CN110256549A - Plant disease-resistant Protein G hWRKY40 and encoding gene and its application - Google Patents
Plant disease-resistant Protein G hWRKY40 and encoding gene and its application Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
Abstract
A kind of protein of disclosure of the invention, entitled GhWRKY40, for albumen shown in such as (a) or (b) or (c): (a) amino acid sequence is protein shown in sequence 2 in sequence table；(b) fused protein that the N-terminal of protein shown in sequence 2 or/and C-terminal connection label obtain in sequence table；Protein shown in by (a) or (b) by replacement, missing or/and the insertion of one or more amino acid it is derivative obtain still there is GhWRKY40 functional transcription factor or active protein.The beneficial effects of the present invention are: the transcriptional expression of GhWRKY40 regulation specific gene; cause the change of plant physiology and biochemistry; increase plant to pathogen invasion resistance, to protect the growth and development of plant, is of great significance improving plant to the application in terms of pathogen response.
Description
Technical field
The invention belongs to technical field of biotechnology, and in particular to a kind of plant disease-resistant Protein G hWRKY40 and encoding gene
And its application.
Background technique
Cotton belongs to Malvaceae cotton, and cultivar has 4 kinds, including upland cotton, sea island cotton, cotton and Asiatic cotton,
Wherein upland cotton is main breed.Cotton is important industrial crops, and fiber is the important source material of textile industry, and cottonseed oil is
One of the main source of food and chemical industry oil, Cottonseed Meal are the albumen adding ingredients of the ruminant feeds such as cattle and sheep.However cotton
Colored yield and quality is influenced by various biotics and abiotic stress, and wherein cotton verticillium wilt seriously restricts cotton
Production.Verticillium wilt is a kind of vascular bundle diseases of soil-borne, and pathogen is verticillium dahliae, which belongs to Fungi Imperfecti Asia
Trolley branch Pseudomonas.Pathogen is invaded from cotton root, and the fiber production and quality of cotton can be seriously affected after the onset of plant.With
Cotton verticillium wilt distribution is wide, and harm is serious, and route of transmission is mostly long with life span.In order to solve this stubborn problem, cultivate
Disease-resistant variety is most economical effective method.However, the difficult point of cotton disease resistance breed of variety is the acquisition of Resistant gerplasm resource.
Currently, due to the germ plasm resource for lacking anti pathologic immunity, so being difficult effectively to cultivate disease-resistant variety using conventional breeding methods.Cause
This, excavates disease-resistant related gene using technique for gene engineering, formulates disease-resistant varieties, becomes current and solves cotton verticillium wilt harm
Important research direction.In short, the disease resistance of raising cotton is for the safety in production of cotton and people's good life with important
Meaning.
The excavation of plant disease resistance genes relates generally to the regulatory factor of some controlling gene expression, mainly includes modulin
And transcription factor.Wherein transcription factor is also known as trans-acting factor, specifically binding to eukaryotic gene promoter region cis acting
On element, and activate or inhibit the transcription of downstream gene.These transcription factors mainly include NAC, MYB, bZIP, WRKY etc., he
Play an important role in plant growth and development and defense response.
Based on this, a kind of transcription factor that raising plant acts on pathogen response is studied, it is anti-yellowing especially for cotton
The transcription factor of disease of withering reaction is just particularly important.
Summary of the invention
In order to make up for the deficiencies of the prior art, it improves plant to act on the response of pathogen, the present invention provides Genes For Plant Tolerances
Sick Protein G hWRKY40 and encoding gene and its application.
The present invention provides a kind of protein, entitled GhWRKY40, for albumen shown in such as (a) or (b) or (c):
(a) amino acid sequence is protein shown in sequence 2 in sequence table；
(b) fused protein that the N-terminal of protein shown in sequence 2 or/and C-terminal connection label obtain in sequence table；
(c) by (a) or (b) shown in protein spread out by replacement, missing or/and the insertion of one or more amino acid
What life obtained still has GhWRKY40 functional transcription factor or active protein.
In order to make protein shown in above-mentioned (a) convenient for purifying, can in sequence table protein shown in sequence 2 amino
End or carboxyl terminal connect upper label as shown in Table 1.
