CN109112149A - Regulate and control cotton Calcium-dependent protein kinase GhCPK33 gene and the application of cotton verticillium wilt resistance - Google Patents
Regulate and control cotton Calcium-dependent protein kinase GhCPK33 gene and the application of cotton verticillium wilt resistance Download PDFInfo
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Abstract
The invention belongs to field of plant genetic project technology, and in particular to the cotton Calcium-dependent protein kinase GhCPK33 gene of regulation cotton verticillium wilt resistance and application.The present invention by upland cotton be inoculated with verticillium wilt pathogen after, it is analyzed using different time points RAN-Seq data, screening obtains a kind of protein kinase G hCPK33 gene that calcium ion is relied on by verticillium wilt inducing expression, and the nucleotide sequence of the gene is as shown in SEQ ID NO:1；The protein sequence of gene coding is as shown in SEQ ID NO:2.By VIGS system, the corresponding gene of silencing, is inoculated with verticillium wilt pathogen, function of the Rapid identification gene in cotton verticillium wilt resistance in cotton.Play negative regulation in the resistance for the gene pairs cotton verticillium wilt that the present invention separates, the silencing gene can significantly improve cotton to the resistance of verticillium wilt pathogen.
Description
Technical field
The invention belongs to plant genetic engineering fields, and in particular to the cotton calcium dependent protein of regulation cotton verticillium wilt resistance
Kinases GhCPK33 gene and application.
Background technique
Cotton is the important industrial crops in China, and verticillium wilt is known as the title of cotton " cancer ", seriously endangers the production of cotton
Amount and fiber quality.In recent years, verticillium wilt occurred year after year, caused underproduction 10-20%, some areas throughout the year to Cotton in China production
Up to 70% when falling ill serious, it has also become restrict one of the major obstacle of Cotton in China stable high yield.Cotton verticillium wilt is by big
Soil transmissibility vascular bundle diseases caused by beautiful Verticillium dahliae, currently, single-minded targetedly special efficacy chemical agent not yet, biological control expense
With height, and effect is undesirable, and therefore, breeding resistant variety is main path (Wang Sheng just etc., the resisting verticillium new product for preventing and treating the disease
Cotton KV-3 genetics of resistance characteristic research Cotton Science, 2010,22 (5): 501~504) are planted in system.Research shows that upland cotton is to Huang
The resistance basic expressions for disease of withering are susceptible or resistance to disease, and it is minor-polygene site that the disease-resistant site carried is also mostly, and by ring
Border influences big, it is difficult to be polymerize, therefore the kind for producing upper highly resistance cotton verticillium wilt not yet is widely applied.
Ca2+As second messenger, activating in corresponding disease-resistant response after plant perceives pathogen plays important angle
Color.Studies have shown that most first occurred physiological change just includes intracellular in plant during verticillium wilt pathogen invades host
Ca2+The Rapid Accumulation of concentration.Classical plant congenital immunity model (immune (PTI) that pathogen associated molecular pattern induces and
Immune (ETI) that effector induces) all by adjusting Ca intracellular2+Concentration, to activate the immune response in downstream to resist cause of disease
The invasion of bacterium.It is now recognized that PTI reaction will lead to plant Ca intracellular2+Concentration is instantaneous and quickly accumulates, and ETI is then induction born of the same parents
Interior relative durations and stable calcium ion concentration increases.Mainly there are calmodulin (CaMs), calmodulin-like protein in plant
(CMLs), four kinds of Ca of calcineurin (CBLs) and Calcium-dependent protein kinase (CPKs)2+Sensor protein (Ca2+-binding
Sensory proteins), wherein calcium dependent protein kinases (calcium-dependent protein kinase, CPKs)
Research it is relatively more.The structure of calcium dependent protein kinases can be divided into four parts: the variable region of N-terminal, serine/threonine
Protein kinase area, from inhibition zone and calmodulin similar to area.In no Ca2+In the presence of, it is located at kinases area and calmodulin
Between similar area from the active site that inhibition zone is integrated to kinases area, inhibit the activity of kinases.And Ca2+In conjunction with can cause
The similar area's conformation of calmodulin changes, to make to discharge on the active site from inhibition zone from kinases area and make kinase activation
(the CDPK/SnRK protein kinase family laser biology journal in the plant such as Wu Junyan, Wang Maoguang, Wu Nengbiao, 2004,13
(4):265-269)。
The function of calcium dependent protein kinases is constantly mined in recent years, research shows that CPKs is not only involved in the growth of plant
The regulation of development also participates in plant to response (Kawamoto N, Sasabe M, the Endo M, et of biology and abiotic stress
al.Calcium-dependent protein kinases responsible for the phosphorylation of a
bZIP transcription factor FD crucial for the florigen complex
formation.Scientific reports,2015,5:8341；Munemasa S,Hossain M A,Nakamura Y,et
al.The Arabidopsis calcium-dependent protein kinase,CPK6,functions as a
positive regμlator of methyl jasmonate signaling in guard cells.Plant
physiology,2011,155(1):553-61；Wei S,Hu W,Deng X,et al.A rice calcium-
dependent protein kinase OsCPK9 positively regμlates drought stress tolerance
and spikelet fertility.BMC plant biology,2014,14(1):133；Fedorowicz-Stronska
O,Koczyk G,Kaczmarek M,et al.Genome-wide identification,characterisation and
expression profiles of calcium-dependent protein kinase genes in barley
(Hordeum vμlgare L.).Journal of applied genetics,2017,58(1):11-22)。Coca
Arabidopsis is participated in the Resistant reaction of pathogen Deng discovery AtCPK1 gene, and the exciton of fungi can induce the rapid table of AtCPK1
It reaches, compared with wild type, the mutant arabidopsis of AtCPK1 is more sensitive to pathogen, and after overexpressing AtCPK1, transgenosis is quasi-
Southern mustard accumulates a large amount of salicylic acid, and the acid mediated resistance signal path duration activation of bigcatkin willow shows the enhancing of pathogen resistance
(Coca M,San Segundo B.AtCPK1 calcium-dependent protein kinase mediates
pathogen resistance in Arabidopsis.Plant journal,2010,63(3):526-40).Rice
OsCPK10 gene is by rice blast fungus inducing expression, and compared with wild type, the rice plant for overexpressing OsCPK10 is shown as to rice blast
The enhancing of bacterium resistance；Heterologous transformant arabidopsis shows to enhance (Fu L, Yu X, An to the resistance of pseudomonas syringae
C.Overexpression of constitutively active OsCPK10 increases Arabidopsis
resistance against Pseudomonas syringae pv.tomato and rice resistance against
Magnaporthe grisea.Plant physiology and biochemistry,2013,73:202-210).The above knot
Fruit shows that calcium dependent protein kinases take part in plant in the Resistant reaction of pathogen and playing crucial regulating and controlling effect.
Virus induced gene silencing (Virus-induced gene silencing, VIGS) technology develops fast in recent years
Speed, it is advantageous that it is easy to operate, and multiple genes can be operated simultaneously, more and more researchers are using the system come quickly
Identify the function of gene.Before the present invention proposes, applicant is by being inoculated with verticillium wilt (Verticillium to upland cotton YZ1
Dahliae) different time points RAN-Seq data are analyzed after V991, have been screened a series of by verticillium wilt inducing expression
Rely on the protein kinase of calcium ion.It identifies to obtain GhCPK33 gene by VIGS system, finds the GhCPK33 gene in cotton
To negative regulation is played the role of in the resistance of verticillium wilt, the silencing gene can significantly improve cotton to the resistance of verticillium wilt pathogen.
