invention in the overexpression rice plant (T2 generation), and wherein WT refers to the wild-type contrast, and 1-7,3-9,9-8,10-2,11-6,12-2 and 16-1 represent that 7 representational T2 are for transgenic rice plant.
Concrete experimentation is as follows:
The complete encoding sequence of gene cDNA of the present invention is inserted in the pCambia1301 carrier of a reconstruction, place under the control of corn ubiquitin gene promoter, make up overexpression vector (ubiquitin::WRKY), see Fig. 3, then overexpression vector is changed in the callus of paddy rice (elegant water 11) with agriculture bacillus mediated conversion method.Concrete grammar is: ripe rice paddy seed carries out surface sterilization after 1 minute with 70% ethanol, sterilizes 30 minutes with the clorox of 20% (v/v), cultivates (MS interpolation 0.5g L on the NBI substratum
-1Glutamine, 0.5g L
-1Prolineamide, 2.0mg L
-12,4-D; PH 5.8), grow callus after about 14 days, callus is peeled, put on the new NBI substratum and cultivate, can infect after 4 days; The agrobacterium liquid that will contain ubiquitin::WRKY is evenly coated on the solid YEP flat board to be cultivated 2-3 days, scraped bacterium with aseptic spoon from flat board and was total to substratum (MS+2g L in liquid
-1Inositol+0.3g L
-1Casein hydrolysate+100 μ M Syringylethanones) in to cell concn be 3 * 10
9Cfu mL
-1, callus is put into bacterium infected 20 minutes, callus is poured on the bacterium liquid that blots the surface on the clean filter paper, move on on the Nbco substratum and cultivate (the NBI substratum adds 100 μ M Syringylethanones); Cultivate after 2 days, callus is transferred to the last cultivation of screening culture medium NBs, and (the NBI substratum adds 500mg L
-1Cephamycin, 30mg L
-1Totomycin); By the time callus goes out green back (about 30 days) and moves to division culture medium and (add 2.0mg L in the MS substratum
-16-BAP, 0.5mg L
-1Naphthylacetic acid, 30g L
-1Sorbyl alcohol and 30mg L
-1Totomycin) goes up cultivation.The seedling that obtains be T0 for the transfer-gen plant kind in the greenhouse, T0 is T1 generation for the plant that the seed of transfer-gen plant selfing gained grows up to, T1 is T2 generation for the plant that the seed of transfer-gen plant selfing gained grows up to.The Northern analysis revealed, T2 obviously strengthens than wild-type for expression of gene of the present invention in the plant, shown in the B part of Fig. 3.
The disease-resistant evaluation of the overexpression transfer-gen plant of gene of the present invention adopts T2 for transgenic rice plant.Fig. 4 is the anti-banded sclerotial blight analysis chart of overexpression rice plant (T2 generation) of gene of the present invention, and WT refers to the wild-type contrast among the figure, and #3-9 and #10-2 represent that 2 representational T2 are for transgenic rice plant.As seen from Figure 4, the transfer-gen plant of overexpression obviously strengthens the resistance bacterium of sheath blight fungus.
Claims (7)
1. coding rice transcription factor WRKY proteic gene is characterized in that being following (a) or (b) or (c) or (d) described nucleotide sequence:
(a) nucleotide sequence of SEQ ID No:1 in the sequence table;
(b) nucleotide sequence of the amino acid residue sequence of SEQ ID No:2 in the code sequence tabulation;
(c) with sequence table in the homology of nucleotide sequence 90% or more of SEQ ID No:1, and the proteinic nucleotide sequence of identical function of encoding;
(d) nucleotide sequence of the nucleotide sequence hybridization that under the rigorous condition of height, can limit with the SEQ ID No:1 in the sequence table;
The rigorous condition of described height is in the solution of 0.1 * SSPE or 0.1 * SSC, 0.1% SDS, 65
oHybridize under the C and wash film.
2. rice transcription factor WRKY albumen is following (a) or (b) described amino acid residue sequence:
(a) the described amino acid residue sequence of SEQ ID No:2 in the sequence table;
(b) amino acid residue sequence in (a) is formed through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation have rice transcription factor WRKY protein function by (a) deutero-amino acid residue sequence;
The replacement of described one or several amino-acid residue and/or disappearance and/or interpolation are meant replacement and/or the disappearance and/or the interpolation of no more than ten amino-acid residues.
3. recombinant expression vector that contains the proteic gene of rice transcription factor WRKY of encoding according to claim 1.
4. transgenic cell line that contains the proteic gene of rice transcription factor WRKY of encoding according to claim 1.
5. engineering bacteria that contains the proteic gene of rice transcription factor WRKY of encoding according to claim 1.
6. the application of the proteic gene of rice transcription factor WRKY in cultivating the resistance plant of encoding according to claim 1.
7. according to the application of the proteic gene of the described coding rice transcription factor of claim 6 WRKY in cultivating the resistance plant, it is characterized in that: described resistance plant is a paddy rice.
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| CN102816773A (en) * | 2012-08-30 | 2012-12-12 | 中山大学 | Application of OsWRKY28 transcription factor gene of rice in improvement of plant disease resistance |
| CN109735550A (en) * | 2019-01-18 | 2019-05-10 | 上海交通大学 | A kind of nucleotide sequence and its application in raising plant secretion type trichome density |
| CN111995668A (en) * | 2020-07-27 | 2020-11-27 | 安徽农业大学 | Corn WRKY transcription factor ZmWRKY112 and coding gene and application thereof |
| CN112725351A (en) * | 2021-03-23 | 2021-04-30 | 上海师范大学 | Application of gene OsWRKY43 in resisting bacterial blight of rice |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102816773A (en) * | 2012-08-30 | 2012-12-12 | 中山大学 | Application of OsWRKY28 transcription factor gene of rice in improvement of plant disease resistance |
| CN102816773B (en) * | 2012-08-30 | 2014-01-29 | 中山大学 | Application of OsWRKY28 transcription factor gene of rice in improvement of plant disease resistance |
| CN109735550A (en) * | 2019-01-18 | 2019-05-10 | 上海交通大学 | A kind of nucleotide sequence and its application in raising plant secretion type trichome density |
| CN109735550B (en) * | 2019-01-18 | 2022-11-11 | 上海交通大学 | Nucleotide sequence and application thereof in improving density of plant secretory glandular hairs |
| CN111995668A (en) * | 2020-07-27 | 2020-11-27 | 安徽农业大学 | Corn WRKY transcription factor ZmWRKY112 and coding gene and application thereof |
| CN112725351A (en) * | 2021-03-23 | 2021-04-30 | 上海师范大学 | Application of gene OsWRKY43 in resisting bacterial blight of rice |
| CN112725351B (en) * | 2021-03-23 | 2022-11-11 | 上海师范大学 | Application of gene OsWRKY43 in resisting bacterial blight of rice |
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