The sequence of 1 label of table
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (usually 5) | RRRRR |
| Poly-His | 2-10 (usually 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
(b) or (c) shown in protein can be artificial synthesized, can also first synthesize its encoding gene, then carry out biological table
It reaches.
(b) or (c) shown in protein encoding gene can by will in sequence table in base sequence shown in sequence 1 lack
The codon of one or several amino acid residues is lost, and/or carries out the missense mutation of one or several base-pairs, and/or at it
The coded sequence that the end 5' and/or 3, end connect label shown in table 1 obtains.
It is following (1) into (12) the present invention also provides biomaterial relevant to the protein G hWRKY40
It is any:
(1) nucleic acid molecules of protein described in claim 1 are encoded；
(2) contain the expression cassette of (1) described nucleic acid molecules；
(3) contain the recombinant vector of (1) described nucleic acid molecules；
(4) contain the recombinant vector of (2) described expression cassette；
(5) contain the recombinant microorganism of (1) described nucleic acid molecules；
(6) contain the recombinant microorganism of (2) described expression cassette；
(7) contain the recombinant microorganism of (3) described recombinant vector；
(8) contain the recombinant microorganism of (4) described recombinant vector；
(9) contain the transgenic plant cells system of (1) described nucleic acid molecules；
(10) contain the transgenic plant cells system of (2) described expression cassette；
(11) contain the transgenic plant cells system of (3) described recombinant vector；
(12) contain the transgenic plant cells system of (4) described recombinant vector.
The biomaterial, nucleic acid molecules described in (1) are DNA molecular shown in following (a1) or (b1) or (c1):
(a1) nucleotide sequence is DNA molecular shown in sequence 1 in sequence table；
(b1) one or more alkali are carried out on the basis of nucleotide sequence is the nucleotide sequence shown in sequence 1 in sequence table
Replacement, missing or/and the insertion of base and it is derivative obtain DNA molecular, and the encoded protein of the DNA molecular is claim 1
The protein；
(c1) nucleotide sequence hybridization limited under strict conditions with (a1) or (b1), and encode described in claim 1
The DNA molecular of protein.
Wherein, sequence 1 is a kind of cotton transcription factor encoding gene cloned from cotton leaf in sequence table, name
For GhWRKY40, open reading frame 981bp, the sequence being made of 326 amino acid residues is encoded, particular sequence is in sequence table
Sequence 2.The transcriptional expression for regulating and controlling specific gene, causes the change of plant physiology and biochemistry, increases plant to pathogen invasion resistance,
To protect the growth and development of plant.
The present invention also provides the protein or the biomaterial to improve plant in pathogen response
Using.
The present invention also provides the protein or the biomaterial to improve plant to vascular bundle parasitism cause of disease
Application in bacterium response.
The present invention also provides the protein or the biomaterial to improve plant to verticillium dahliae response
In application.
In above-mentioned application, the plant is cotton.
The invention has the beneficial effects that: the present invention is designed specific primer and is cloned from cotton leaf using round pcr
The gene, is named as GhWRKY40.It finds that the gene is expressed in the root, stem and leaf of cotton using qPCR analysis, is excellent in root
Gesture expression, the expression quantity in leaf are lower；GhWRKY40 expression simultaneously is by verticillium dahliae, jasmonic and salicylic lures
It leads.GhWRKY40 gene silencing plant is obtained by Gene Silencing technology.Big beautiful wheel is inoculated with to gene silencing plant
Branch bacterium carries out Disease-resistance Analysis, and the diseased plant rate and disease of silencing plant, which refer to, as the result is shown is below adjoining tree, shows silencing
GhWRKY40 gene can be improved cotton plant disease resistance.Therefore GhWRKY40 is the disease-resistant transcription factor of a negative regulation, Ke Yizuo
For the candidate gene of verticillium wilt resistance of cotton by same breeding, there is preferable application potential.The transcription table of GhWRKY40 regulation specific gene
It reaches, causes the change of plant physiology and biochemistry, increase plant to pathogen invasion resistance, so that the growth and development of plant is protected,
Plant is improved to be of great significance to the application in terms of pathogen response.