The result shows that: compared with the control, the plant of the VIGS silencing gene is more disease-resistant, the accumulation of jasmine acid content, jasmonate acid signal road
The activation of diameter duration, this is to cultivate that there is the new cotton variety of verticillium wilt pathogen resistance to have great importance.
Summary of the invention
The purpose of the invention is to provide the protein kinase gene GhCPK33 that a cotton calcium ion relies on, the genes
It screening and identification and is cloned from the RNA-Seq data of cotton inoculation verticillium wilt pathogen V991, protein kinase gene GhCPK33 has such as
Nucleotide sequence shown in SEQ ID NO:1, or at least 50% homology sequence and above-mentioned DNA fragmentation coding albumen
Or the albumen with the same function by transformation modification.
Another object of the present invention is the protein kinase gene GhCPK33 for being the provision of the dependence of a cotton calcium ion
In the application for participating in verticillium wilt resistance of cotton by same, by GhCPK33 gene, SEQ ID NO:1 or the homologous gene being equal with its function
Inhibit expression, a kind of isolated protein, with amino acid sequence at least 50% shown in SEQ ID NO:2 in cotton plants
The sequence of homology, to achieve the purpose that regulate and control cotton to resistance to verticillium wilt, to be applied to the training of verticillium wilt resistance of cotton by same strain
It educates, at the same time as the marker gene of resistance to verticillium wilt cotton
The present invention passes through to different time points after upland cotton YZ1 inoculation verticillium wilt (Verticillium dahliae) V991
RAN-Seq data are analyzed, and screening obtains a series of protein kinases that calcium ion is relied on by verticillium wilt inducing expression.Pass through
VIGS system, the corresponding gene of silencing in cotton, and be inoculated with verticillium wilt pathogen, Rapid identification its in cotton verticillium wilt resistance
Function.Wherein, the GhCPK33 gene identified plays the role of negative regulation in resistance of the cotton to verticillium wilt, i.e. silencing should
Gene can significantly improve cotton to the resistance of verticillium wilt pathogen.The invention further relates to contain the gene or its homologous gene piece
Application of the carrier and the design gene or its functional analogue enhancing plant of section to resistance to verticillium wilt in agricultural production.
Technical scheme is as follows:
Applicant clones to obtain a kind of cotton Calcium-dependent protein kinase GhCPK33 gene of regulation cotton verticillium wilt resistance,
Its nucleotide sequence is as shown in SEQ ID NO:1.
The protein sequence of the cotton Calcium-dependent protein kinase GhCPK33 gene coding of above-mentioned regulation cotton verticillium wilt resistance
As shown in SEQ ID NO:2.
Calcium-dependent protein kinase GhCPK33 gene of the invention can be in the application in regulation cotton verticillium wilt resistance.
A kind of Calcium-dependent protein kinase GhCPK33 gene coded sequence of the invention is in regulation cotton verticillium wilt resistance
Using.
Applicant is prepared for a kind of inhibition expression vector TRV:GhCPK33, and the carrier contains claims 1 or 2 institute
The nucleotide fragments one of stated.
The expression vector is virus-mediated gene silencing vector, obtains gene silencing by transgene method
Plant.
The present invention provides a kind of method of Rapid identification gene function, the method is by above-mentioned inhibition expression vector
Host cell is imported, rapidly by target gene silencing.
More detailed technical solution is as described below.
The previous work of applicant is inoculated with after verticillium wilt (Verticillium dahliae) V991 not to upland cotton YZ1
It is analyzed with time point RAN-Seq data, has screened a series of egg of calcium ion dependent forms by verticillium wilt inducing expression
White kinases.By VIGS system, the corresponding gene of silencing in cotton, and be inoculated with verticillium wilt pathogen, Rapid identification its in cotton yellow
Function in disease of withering resistance.Wherein, the GhCPK33 gene identified can negative regulation cotton to the resistance of verticillium wilt, i.e. silencing
The gene can significantly improve cotton to the resistance of verticillium wilt pathogen.Choose other representative plants carry out phylogenetic analysis and
Bioinformatic analysis, the results showed that the sequence and Asiatic cotton (Gossypium arboretum) and Lei Mengdeshi cotton
GhCPK33 the most homologous (see Fig. 1) in (Gossypium raimondii).From extraction blade RNA in upland cotton ' YZ1 '
(Deng F,Tu L,Tan J,et al.GbPDF1 is involved in cotton fiber initiation via
The core cis-element HDZIP2ATATHB2.Plant Physiol, 2012,158:890-904), utilize reverse transcription
Its reverse transcription is synthesized cDNA, reaction condition are as follows: 65 DEG C by enzyme Superscript III (being purchased from Invitrogen company, the U.S.)
5min, 50 DEG C of 60min, 70 DEG C of 10min.Using the genomic information of upland cotton TM-1, amplimer GhCPK33-F (5 ' is synthesized
ATGGGTTCTTGCCTGACGAAAAGC 3 ') and GhCPK33-R (5 ' CTAAAAGAGTTGTGTGTGTTGGG 3 ') amplify
The cDNA overall length (1614bp) of GhCPK33 gene.PCR reaction condition are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of 30sec, 59 DEG C
30sec, 72 DEG C of 1min 50sec, 32 circulations；72 DEG C of extension 7min.The PCR product that amplification obtains is connected into pGEM-T carrier
(be purchased from Promega company, the U.S.), screening positive clone is simultaneously sequenced, and obtains required gene ORF, a kind of isolated gene
ORF, sequence are nucleotide sequence shown in SEQ ID NO:1.By BlastX (http: //
Www.ncbi.nlm.nih.gov) determine that the protein sequence of 537 amino acid corresponding to ORF is same with GrCPK33 albumen
Source.A kind of isolated protein, sequence are and amino acid sequence shown in SEQ ID NO:2.
BamHI and KpnI restriction enzyme site is added respectively according to the ORF design primer of GhCPK33, and at primer both ends
The primer is named as VGhCPK33-F (5' respectively by infusion recombination sequence
) and VGhCPK33-R (5' AGAAGGCCTCCATGGGGATCCGCCTGACGAAAAGCAAAGACTC3'
GAGACGCGTGAGCTCGGTACCAGGCTGTCCACTCAAATGCTG 3'), it is carried out by template of the cDNA of GhCPK33 gene
PCR amplification, obtained PCR product are building up on pTRV2 in 400bp or so by infusion recombining reaction, specific method ginseng
Examine document (Gao X, Wheeler T, Li Z, Kenerley CM, He P, Shan L.Silencing GhNDR1 and
GhMKK2 compromises cotton resistance to Verticillium wilt.Plant journal.2011,
66:293-305), to obtain the carrier of VIGS.
Utilize primer RT-GhCPK33-F (5'TCCACCACATCCCTGAAAAACC3') and RT-GhCPK33-R (5'
AAATGGAGGCACACCACTGAGT3') it is carried out tissue expression pattern analysis (Fig. 2), the results showed that the gene is in cotton
There is expression in each tissue.Further, using primer qGhCPK33-F (5'TCCACCACATCCCTGAAAAACC3') and
QGhCPK33-R (5'CAATACAAAGATAAGTCACAC3') carries out inducing expression mode point to it, obtains knot as described in Figure 3
Fruit.Shown using pathogenic bacterium inducing expression pattern, GhCPK33 gene is expressed by verticillium wilt pathogen inducible up regulation (see Fig. 3；Cause of disease
Bacterium inducing expression mode shows that GhCPK33 is inhibited to express by salicylic acid, is expressed by jasmonic inducible up regulation, is not had to hydrogen peroxide
It obvious responses to and (schemes to-D to scheme see the B in Fig. 3).
Using the Plant Transformation of mediated by agriculture bacillus, with injection method, by the VIGS carrier built, (target gene bacterial strain is
TRV:CPK33, control strain TRV:00 and silencing effect indicator strain are TRV:CLA1, and (Fig. 4) imported into upland cotton
In the cotyledon of YZ1, injection VIGS carrier after two weeks, detects the expression quantity of GhCPK33, and confirmation VIGS effect has generated (Fig. 5).
Using the method for hurting piece-root grafting bacterium, being inoculated with verticillium wilt pathogen V991 spore liquid, (concentration is about 105/ml).Start to send out after general inoculation 7d
Disease, and counting disease index after the onset, specific method with reference to horse equality Method for Identification of Cotton Verticillium Wilt Resistance (horse equality,
A kind of quick inoculation technique of new cotton verticillium wilt and its application in pathogen pathogenicity and host resistance identification, plant
Pathology journal, 2004,34 (6): 536-541).Disease refers to that statistical result shows that the plant resistance of TRV:GhCPK33 is apparently higher than pair
According to (Fig. 6), jasmonic synthesis and downstream responses related gene TRV:GhCPK33 the obvious up-regulated expression of plant (Fig. 7).This
Outside, applicant has also carried out the mirror of botrytis cinerea (Botrytis cinerea) resistance to the plant of TRV:00 and TRV:GhCPK33
It is fixed.The results show that compared with the control, the plant of TRV:GhCPK shows to significantly increase the resistance of botrytis cinerea, Spot expansion
Area is substantially reduced (Fig. 8).Illustrate GhCPK3 negative regulation cotton to the Resistant reaction of verticillium wilt pathogen and botrytis cinerea.To TRV:00
Hormone is extracted respectively with TRV:GhCPK33 root and blade, and hormone-content is shown, jasmonic and jasmine in TRV:GhCPK33 plant
Sour isoleucine content is significantly higher than control (Fig. 8), illustrates the synthesis of the gene negative regulation jasmonic, that is, inhibits the table of the gene
Danone enough promotes the accumulation of jasmonic and its derivative content in plant.Therefore GhCPK33 may be by regulation jasmonic
Synthesis regulate and control cotton to the resistance of verticillium wilt pathogen and botrytis cinerea, the application of the gene is for cultivating cotton disease resistance strain tool
There is important meaning.
Advantages of the present invention:
(1) when the present invention is by being inoculated with different after verticillium wilt (Verticillium dahliae) V991 to upland cotton YZ1
Between point RAN-Seq data analyzed, in conjunction with VIGS technology, screening and cloning simultaneously identifies GhCPK33 gene to be that cotton is anti-yellowing withers
The negative regulatory factor of disease.GhCPK33 mainly adjusts cotton to the resistance of verticillium wilt pathogen by the synthesis of regulation jasmonic.It utilizes
CRISPR/Cas9 system (Wang P C, Zhang J, Sun L, et al.High efficient multi-sites
genome editing in allotetraploid cotton(Gossypium hirsutum)using CRISPR/Cas9
System.Plant Biotechnology Journal, 2017,16 (1): 137-150), the base can be knocked out by genetic transformation
Because the New cotton line of resisting verticillium can be obtained, the marker gene of Cotton Resistance material can also be used as.
(2) although having cloned multiple CPKs genes in arabidopsis, and functional verification has been carried out to it, existed at present
Also seldom to the research report of CPKs in cotton, also CPKs is able to participate the immune anti-of plant in few document support cottons
Ying Zhong, the GhCPK33 gene that the present invention clones enrich the research in cotton to this genoid.
(3) cotton of GhCPK33 expression is inhibited to significantly enhance cotton for verticillium wilt, the resistance of gray mold.Explanation
GhCPK33 gene has participated in plant in the response of the resistance of verticillium wilt pathogen, and plays important role.Pass through regulation
The expression of GhCPK33 gene can adjust the accumulation of resistance factor in plant, to improve plant to the resistance of verticillium wilt pathogen.
(4) the jasmonic synthesis in the plant of GhCPK33 gene expression is inhibited to dramatically increase using VIGS carrier, and adjoint
The activation in the acid mediated resistance signal path of jasmine, illustrate inhibit GhCPK33 expression plant pair verticillium wilt pathogen and gray mold
Bacterium shows resistance enhancing and depends on jasmonic path.
(5) cotton material of breeding resisting verticillium is paid attention to by cotton breeder always, and the discovery and utilization of GhCPK33 will
Help to solve to produce the rare of upper verticillium wilt resistance of cotton by same breeding resources.As producing output of cotton caused by upper verticillium wilt at present
It loses huge, so that breeding resistant variety is particularly important, will be helpful to cultivate high resistance to verticillium wilt using GhCPK33 gene
New cotton variety.
Detailed description of the invention
Fig. 1: GhCPK33 phylogenetic tree analysis figure.It chooses other representative plants and carries out phylogenetic analysis and biology
Bioinformatics analysis, the results showed that the sequence and Lei Mengdeshi cotton (Gossypium raimondii) GhCPK33 are the most homologous, with
This unnamed gene is GhCPK33 gene by the CPK33 homology highest in arabidopsis CPK protein family, applicant.
Expression pattern analysis of Fig. 2: the GhCPK33 gene in each tissue of cotton.GhCPK3 is in each tissue of cotton
There is expression.Description of symbols: each tissue in Fig. 2 is followed successively by: root (root)；Stem (stem)；Blade (leaf)；Petal
(flower)；Anther (anther)；5 days ovules of Post flowering (5d ovule).GhUB7 is reference gene.
Fig. 3: GhCPK33 Primary structure pattern analysis.A figure in Fig. 3 is GhCPK33 gene in inoculation verticillium wilt pathogen
6h, 12h, for 24 hours after, compared with the control, by apparent inducing expression.Figure GhCPK33 gene in Fig. 3 is lured by jasmonic processing
Lead up-regulated expression.C figure in Fig. 3 is GhCPK33 gene by salicylic acid processing downward expression.DGhCPK33 gene pairs in Fig. 3
Hydrogen peroxide treatment does not respond to.
Fig. 4: VIGS vector construction schematic diagram.Description of symbols: LB, T-DNA left margin；35S, Caulimovirus 35S
Promoter；Cp, capsid protein；RdRP, RNA dependent form RNA polymerase；Mp, transport protein；16K, 16-kD are rich in cysteine
Albumen；MCS, multiple cloning sites；T, terminator；RB, T-DNA right margin.