Detailed description of the invention
Fig. 1 is cotton GhWRKY40 and other plant WRKY40 phylogenetic tree analyzes comparison diagram, and At indicates quasi- south in figure
Mustard, Mt indicate that clover, Tc indicate that cocoa, Pt indicate that comospore poplar, Bn indicate that rape, Gm indicate that soybean, Ha indicate sunflower, Br table
Show that Chinese cabbage, Fm indicate that Manchurian ash, Gh indicate cotton；
Fig. 2 is specific expressed analysis chart of the GhWRKY40 in cotton different tissues organ；
Fig. 3 is expression analysis schematic diagram of the cotton GhWRKY40 gene under SA processing；
Fig. 4 is expression analysis schematic diagram of the cotton GhWRKY40 gene under JA processing；
Fig. 5 is infected the expression analysis schematic diagram of rear GhWRKY40 in different time points by verticillium dahliae for cotton；
Fig. 6 is that PYL-156-WRKY40 silent carrier constructs schematic diagram；
Fig. 7 is GhPDS silencing plant leaf albefaction phenotypic map；
Fig. 8 is that cotton GhWRKY40 gene silencing analyzes schematic diagram；
Fig. 9 is cotton GhWRKY40 gene silencing plant and adjoining tree incidence schematic diagram；
Figure 10 is cotton GhWRKY40 gene silencing plant and adjoining tree diseased plant rate figure；
Figure 11 is cotton GhWRKY40 gene silencing plant and adjoining tree disease index figure.
Specific embodiment
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, to the technology in the embodiment of the present invention
Scheme is clearly and completely described, it is clear that and described embodiments are some of the embodiments of the present invention, rather than whole
Embodiment.
Embodiment 1
1. experimental material and reagent
Cotton material is nakamise 35, is purchased from Shanxi Agricultural academy of sciences Cotton Research Institute seedling company；Escherichia coli ' DH5
α ' competent cell and Agrobacterium tumefaciens strain ' GV3101 ' are saved by Research of Plant Genomics National Key Laboratory；Verticillium wilt pathogen
Bacterial strain (verticillium dahliae, V.dahliae) ' V991 ' is presented by Plant Protection institute, Chinese Academy of Agricultral Sciences letter Gui Liang researcher
It gives；Various restriction enzymes and Buffer are purchased from TaKaRa company, and pillar plant total serum IgE separation and Extraction purification kit is purchased from
Shanghai Sheng Gong bioengineering limited liability company, Plasmid Miniprep Kit (upgrade version centrifugal column type) are raw purchased from Shanghai JaRa
Object Engineering Co., Ltd；Taq DNA polymerase, dNTPs, Taq Buffer, T4DNA ligase, PCR purification and recovery kit,
PCR gel extraction kit, cloning vector pEASY-T1 kit, EasyScript One-Step gDNA Removel and
CDNASynthesis SuperMix kit, TransStart Top Green qPCR SuperMix kit are purchased from north
Jing Quanshijin Bioisystech Co., Ltd.
2. experimental method and interpretation of result: the analysis of cotton GhWRKY40 gene cloning and expression
(1) extraction of total serum IgE and the synthesis of the first chain of cDNA
Cotton seeds are impregnated with sterile water, are placed in 37 DEG C of soaked overnights, is then displaced in culture box, is placed in 25 DEG C
48h is handled in illumination box, the photoperiod is 12h illumination 8h dark, it is displaced in water-filled culture box after waiting germinations,
It is placed into incubator and is cultivated.Cotton seedling grows the root, stem and leaf sample for winning cotton seedling after true leaf respectively, uses
Pillar plant total serum IgE separation and Extraction purification kit extracts cotton RNA.The synthesis of the first chain of cDNA is referring to EasyScript
One-Step gDNA Removal and cDNA Synthesis SuperMix kit illustrates to carry out.
(2) cotton GhWRKY40 gene cloning
Design primer, primer GhWRKY40-F:5 '-ATGAATATATTAGATCTGGGTCTTTGC-3 '；GhWRKY40-
R:5 '-ATTGGAATTTGTTGAAAACATTCTTC-3 '.All primers of this experiment are by Beijing Zi Xi Biotechnology Co., Ltd
Synthesis.PCR amplification is carried out by template of the cDNA of cotton leaf, PCR product is connected in carrier T and is sequenced, is sequenced by north
Bioisystech Co., Ltd, Jing Qing section completes.The result shows that the clip size and expected in the same size, the segment overall length that obtain
981bp, containing the TGA since ATG terminates complete ORF reading frame, encodes 326 amino acid residues, and molecular weight is
44.8KDa, isoelectric point pI=5.21 (referring to sequence 1 and sequence 2 in sequence table).