GhCPK33 expression quantity detects Fig. 5 VIGS after two weeks.Description of symbols: after VIGS after two weeks, young tender leaf is selected
Piece and root system are material, extract RNA, detect the expression quantity of target gene.GhUB7 is reference gene.
The Resistance Identification of Fig. 6: TRV:GhCPK33 plant pair verticillium wilt.Description of symbols: determine that GhCPK33 successfully sinks
After silent, it is inoculated with verticillium wilt pathogen V991, the plant occurring degree of TRV:GhCPK33 illustrates to inhibit significantly lower than control (TRV:00)
After the expression of GhCPK33, the resistance of plant pair verticillium wilt pathogen enhances.Description of symbols: disease refers to statistical result and Phenotypic Observation
It is consistent.
The Resistance Identification of Fig. 7: TRV:GhCPK33 plant pair botrytis cinerea.After confirming target gene GhCPK33 success silencing,
Choose the blade inoculation botrytis cinerea of adjoining tree and TRV:GhCPK33 plant same position.As the result is shown: TRV:GhCPK33's
Mycelia expanding area is significantly less than control, illustrates after inhibiting GhCPK33 expression, the resistance of plant pair botrytis cinerea enhances.
In Fig. 8: TRV:00 and TRV:GhCPK33 plant jasmonic synthesis and downstream responses path on marker gene expression
Analysis.Material using Fig. 5 is the key gene of template, the synthesis of detection jasmonic and downstream responses path, as the result is shown: inhibiting
After GhCPK33 expression, the key gene in jasmonic synthesis and downstream responses path is activated, and illustrates that GhCPK33 can join
With the synthesis of regulation JA.
The detection of jasmine acid content in Fig. 9: TRV:00 and TRV:GhCPK33 plant.TRV:00 and TRV is selected respectively:
The blade and root system of the same area of GhCPK33 extract plant hormone as material, and measurement result shows to inhibit GhCPK33 table
After reaching, the content of jasmonic and jasmonic isoleucine in blade and root system is significantly increased, thus it is speculated that GhCPK33 gene is negative
Regulate and control the biosynthesis of jasmonic in cotton body.
Specific embodiment
Following embodiment defines the present invention, and describing the present invention in separation clone includes that GhCPK33 gene is complete
The DNA fragmentation of coding section, and the method for verifying GhCPK33 gene function.According to description below and these embodiments, originally
Field technical staff can determine essential characteristic of the invention, and without departing from the spirit and scope of the invention, can
To make various changes and modifications to the present invention, so that it is applicable in different purposes and condition.
The separation of embodiment 1:GhCPK33 gene is cloned
1, gene order obtains
The applicant's previous work is by analysis upland cotton YZ1 (kind comes from the Chinese Academy of Agriculture Science and Technologys Cotton Research Institute)
Expression modal data after inoculation verticillium wilt pathogen V991 sets out, and in conjunction with VIGS method, has identified a negative regulation cotton verticillium wilt
The gene of resistance.Sequence alignment is carried out in ncbi database, finds the gene and Asiatic cotton (Gossypium arboretum)
It is high with the GhCPK33 genetic homology in Lei Mengdeshi cotton (Gossypium raimondii), therefore separated gene is ordered
Entitled GhCPK33 gene (see Fig. 1).
Utilize primer GhCPK33-full-F (5 ' ATGGGTTCTTGCCTGACGAAAAGC3 ') and GhCPK33-full-R
(5 ' CTAAAAGAGTTGTGTGTGTTGGG3 ') uses the cDNA of upland cotton YZ1 as the full length sequence of template clone GhCPK33.
PCR reaction condition are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of 30sec, 59 DEG C of 30sec, 72 DEG C of 1min 50sec, 32 circulations；72℃
Extend 7min.The PCR product that amplification obtains is connected into pGEM-T carrier (purchased from Promega company, the U.S.), screening positive clone
And be sequenced, required gene ORF is obtained, sequence is nucleotide fragments shown in SEQ ID NO:1.Pass through BlastX
(http://www.ncbi.nlm.nih.gov) determines that 537 amino acid are (see 1- in SEQ ID NO:1 sequence corresponding to ORF
Amino acid sequence shown in 1614bp), the protein sequence of GhCPK33 gene coding is as shown in sequence table SEQ ID NO:2.
2, expression analysis
A. expression of the gene in each tissue of upland cotton is detected using semiquantitive PCR method, and (result is shown in
Fig. 2).Using upland cotton YZ1 as material, the RNA of following 6 different tissues is extracted, detects GhCPK33 with the method for semiquantitive PCR
Expression.6 tissues chosen are respectively: root (root)；Stem (stem)；Blade (leaf)；Petal (flower)；Anther
(anther)；5 days ovules (5d ovule).The extracting method of cotton total serum IgE is according to (the An improved simple such as Zhu
protocol for isolation of high quality RNA from Gossypium spp.suitable for
CDNA library construction, Acta Agronomica Sinica.2005,31.1657-1659) text delivered
It offers, RNA is rear after the completion of extracting to use DNaseI (being purchased from Promega company) processing, and RNA integrality passes through 1.2% (w/v) agarose
Glue (EtBr) electrophoresis detection (5V/cm).The measurement of nucleic acid concentration is enterprising in Beckman DU800 spectrophotometer
Row.For 260/280 ratio of RNA between 1.9 to 2.1, RNA of 260/230 ratio greater than 2.0 is used for the analysis of next step.cDNA
Synthesis be using 3 μ g total serum IgEs as template, with 1 μ l 500 μ g/ml oligo-dT (15) primer (be purchased from Promega company, beauty
State), DEPC-water mixing, 15 μ l of total volume；70 DEG C of denaturation 5min, are put in and are quenched 5min on ice；Add 5 × M-MLV
Buffer5 μ l,Ribonuclease Inhibitor (Promega) 1 μ l, M-MLVRT 1 μ l, 1.25 μ l 10mM
1.75 μ l of dNTP, DEPC-water mixing, total volume are 25 μ l；Then in 42 DEG C of 70min, 70 DEG C of 10min, every part of cDNA are dilute
It is saved at -20 DEG C after releasing to 250 μ l stand-by.Using the cDNA of above-mentioned reverse transcription synthesis as template, with primer RT-GhCPK33-F
(5'TCCACCACATCCCTGAAAAACC3') and RT-GhCPK33-R (5'AAATGGAGGCACACCACTGAGT3') are right
GhCPK33 gene carries out the PCR amplification of specificity, at the same with primer UB7-F (5'GAAGGCATTCCACCTGACCAAC3') and
UB7-R (5'CTTGACCTTCTTCTTCTTGTGCTTG3') does cotton GhUB7 (the GenBank number of logging in: DQ116441) gene
Specific amplified (the long 198bp of amplified production) is used as internal reference, carries out semi-quantitative analysis (see Fig. 2).With primer qGhCPK33-F (5'
TCCACCACATCCCTGAAAAACC3') and qGhCPK33-R (5'CAATACAAAGATAAGTCACAC3') is to GhCPK33 gene
Carry out the PCR amplification of specificity.Primer UB7-F and UB7-R is used to do specific amplified as internal reference to cotton GhUB7 gene simultaneously,
To carry out relative quantitative assay as internal reference.GhCPK33 gene is lured by verticillium wilt and various HORMONE TREATMENTs with quantitative PCR
Lead expression pattern analysis.
B. Huang is withered bacterium perception kind ' YZ1 ' as research object, since it is considered that earth culture can be to children when lifting seedlings by applicant
There is certain injury in seedling root, so selection water planting.Upland cotton presprouting of seeds, plant when it puts out new shoots 1-2cm to leech
Shi Zhong is moved it in nutrient solution when it just breaks ground.After growth of seedling to two panels true leaf is completely open and flat, inoculation is activated
V991 spore liquid (105A/mL), the distillation water process of same volume is compareed, then 0.5h, 2h, 6h after inoculation,
12h, for 24 hours point in time sampling.The total serum IgE that root system is extracted according to above-mentioned method, the verticillium wilt pathogen for GhCPK33 gene induce
Expression analysis (the A figure in Fig. 3).Quantitative PRC is the results show that GhCPK33 gene is expressed by the inducible up regulation of verticillium wilt pathogen.
C. salicylic acid, jasmonic and hydrogen peroxide play extremely important effect during plant is to anti-microbial pathogen,
The accumulation of signal transduction and downstream resisting substance in mediated cell.Therefore the sound of these substances of GhCPK33 gene pairs is analyzed
Situation is answered, can speculate the signal path that GhCPK33 may be participated in advance.With control and hormon processing (concentration for the treatment of:
200 μM of methyl jasmonates, 2mM salicylic acid and 1mM hydrogen peroxide) afterwards plant root be set as sample, sampling time point
0.5h, 1h and 3h extract root system total serum IgE according to the method described above.Expression of results after GhCPK33 HORMONE TREATMENT analysis shows,
GhCPK33 gene is lowered expression by salicylic acid processing by jasmonic processing inducible up regulation expression, not bright to hydrogen peroxide treatment
Aobvious response.
Embodiment 2: gene silencing is carried out using VIGS method
1. nursing young plants in hothouses
The cotton line YZ1 seed for choosing full seed impregnates 2h with 25 DEG C of warm water, is then laid in be placed on and soak
On gauze, one layer of wet gauze is covered again above, be placed on 28 DEG C of incubator vernalization.When seed root long is to 1-2cm,
It is transplanted to the plantation in Nutrition Soil (volume ratio, Nutrition Soil: vermiculite=3:1).The young root of seed is gently put into Nutrition Soil, on
Face covers the Nutrition Soil of 1-2cm thickness again, is then covered with preservative film, cultivates under light, and set temperature is 28 DEG C, light application time 16h,
After young plant is broken ground, film is taken off.It can be injected when the cotyledon of young plant is fully deployed.
2. the activation and preservation of Agrobacterium
2.1 prepare:
Agrobacterium strains are GV3101, prepare sterile triangular flask, and LB (solid, liquid) culture medium (contains kanamycins 50mg/L),
Sterile glycerol, sterile pipette tips, sterile 1.5mL centrifuge tube.
2.2 operations:
2.2.1 it activates, suspends:
In the assistant carrier pTRV1 (Holland for being stored in -70 DEG C of to cotton seedling progress VIGS processing first 2 days, going bail for respectively
The laboratory Bart P.H.J.Thomma), empty carrier pTRV2 (laboratory Dutch Bart P.H.J.Thomma) and gene TRV
50 μ L of recombinant vector bacterium solution, is placed in 2ml sterile centrifugation tube, and 500 μ l sterilizing LB is added (containing 50 μ g/ml's into centrifuge tube
Kanamycins and rifampin) culture solution, in 28 DEG C of shaking table 200r/min activation cultures.By activated strain inoculated in containing 50ml
In the sterilizing triangular flask of LB culture solution (kanamycins and rifampin containing 50 μ g/ml), 28 DEG C are placed in, the training of 200r/min shaking table
Support 16h.When shaking table culture Agrobacterium bacterium solution to OD600 value is 0.8 or so, bacterium solution is collected in the 10ml that sterilization treatment is crossed and is centrifuged
Guan Zhong is centrifuged 10min in 3000r/min.After removing the supernatant in centrifuge tube, bacterium solution is readjusted using VIGS re-suspension liquid
OD600 value to 1.0, and by the resuspended bacterium solution of each gene TRV recombinant vector and pTRV2 empty carrier respectively with assistant carrier pTRV1
Resuspended bacterium solution mixes in equal volume, places 3h at room temperature or is placed in 28 DEG C of shaking table 200r/min activation culture 1h.Formula of liquid (body is resuspended
Product is based on 1L): magnesium chloride: 2.03g；Morpholino b acid: 2.135g；Acetosyringone: 0.03g.
2.2.2 the preservation of bacterial strain:
It is connected to 150rpm in LB liquid medium from picking single colonie in culture dish, 26 DEG C are shaken 48h, by bacterium solution and glycerol body
Product is than being that the mixing of 1.5mL centrifuge tube, -70 DEG C of preservations are added in 1:1.
2.3. it disseminates, co-cultures:
When carrying out VIGS processing, first with 1ml pipette tips in the Cotton Seeding Cotyledon back side, 3-4 aperture of slight point.Then
The bacterium solution handled well is injected into cotton cotyledon along aperture with 1ml syringe, until entire cotyledon shows water stain shape.To children
It is unified to water after seedling VIGS is disposed, and covered the cotton seedling handled well with black plastic film, it is protected from light culture for 24 hours.
VIGS is handled seedling cotyledon second day, and black plastic film is opened, and keeping condition of culture is 25 DEG C, illumination 120 μ EM-2/S, 16h
Illumination/8h is dark.
The monitoring of 3.GhCPK33 Gene silencing efficacy
Cotton seedling is handled 10 days or so through VIGS, i.e., when chlorosis blade occur white for the blade of positive control TRV:GbCLA
When change, the young leaflet tablet of control empty carrier TRV:00 and TRV:GhCPK33 plant is taken respectively, as detection gene.PCR detection knot
Fruit shows, when the 12nd day after VIGS processing, GhCPK33 is significantly lower than control in the expression quantity of interference plant, therefore GhCPK33 exists
Expression in sea island cotton blade is successfully suppressed (Fig. 6).The key gene (Fig. 9) in the path JA, discovery are also had detected simultaneously
After inhibiting GhCPK33 expression, the key gene in the path JA is activated, and illustrates that GhCPK33 can regulate and control the synthesis of JA.
4. the activation and culture of verticillium wilt pathogen
Verticillium wilt pathogen is set on PDA plate and (is formulated as follows: weighing 200g potato, clean peeling chopping, add water 1000mL
Boil 15min, filtered through gauze, then plus 15g glucose and 7g agar, filtered through gauze while hot, exists according to a conventional method after completely dissolution
121 DEG C, high pressure steam sterilization under 103.4kPa) it is activated one week in 25 DEG C of dark culture casees, fungus block ddH is taken with transfer needle2O concussion
It suspends, then takes in the tiling to new PDA plate of 1mL bacterium solution, use ddH after five days2O washes lower spore from plate, and concentration
It is adjusted to 106spores/mL。
5. verticillium wilt pathogen is inoculated with
It when cotton seedling grows to two leaves wholeheartedly, chooses and grows consistent seedling, cut off its a small amount of root system with scissors, carry out
Hurt root processing, then it is 10 that root system, which is immersed in concentration,51min in spores/mL verticillium wilt pathogen (V991) spore liquid, then will connect
For the plantlet of transplant planted into Nutrition Soil, every alms bowl plants 4 plants.