(3) GhWRKY40 gene order systematic evolution tree constructs
The amino acid sequence of the WRKY40 of different plants is chosen in NCBI, they are respectively: arabidopsis AtWRKY40
(Arabidopsis thaliana, NP_178199.1), clover MtWRKY40 (Medicago truncatula, XP_
013465603.1), cocoa TcWRKY40 (Theobramo cacao, EOY26052.1), comospore poplar PtWRKY40 (Populus
Trichocarpa, XP_002303852.3), rape BnWRKY40 (Brassica napus, NP_001303203.1), soybean
GmWRKY40 (Glycine max, XP_003530265.1), sunflower HaWRKY40 (Helianthus annuus, XP_
021984272.1), Chinese cabbage BrWRKY40 (Brassica rapa, NP_001288924.1), Manchurian ash FmWRKY40
(Fraxinus mandshurica, AMR43674.1).They are subjected to system with cotton GhWRKY40 using MEGA5.2 program
The building of chadogram, the result is shown in Figure 1, it can be seen from the figure that GhWRKY40 and cocoa (Theobramo cacao) WRKY40 egg
The white affiliation with height belongs to IIa. class WRKY.Simultaneously it can also be seen that WRKY in plant there are biggish variation, show
It executes different physiology and biochemical function in plant.
(4) expression analysis of GhWRKY40 gene under the conditions of different tissues and different disposal
(4.1) acquisition and processing setting of sample
V.dahliae connects bacterium: the cotton seeds for sending out bud good being planted into water planting culture box, illumination 12h, dark are placed in
It is cultivated in 8h, 25 DEG C of incubator, chooses the consistent and cotton seedling with two panels true leaf of growing way and carry out different disposal.To cotton
Strain main root is put into after uniformly being hurt root processing containing 10650min is impregnated in a/mL V.dahliae spore liquid, is then turned again
Into water planting box, it is placed in and continues to cultivate in incubator.Cotton plant root sample (0,6,12,24,36 and are taken according to corresponding time point
48h), it is stand-by that -80 DEG C of refrigerators are stored in.
HORMONE TREATMENT: handling above-mentioned similar cotton seedling, and the method for processing is carried out according to report.It will be configured
Jasmonic (JA) and salicylic acid (SA) mother liquor are added separately in the culture box containing Hoaglangs nutrient solution, make its final concentration point
Not Wei 0.1mmol/L and 2mmol/L, take cotton plant root sample (0,6,12,24,36 and 48h) according to corresponding time point, be stored in-
80 DEG C of refrigerators are stand-by.
(4.2) Quantitative Real-time RT-PCR (qRT-PCR) method
Using qRT-PCR method, detects expression and its pathogen of the GhWRKY40 in Levant Cotton Root, stem and leaf and swash
Expression after element induction, expands the primer of GhWRKY40 are as follows: qGhWRKY40-F:5 '-CAGCAACAACAACA
ATGGG-3'；QGhWRKY40-R:5 '-TAACAGGGCAACTTGGTGC-3.' using cotton UBQ7 as internal reference base
Cause, amplimer are as follows: UBQ7-F:5 '-GAAGGCATTCCACCTGACCAAC-3 '；UBQ7-R:5'-
CTTGACCTTCTTCTTCTTGTGCTTG-3'.Reaction system used in qRT-PCR is 20 μ L, with the cotton sample after different disposal
This cDNA is template, and specific reaction system is configured according to kit operation instruction.Amplification condition are as follows: 95 DEG C, 5min；95
DEG C, 10s；60 DEG C, 30s；72 DEG C, 30s；40 circulations.Using UBQ7 gene as internal reference, biological experiment is repeated 3 times.The phase of gene
2 are passed through to expression quantity–ΔΔCTMethod calculates.
(4.3) organizing specific expression is analyzed
The root of cotton, the total serum IgE of stem and foliage organ are extracted, reverse transcription is carried out at cDNA, using the cDNA as template
QRT-PCR analysis, the results showed that GhWRKY40 gene is expressed in these histoorgans, and referring to fig. 2, but it is in root
Expression quantity is higher, and stem takes second place, and the expression quantity in leaf is minimum, and implying the function of the gene, there may be tissue specificities.
(4.4) hormon handles lower GhWRKY40 expression analysis
Cotton is handled using SA and JA, respective treated RNA is extracted and reverse transcription carries out qRT-PCR at cDNA and divides
Analysis, the results showed that SA and JA can induce GhWRKY40 expression and reach most after SA processing in 1.5 hours GhWRKY40 expression quantity
Greatly, it is hereafter gradually reduced, in addition to 12 hours lower than control, the expression quantity of other times is above control, referring to Fig. 3.At JA
After reason, reach first peak in 1.5 hours GhWRKY40 expression quantity, but minimum in 3 hours expression quantity, expression quantity reaches within 12 hours
To maximum, second peak, bimodal trend is presented in overall say, referring to fig. 4.Illustrate the gene pairs both hormones all there is
Responsing reaction, but the mode of response has differences.
(4.5) cotton GhWRKY40 inducing expression is analyzed in verticillium dahliae invasion
In order to analyze the disease resistance whether GhWRKY40 gene participates in cotton, with the spore liquid of verticillium dahliae to cotton seedling into
Row connects bacterium, extracts Levant Cotton Root RNA and reverse transcription is analyzed using the cDNA as template by qRT-PCR method at cDNA
The response of GhWRKY40 gene pairs verticillium dahliae, the results showed that its expression quantity shows first to increase the trend reduced afterwards, connects bacterium 6
Hour its expression quantity has almost no change, however its expression quantity acutely increases at 12 hours, reaches maximum value, hereafter gradually drops
Low, in addition to 48 hours lower than control, the expression quantity of other times is obviously higher than control, referring to figure Fig. 5.Show GhWRKY40
Induced strong of the gene by verticillium dahliae may participate in cotton to the resistance of verticillium wilt.
Embodiment 2
1. experimental material and reagent
With embodiment 1.
2. experimental method and interpretation of result: the Analysis of Resistance of GhWRKY40 gene silencing plant pair verticillium dahliae
(1) cultivation (1.1) of Gene Silencing (virus-induced gene silencing, VIGS) plant
The building of viral silent carrier pYL-156-GhWRKY40
Viral silencing expression vector pYL-156 teaches Hui Zeng by Tsinghua University Liu Yule.First with pcr clone
The target fragment of GhWRKY40 gene, amplification the primer are VGhWRKY40-F:5 '-CGGGATCCGAATACTGAGAAAGAA
CTTAGTCCTAC-3'；VGhWRKY40-R:5 '-GGGGTACC(underscore is respectively AACCGGAGTCGAACCAACAG-3 '
BamH I and Kpn I restriction enzyme site).With restriction enzyme BamH I and Kpn I digestion pYL-156 carrier and amplified production,
Then it is attached reaction, segment is inserted between BamH I and the Kpn I site of pYL-156 carrier and is built into viral silencing
Carrier pYL-156-GhWRKY40, referring to Fig. 6.Successfully virus silent carrier pYL-156-GhWRKY40 will be constructed and convert large intestine
After bacillus DH5 α, positive colony is selected, carries out BamH I and Kpn I digestion and sequencing identification, the results showed that obtained
Section is the target fragment of the GhWRKY40 gene to be cloned.
(1.2) culture of engineering Agrobacterium
Correctly viral silent carrier pYL-156-GhWRKY40, assistant carrier pYL-192, positive control will have been constructed to carry
Body pYL-156-PDS (preservation of Research of Plant Genomics National Key Laboratory) and negative control vector pYL-156 is turned with electric shocking method
Change into Agrobacterium GV3101, is sieved through kanamycins (50mg/mL), gentamicin (50mg/mL) and rifampin (25mg/mL)
Positive colony is selected in choosing, carries out PCR and sequence verification, the results showed that viral silent carrier pYL-156-GhWRKY40, auxiliary carry
Body pYL-192, positive control vector pYL-156-PDS and negative control vector pYL-156 have been transferred to Agrobacterium GV3101.