6. Disease investigation
Connect bacterium it is latter week left and right according to cotton verticillium wilt seedling stage grade scale investigate and records disease index, calculating the state of an illness refer to
Number, wherein (Zhang Xinghua etc., Resistance Strain of Cotton is withered, verticillium wilt progress and its resists by 5 grades of systems note of greenhouse cotton seedling morbidity for disease index
Property identification method；Agriculture in Jiangxi journal, 2008,20 (3): 43-49).Specific grade scale is as follows:
0 grade: appearance is without symptom.
1 grade: 25% or less diseased plant blade shows symptom.
2 grades: blade 25%-50% shows symptom.
3 grades: 50% or more blade shows symptom, has a small number of blades to wither and fall.
4 grades: blade is complete withered or falls off, and productivity is very low.
Diseased plant rate (%)=(morbidity total strain number ÷ investigates total strain number) × 100%.
Disease refer to (disease index)=[diseased plant numbers at different levels respectively multiplied by the sum of corresponding series ÷ (investigation total strain number × superlative degree
Number)] × 100.
Bacterium is connect the result shows that the strain of TRV:GhCPK33 refers to compared with control material TRV:00 morbidity phenotype, disease contains with pathogen
Amount significantly reduces (Fig. 7), illustrates that GhCPK33 gene can participate in regulation cotton to the disease resistance response of verticillium wilt, and play negative tune
The effect of control.
Embodiment 5: transgenic line gray mold inoculated identification
1, the activation and culture of ash arrhizus bacteria
Take one piece of about 1cm2Botrytis cinerea (strain number BC17, Hua Zhong Agriculture University professor Li Guoqing give, and are conventional
Grey mold bacteria strain carries out the test and is not specifically addressed by using special strain therefore) fungus block, it is inoculated on new PDA plate, 25 DEG C are dark
Culture 5 days or so, is covered with entire culture dish to mycelia, can connect bacterium processing.
2, ash arrhizus bacteria is inoculated with
After the expression quantity decline for detecting GhCPK33, after determining that gene GhCPK33 is silenced, 3 plants or so differences are chosen
The blade for handling cotton plant same portion is placed on and is distilled on the filter paper of water saturates that (filter paper is placed on Bai Panli, and filter paper in advance
Have one layer of blotting paper being equally saturated between white disk), the consistent fungus block of growing way is chosen with the punch that diameter is 0.5cm,
It is inoculated on excised leaf, the one side with mycelia directly contacts blade face.It keeps humidity 70% or so, 25 DEG C of temperature, secretly trains
After supporting 3d, (Fig. 8) is taken pictures to the scab on blade.The result shows that connecing bacterium the result shows that inhibiting the strain of GhCPK33 expression
It is substantially reduced compared with control material Spot expansion area, illustrates that the gene can participate in regulation cotton to the disease resistance response of gray mold,
And play the role of negative regulation.
3, the measurement of hormone-content
Method for extracting (Liu of the extracting method of jasmonic and jasmonic isoleucine in cotton plants referring to Liu et al.
Et al 2012), and be modified slightly.Specific steps are as follows: the cotton tissue sample for taking 0.1g or so, with liquid nitrogen grinding at powder.
The extraction buffer (methanol: double pure water: glacial acetic acid, 80:19:1, V:V:V) of 750 μ l pre-cooling is added in every part of sample.Shake manually
It swings after shaking up, is protected from light and acutely vibrates 12h or so in 4 DEG C of concussion instruments.After homogenate sufficiently extracts, 4 DEG C, 12000rpm centrifugation
Supernatant is transferred to a new centrifuge tube by 15min, and 250 μ l extraction buffers are added into former centrifuge tube, and continuation is protected from light at 4 DEG C
2h is shaken, then 4 DEG C, 12000rpm is centrifuged 15min, draws supernatant in centrifuge tube before.The total supernatant that will be obtained twice
It is dried up with nitrogen evaporator, is then dissolved with the methanol of 300 μ l 10%, 4 DEG C are protected from light concussion 2h or so.Finally by the hormone liquid of dissolution
Body is through diameter 13mm, in filter membrane (Nylon 66) filtering to brown vial for surveying hormone of 0.22 μm of aperture, be stored in 4 DEG C it is standby
With.Last extract measures jasmonic and the different bright ammonia of jasmonic with HPLC-MS/MS (1200LLC-MS system, Varian)
The content of acid.Jasmine acid content can not be measured very little when measurement, only the content (Fig. 9) of jasmonic isoleucine.It can be with by Fig. 9
Find out, after inhibiting GhCPK33 expression, the content of JA-Ile is than compareing significant raising, explanation in cotton plants root and blade
GhCPK33 gene is the synthesis of negative regulation JA-Ile.
Sequence table
<110>Hua Zhong Agriculture University
<120>regulate and control cotton Calcium-dependent protein kinase GhCPK33 gene and the application of cotton verticillium wilt resistance
<141> 2018-02-12
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1614
<212> DNA
<213>cotton (Gossypium hirsutum)
<220>
<221> gene
<222> (1)..(1614)
<220>
<221> CDS
<222> (1)..(1614)
<400> 1
atg ggt tct tgc ctg acg aaa agc aaa gac tcg aag cca aag cat aat 48