(1.3) cultivation of GhWRKY40 gene silencing plant
The above-mentioned correct engineering Agrobacterium of verifying be added to (50mg/mL) containing kanamycin, gentamicin (50mg/mL) and
In 28 DEG C in the LB liquid medium of rifampin (25mg/mL), 200rpm is incubated overnight, and culture solution removes supernatant after being centrifuged,
Thallus is resuspended with MMA (10mM MES, 10mM MgCl2,200mM AS) solution, being adjusted to final concentration OD600 is 1.2, is put into black
Dark place stands 2-3 hours, then by the agrobacterium suspension containing pYL-156-GhWRKY40, pYL-156-PDS, pYL-156
It is uniformly mixed respectively with the agrobacterium suspension containing pYL-192 by 1:1 spare.After cotton two panels cotyledon is fully deployed, it can use
It is infected in Agrobacterium injection；Wound is gently first marked at the cotyledon back side with syringe needle, then removes the asepsis injector of syringe needle with 1mL
By bacterium solution, from the wound at the cotyledon back side, injection is entered, and infects full wafer cotyledon all, the plant after injection is put into
12h is handled at dark, is then put into illumination box and is cultivated.
(1.4) GhWRKY40 gene silencing is analyzed
The plant that positive control PDS gene is silenced shows as leaf chlorosis, generally as Gene Silencing
After the silencing phenotype of marker gene occurs, it is heavy to show that TRV system has successfully carried out gene in cotton plants for marker gene
It is silent.After Agrobacterium infects plant 14 days, cotton plants blade of the injection containing pYL-156-PDS Agrobacterium shows albefaction table
Type, the blade of injection empty carrier (pYL-156) negative control plant are still green, referring to Fig. 7, illustrate that TRV system is planted in cotton
Gene silencing is successfully carried out in strain, implies that target gene has been silenced.At this point, being detected using qPCR technology
Expression of the GhWRKY40 gene in silencing plant and adjoining tree, the results showed that the gene expresses water in silencing plant
It is flat to have dropped about 60%, referring to Fig. 8.
(2) Analysis of Resistance of GhWRKY40 silencing plant pair verticillium dahliae
It chooses the preferable plant progress verticillium dahliae of silencing efficiency and connects bacterium processing, after connecing bacterium 20 days, GhWRKY40 silencing
Plant shows apparent yellowing leaf, and wilting, which falls off, waits disease symptoms, and adjoining tree shows stronger disease resistance, with
GhWRKY40 silencing plant is lighter compared to yellowing leaf, fallen leaves and disease symptom, referring to Fig. 9.The hair of GhWRKY40 silencing plant
Sick rate and disease index are about lower by 25% than adjoining tree respectively and 50%, referring to Figure 10 and Figure 11, show GhWRKY40 silencing
Plant pair verticillium dahliae has stronger resistance.Illustrate GhWRKY40 negative regulation cotton to the disease resistance of verticillium dahliae.
It should be appreciated that described above, the specific embodiments are only for explaining the present invention, is not intended to limit the present invention.By
Spiritual changes and variations that derived from of the invention are still in the protection scope of this invention.
Sequence table
<110>nine Sheng Hezhong industry limited liability companies
<120>plant disease-resistant Protein G hWRKY40 and encoding gene and its application
<130> 2019
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 981
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 1
atgaatatat tagatctggg tctttgctta aagattgaaa aaaaagctat gaattcttct 60
tcttcttcat gggttgattc actggatctt aatgcttctt caacaaagtc ccttcaactt 120
gaaactcctg taagtcatcc taataccagc aacagcttaa ttgtatttgg aaggaaactt 180
tcagtcaaag aagagagtgg tgctttagtg gaggaactga accgcgtaaa tgcagaaaat 240
aagaagctaa ctgagatgtt aaaagcaatg tgtgagagct ataatgcatt gcaaagccag 300
ttagtggatt tgatgaacaa gaatactgag aaagaactta gtcctacaaa gaaaagaaaa 360
tcagaaacca gcaacaacaa caatgggaat atcattggga attcagagag tagttcaact 420
gatgaagaag aatcatgtaa gaaacctaga gaagaaatca tcaaagccaa aatttcaaga 480
gcttatgtca ggactgaact atctgatacc agccttgttg taaaggatgg atatcaatgg 540
aggaaatatg ggcaaaaagt caccagggat aatccctgtc caagagctta tttcaagtgt 600
tcttttgcac caagttgccc tgttaaaaag aaggtgcaaa gaagtgtgga tgatcaatca 660
gttctagttg caacatatga aggtgaacat aatcatcttc ccccttctca aatcgaggca 720
acatcgggtt cgagccgcct tggttcggtc cctgttggtt cgactccggt taagtcatcc 780
agtccaacca ttactctaga cctcaccaat tcaatcaagt caagtgatga agctagaaac 840
tcaaagccaa aactggattc accagaagct acgcaatatt tggtggaaca tatggcatct 900
tctttaacta aagaccctaa tttcactgca gcactggcag ctgctatttc aggaagaatg 960
ttttcaacaa attccaattg a 981
<210> 2
<211> 326
<212> PRT
<213>artificial sequence (Artificial sequence)
<400> 2
Met Asn Ile Leu Asp Leu Gly Leu Cys Leu Lys Ile Glu Lys Lys Ala
1 5 10 15
Met Asn Ser Ser Ser Ser Ser Trp Val Asp Ser Leu Asp Leu Asn Ala
20 25 30
Ser Ser Thr Lys Ser Leu Gln Leu Glu Thr Pro Val Ser His Pro Asn
35 40 45
Thr Ser Asn Ser Leu Ile Val Phe Gly Arg Lys Leu Ser Val Lys Glu
50 55 60
Glu Ser Gly Ala Leu Val Glu Glu Leu Asn Arg Val Asn Ala Glu Asn
65 70 75 80
Lys Lys Leu Thr Glu Met Leu Lys Ala Met Cys Glu Ser Tyr Asn Ala
85 90 95
Leu Gln Ser Gln Leu Val Asp Leu Met Asn Lys Asn Thr Glu Lys Glu
100 105 110
Leu Ser Pro Thr Lys Lys Arg Lys Ser Glu Thr Ser Asn Asn Asn Asn
115 120 125
Gly Asn Ile Ile Gly Asn Ser Glu Ser Ser Ser Thr Asp Glu Glu Glu
130 135 140
Ser Cys Lys Lys Pro Arg Glu Glu Ile Ile Lys Ala Lys Ile Ser Arg
145 150 155 160
Ala Tyr Val Arg Thr Glu Leu Ser Asp Thr Ser Leu Val Val Lys Asp
165 170 175
Gly Tyr Gln Trp Arg Lys Tyr Gly Gln Lys Val Thr Arg Asp Asn Pro
180 185 190
Cys Pro Arg Ala Tyr Phe Lys Cys Ser Phe Ala Pro Ser Cys Pro Val
195 200 205
Lys Lys Lys Val Gln Arg Ser Val Asp Asp Gln Ser Val Leu Val Ala
210 215 220
Thr Tyr Glu Gly Glu His Asn His Leu Pro Pro Ser Gln Ile Glu Ala
225 230 235 240
Thr Ser Gly Ser Ser Arg Leu Gly Ser Val Pro Val Gly Ser Thr Pro
245 250 255
Val Lys Ser Ser Ser Pro Thr Ile Thr Leu Asp Leu Thr Asn Ser Ile
260 265 270
Lys Ser Ser Asp Glu Ala Arg Asn Ser Lys Pro Lys Leu Asp Ser Pro
275 280 285
Glu Ala Thr Gln Tyr Leu Val Glu His Met Ala Ser Ser Leu Thr Lys
290 295 300
Asp Pro Asn Phe Thr Ala Ala Leu Ala Ala Ala Ile Ser Gly Arg Met
305 310 315 320
Phe Ser Thr Asn Ser Asn
325
Claims (7)
1. a kind of protein, entitled GhWRKY40, for albumen shown in such as (a) or (b) or (c):
(a) amino acid sequence is protein shown in sequence 2 in sequence table；
(b) fused protein that the N-terminal of protein shown in sequence 2 or/and C-terminal connection label obtain in sequence table；
(c) by (a) or (b) shown in protein it is derivative by the replacements of one or more amino acid, missing or/and insertion
That arrives still has GhWRKY40 functional transcription factor or active protein.