Met Gly Ser Cys Leu Thr Lys Ser Lys Asp Ser Lys Pro Lys His Asn
1 5 10 15
ggg tat gga tca gga cca aca act acg gct gct gtg cac caa caa agg 96
Gly Tyr Gly Ser Gly Pro Thr Thr Thr Ala Ala Val His Gln Gln Arg
20 25 30
tac cag gag cct gtt agg cct gca cca gtt caa tcc caa ttc cgt cac 144
Tyr Gln Glu Pro Val Arg Pro Ala Pro Val Gln Ser Gln Phe Arg His
35 40 45
atc cct gaa aaa cca ggt act caa aca tct tgg aag cca gtg gct cca 192
Ile Pro Glu Lys Pro Gly Thr Gln Thr Ser Trp Lys Pro Val Ala Pro
50 55 60
tca cca agc ccc aag cct gtt gcc cca agg gtt gac acc att ctt ggt 240
Ser Pro Ser Pro Lys Pro Val Ala Pro Arg Val Asp Thr Ile Leu Gly
65 70 75 80
aaa cca ttt gaa gat ata agg gtg cac tac aca att ggt aaa gaa tta 288
Lys Pro Phe Glu Asp Ile Arg Val His Tyr Thr Ile Gly Lys Glu Leu
85 90 95
ggg aaa ggt cag ttt ggt gtg act tat ctt tgt att gaa aat tca act 336
Gly Lys Gly Gln Phe Gly Val Thr Tyr Leu Cys Ile Glu Asn Ser Thr
100 105 110
gga aag caa tat gct tgc aag acc ata tcc aaa aag aaa tta gtc acg 384
Gly Lys Gln Tyr Ala Cys Lys Thr Ile Ser Lys Lys Lys Leu Val Thr
115 120 125
agg aat gat aag gag gat atg aga agg gag ata caa att atg cag cat 432
Arg Asn Asp Lys Glu Asp Met Arg Arg Glu Ile Gln Ile Met Gln His
130 135 140
ttg agt gga cag cct aat att gtg gag ttt aaa ggt gca tat gag gat 480
Leu Ser Gly Gln Pro Asn Ile Val Glu Phe Lys Gly Ala Tyr Glu Asp
145 150 155 160
aag cta tct gtt cac ctt gta atg gaa tta tgt gct ggt ggt gaa ttg 528
Lys Leu Ser Val His Leu Val Met Glu Leu Cys Ala Gly Gly Glu Leu
165 170 175
ttt gat agg att att gcc aag gga cat tat agt gaa agg gca gct gct 576
Phe Asp Arg Ile Ile Ala Lys Gly His Tyr Ser Glu Arg Ala Ala Ala
180 185 190
tcc att tgt agg gca att gtg aat gtt gtc cat gct tgc cat ttt atg 624
Ser Ile Cys Arg Ala Ile Val Asn Val Val His Ala Cys His Phe Met
195 200 205
gga gtg atg cat agg gac ctt aag cct gag aat ttc ttg ttg tct agc 672
Gly Val Met His Arg Asp Leu Lys Pro Glu Asn Phe Leu Leu Ser Ser
210 215 220
aag ggt gag aat gtt ctt ttg aag gcc acc gat ttc ggc ttg tca gtg 720
Lys Gly Glu Asn Val Leu Leu Lys Ala Thr Asp Phe Gly Leu Ser Val
225 230 235 240
ttc ttt gaa gat gga aag gtg tcc aag gat ata gtc ggg agc gcg tac 768
Phe Phe Glu Asp Gly Lys Val Ser Lys Asp Ile Val Gly Ser Ala Tyr
245 250 255
tat gtt gct cct gaa gta tta ctg cgt aaa tat ggg aaa gaa atc gat 816
Tyr Val Ala Pro Glu Val Leu Leu Arg Lys Tyr Gly Lys Glu Ile Asp
260 265 270
att tgg agt gca ggg gtt ata tta tat atc tta ctc agt ggt gtg cct 864
Ile Trp Ser Ala Gly Val Ile Leu Tyr Ile Leu Leu Ser Gly Val Pro
275 280 285
cca ttt tgg gca gaa acg gag aag ggg ata ttc gat gct att tta gaa 912
Pro Phe Trp Ala Glu Thr Glu Lys Gly Ile Phe Asp Ala Ile Leu Glu
290 295 300
gga gaa att gac ttc gaa agt caa cca tgg cct tct att tca gag agt 960
Gly Glu Ile Asp Phe Glu Ser Gln Pro Trp Pro Ser Ile Ser Glu Ser
305 310 315 320
gcc aag gac cta gtg cgc cgg atg tta aca cag gat ccg aaa aag cgg 1008
Ala Lys Asp Leu Val Arg Arg Met Leu Thr Gln Asp Pro Lys Lys Arg
325 330 335
att act tct act caa gtc cta gag cat cca tgg ata aga gag ggt gga 1056
Ile Thr Ser Thr Gln Val Leu Glu His Pro Trp Ile Arg Glu Gly Gly
340 345 350
agt gca tca gac aag cct ata gat agt gca gtc ctt tct aga atg aag 1104
Ser Ala Ser Asp Lys Pro Ile Asp Ser Ala Val Leu Ser Arg Met Lys
355 360 365
caa ttc aga aga atg aac aaa cta agg caa ctt gcc tta aag gtc att 1152
Gln Phe Arg Arg Met Asn Lys Leu Arg Gln Leu Ala Leu Lys Val Ile
370 375 380
gcg gaa aat ctt tct agc gaa gaa atc cag ggt ctg aag caa atg ttt 1200
Ala Glu Asn Leu Ser Ser Glu Glu Ile Gln Gly Leu Lys Gln Met Phe
385 390 395 400
gca aac atc gac act gac aac agt ggc acg atc acc tat gaa gaa ctt 1248
Ala Asn Ile Asp Thr Asp Asn Ser Gly Thr Ile Thr Tyr Glu Glu Leu
405 410 415
aag act gga tta gct cga ctt gga tca aag ctc aca gag gca gag gtg 1296
Lys Thr Gly Leu Ala Arg Leu Gly Ser Lys Leu Thr Glu Ala Glu Val
420 425 430
cag caa cta atg gaa gct gct gac gtg gat gga aat ggt agt att gat 1344
Gln Gln Leu Met Glu Ala Ala Asp Val Asp Gly Asn Gly Ser Ile Asp
435 440 445
tac att gaa ttc atc act gct aca atg cat aga cat agg ctt gaa aga 1392
Tyr Ile Glu Phe Ile Thr Ala Thr Met His Arg His Arg Leu Glu Arg
450 455 460
gat gaa cat ctt tac aaa gct ttt caa cat ttt gat aag gac aac agt 1440
Asp Glu His Leu Tyr Lys Ala Phe Gln His Phe Asp Lys Asp Asn Ser
465 470 475 480
ggg tat atc aca aga gat gag cta gaa gca gcc atg aaa gaa tac gga 1488
Gly Tyr Ile Thr Arg Asp Glu Leu Glu Ala Ala Met Lys Glu Tyr Gly
485 490 495
atg ggt gat gat gac aca atc aag gaa ata ata tcc gaa gtt gac acc 1536
Met Gly Asp Asp Asp Thr Ile Lys Glu Ile Ile Ser Glu Val Asp Thr
500 505 510
gac aac gac ggc aag atc aac tac gag gag ttc cgg gac atg atg aga 1584
Asp Asn Asp Gly Lys Ile Asn Tyr Glu Glu Phe Arg Asp Met Met Arg
515 520 525
agc gga acc caa cac aca caa ctc ttt tag 1614
Ser Gly Thr Gln His Thr Gln Leu Phe
530 535
<210> 2
<211> 537
<212> PRT
<213>cotton (Gossypium hirsutum)
<400> 2
Met Gly Ser Cys Leu Thr Lys Ser Lys Asp Ser Lys Pro Lys His Asn
1 5 10 15
Gly Tyr Gly Ser Gly Pro Thr Thr Thr Ala Ala Val His Gln Gln Arg
20 25 30
Tyr Gln Glu Pro Val Arg Pro Ala Pro Val Gln Ser Gln Phe Arg His
35 40 45
Ile Pro Glu Lys Pro Gly Thr Gln Thr Ser Trp Lys Pro Val Ala Pro
50 55 60
Ser Pro Ser Pro Lys Pro Val Ala Pro Arg Val Asp Thr Ile Leu Gly
65 70 75 80
Lys Pro Phe Glu Asp Ile Arg Val His Tyr Thr Ile Gly Lys Glu Leu
85 90 95
Gly Lys Gly Gln Phe Gly Val Thr Tyr Leu Cys Ile Glu Asn Ser Thr
100 105 110
Gly Lys Gln Tyr Ala Cys Lys Thr Ile Ser Lys Lys Lys Leu Val Thr
115 120 125
Arg Asn Asp Lys Glu Asp Met Arg Arg Glu Ile Gln Ile Met Gln His