2. biomaterial relevant to protein described in claim 1 is any one of following (1) to (12):
(1) nucleic acid molecules of protein described in claim 1 are encoded；
(2) contain the expression cassette of (1) described nucleic acid molecules；
(3) contain the recombinant vector of (1) described nucleic acid molecules；
(4) contain the recombinant vector of (2) described expression cassette；
(5) contain the recombinant microorganism of (1) described nucleic acid molecules；
(6) contain the recombinant microorganism of (2) described expression cassette；
(7) contain the recombinant microorganism of (3) described recombinant vector；
(8) contain the recombinant microorganism of (4) described recombinant vector；
(9) contain the transgenic plant cells system of (1) described nucleic acid molecules；
(10) contain the transgenic plant cells system of (2) described expression cassette；
(11) contain the transgenic plant cells system of (3) described recombinant vector；
(12) contain the transgenic plant cells system of (4) described recombinant vector.
3. biomaterial according to claim 2, which is characterized in that (1) nucleic acid molecules described in be following (a1) or
(b1) or DNA molecular shown in (c1):
(a1) nucleotide sequence is DNA molecular shown in sequence 1 in sequence table；
(b1) one or more bases are carried out on the basis of nucleotide sequence is the nucleotide sequence shown in sequence 1 in sequence table
Replacement lacks or/and is inserted into and derives and obtain DNA molecular, and the encoded protein of the DNA molecular is described in claim 1
Protein；
(c1) nucleotide sequence hybridization limited under strict conditions with (a1) or (b1), and encode albumen described in claim 1
The DNA molecular of matter.
4. protein described in claim 1 or biomaterial described in claim 2 or 3 are improving plant to pathogen response
In application.
5. protein described in claim 1 or biomaterial described in claim 2 or 3 are improving plant to vascular bundle parasitism
Application in pathogen response.
6. protein described in claim 1 or biomaterial described in claim 2 or 3 are improving plant to verticillium dahliae
Application in response.
7. according to any application of claim 4-6, it is characterised in that: the plant is cotton.
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Cited By (4)
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| CN111424038A (en) * | 2020-04-16 | 2020-07-17 | 南京林业大学 | Cymbidium CgWRKY40 gene and application thereof |
| CN112410350A (en) * | 2020-12-14 | 2021-02-26 | 中国农业科学院植物保护研究所 | Upland cotton GhWRKY74 protein and coding gene and application thereof |
| CN112831505A (en) * | 2021-03-16 | 2021-05-25 | 昆明理工大学 | Pseudo-ginseng WRKY transcription factor genePnWRKY15And applications |
| CN115820666A (en) * | 2022-09-30 | 2023-03-21 | 华南农业大学 | Dioscorea composita DcW gene and application thereof in drought stress resistance and salt stress resistance |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111424038A (en) * | 2020-04-16 | 2020-07-17 | 南京林业大学 | Cymbidium CgWRKY40 gene and application thereof |
| CN111424038B (en) * | 2020-04-16 | 2022-04-26 | 南京林业大学 | Cymbidium CgWRKY40 gene and application thereof |
| CN112410350A (en) * | 2020-12-14 | 2021-02-26 | 中国农业科学院植物保护研究所 | Upland cotton GhWRKY74 protein and coding gene and application thereof |
| CN112410350B (en) * | 2020-12-14 | 2021-09-14 | 中国农业科学院植物保护研究所 | Upland cotton GhWRKY74 protein and coding gene and application thereof |
| CN112831505A (en) * | 2021-03-16 | 2021-05-25 | 昆明理工大学 | Pseudo-ginseng WRKY transcription factor genePnWRKY15And applications |
| CN112831505B (en) * | 2021-03-16 | 2023-04-11 | 昆明理工大学 | Pseudo-ginseng WRKY transcription factor gene PnWRKY15 and application thereof |
| CN115820666A (en) * | 2022-09-30 | 2023-03-21 | 华南农业大学 | Dioscorea composita DcW gene and application thereof in drought stress resistance and salt stress resistance |
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|---|---|
| CN110256549B (en) | 2021-04-20 |
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Denomination of invention: Plant disease resistance protein ghwrky40 and its coding gene and its application Effective date of registration: 20220725 Granted publication date: 20210420 Pledgee: Agricultural Bank of China Limited Changji sub branch Pledgor: JOIN HOPE SEED INDUSTRY Co.,Ltd. Registration number: Y2022980011180 |