130 135 140
Leu Ser Gly Gln Pro Asn Ile Val Glu Phe Lys Gly Ala Tyr Glu Asp
145 150 155 160
Lys Leu Ser Val His Leu Val Met Glu Leu Cys Ala Gly Gly Glu Leu
165 170 175
Phe Asp Arg Ile Ile Ala Lys Gly His Tyr Ser Glu Arg Ala Ala Ala
180 185 190
Ser Ile Cys Arg Ala Ile Val Asn Val Val His Ala Cys His Phe Met
195 200 205
Gly Val Met His Arg Asp Leu Lys Pro Glu Asn Phe Leu Leu Ser Ser
210 215 220
Lys Gly Glu Asn Val Leu Leu Lys Ala Thr Asp Phe Gly Leu Ser Val
225 230 235 240
Phe Phe Glu Asp Gly Lys Val Ser Lys Asp Ile Val Gly Ser Ala Tyr
245 250 255
Tyr Val Ala Pro Glu Val Leu Leu Arg Lys Tyr Gly Lys Glu Ile Asp
260 265 270
Ile Trp Ser Ala Gly Val Ile Leu Tyr Ile Leu Leu Ser Gly Val Pro
275 280 285
Pro Phe Trp Ala Glu Thr Glu Lys Gly Ile Phe Asp Ala Ile Leu Glu
290 295 300
Gly Glu Ile Asp Phe Glu Ser Gln Pro Trp Pro Ser Ile Ser Glu Ser
305 310 315 320
Ala Lys Asp Leu Val Arg Arg Met Leu Thr Gln Asp Pro Lys Lys Arg
325 330 335
Ile Thr Ser Thr Gln Val Leu Glu His Pro Trp Ile Arg Glu Gly Gly
340 345 350
Ser Ala Ser Asp Lys Pro Ile Asp Ser Ala Val Leu Ser Arg Met Lys
355 360 365
Gln Phe Arg Arg Met Asn Lys Leu Arg Gln Leu Ala Leu Lys Val Ile
370 375 380
Ala Glu Asn Leu Ser Ser Glu Glu Ile Gln Gly Leu Lys Gln Met Phe
385 390 395 400
Ala Asn Ile Asp Thr Asp Asn Ser Gly Thr Ile Thr Tyr Glu Glu Leu
405 410 415
Lys Thr Gly Leu Ala Arg Leu Gly Ser Lys Leu Thr Glu Ala Glu Val
420 425 430
Gln Gln Leu Met Glu Ala Ala Asp Val Asp Gly Asn Gly Ser Ile Asp
435 440 445
Tyr Ile Glu Phe Ile Thr Ala Thr Met His Arg His Arg Leu Glu Arg
450 455 460
Asp Glu His Leu Tyr Lys Ala Phe Gln His Phe Asp Lys Asp Asn Ser
465 470 475 480
Gly Tyr Ile Thr Arg Asp Glu Leu Glu Ala Ala Met Lys Glu Tyr Gly
485 490 495
Met Gly Asp Asp Asp Thr Ile Lys Glu Ile Ile Ser Glu Val Asp Thr
500 505 510
Asp Asn Asp Gly Lys Ile Asn Tyr Glu Glu Phe Arg Asp Met Met Arg
515 520 525
Ser Gly Thr Gln His Thr Gln Leu Phe
530 535
Claims (7)
1. a kind of cotton Calcium-dependent protein kinase GhCPK33 gene of regulation cotton verticillium wilt resistance, nucleotide sequence such as SEQ
Shown in ID NO:1.
2. a kind of cotton Calcium-dependent protein kinase GhCPK33 gene of regulation cotton verticillium wilt resistance, the albumen of gene coding
Matter sequence is as shown in SEQ ID NO:2.
3. a kind of Calcium-dependent protein kinase GhCPK33 gene as described in claim 1 is in regulation cotton verticillium wilt resistance
Using.
4. a kind of Calcium-dependent protein kinase GhCPK33 gene coded sequence as claimed in claim 2 is in regulation cotton verticillium wilt
Application in resistance.
5. a kind of inhibition expression vector TRV:GhCPK33, it is characterised in that: contain gene of any of claims 1 or 2.
6. inhibition expression vector according to claim 5, it is characterised in that: the expression vector is virus-mediated gene
Silent carrier obtains the plant of gene silencing by transgene method.
7. a kind of method of Rapid identification plant gene function, it is characterised in that: by inhibition expression vector described in claim 5
Host cell is imported, and rapidly makes target gene silencing.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109837297A (en) * | 2019-04-04 | 2019-06-04 | 中国农业科学院植物保护研究所 | GhAGD13 gene relevant to resistance to verticillium wilt and its application |
| CN110004156A (en) * | 2019-04-04 | 2019-07-12 | 中国农业科学院植物保护研究所 | GhCML20 gene relevant to resistance to verticillium wilt and its application |
| CN111197035A (en) * | 2020-01-08 | 2020-05-26 | 西北农林科技大学 | Powdery mildew-resistant grape calcium-dependent protein kinase gene VpCDPK13 and application thereof |
| CN111378684A (en) * | 2020-03-15 | 2020-07-07 | 华中农业大学 | Application of heat-induced gene editing system CRISPR-Cas12b in upland cotton |
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| CN109837297A (en) * | 2019-04-04 | 2019-06-04 | 中国农业科学院植物保护研究所 | GhAGD13 gene relevant to resistance to verticillium wilt and its application |
| CN110004156A (en) * | 2019-04-04 | 2019-07-12 | 中国农业科学院植物保护研究所 | GhCML20 gene relevant to resistance to verticillium wilt and its application |
| CN109837297B (en) * | 2019-04-04 | 2020-11-27 | 中国农业科学院植物保护研究所 | GhAGD13 gene related to verticillium wilt resistance and application thereof |
| CN111197035A (en) * | 2020-01-08 | 2020-05-26 | 西北农林科技大学 | Powdery mildew-resistant grape calcium-dependent protein kinase gene VpCDPK13 and application thereof |
| CN111197035B (en) * | 2020-01-08 | 2022-09-13 | 西北农林科技大学 | Powdery mildew-resistant grape calcium-dependent protein kinase gene VpCDPK13 and application thereof |
| CN111378684A (en) * | 2020-03-15 | 2020-07-07 | 华中农业大学 | Application of heat-induced gene editing system CRISPR-Cas12b in upland cotton |
| CN111378684B (en) * | 2020-03-15 | 2023-06-27 | 华中农业大学 | Application of thermally-induced gene editing system CRISPR-Cas12b in upland cotton |
| CN113106105A (en) * | 2021-05-11 | 2021-07-13 | 中国农业科学院深圳农业基因组研究所 | Cotton gene and its use |
| CN113046375A (en) * | 2021-05-19 | 2021-06-29 | 新疆农业科学院园艺作物研究所 | SpCPK33 gene and application of encoding protein thereof in regulation and control of tomato drought tolerance |
| CN114381466A (en) * | 2022-01-13 | 2022-04-22 | 新疆农业大学 | Coding gene GbC4H of cinnamic acid-4-hydroxylase from cotton and application |
| CN114790449A (en) * | 2022-05-11 | 2022-07-26 | 新疆农业科学院核技术生物技术研究所（新疆维吾尔自治区生物技术研究中心） | Application of calcium-dependent protein kinase gene GhCPK4 in resisting verticillium wilt of plants |
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