CN107501399A - Vitis davidii Foex transcription factor VdWRKY70 and its application in plant resistance to environment stress kind is cultivated - Google Patents
Vitis davidii Foex transcription factor VdWRKY70 and its application in plant resistance to environment stress kind is cultivated Download PDFInfo
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Abstract
The invention belongs to agricultural biological technical field, and in particular to Vitis davidii Foex transcription factorVdWRKY70And its application in plant resistance to environment stress kind is cultivated, the Vitis davidii Foex transcription factorVdWRKY70Coding nucleotide sequence as shown in sequence table SEQ ID NO.1.The present invention has found transgenic arabidopsis pair by Real-Time Fluorescent Quantitative PCR TechniquePstDC3000 has definitely resistance, can cultivate arabidopsis and resistPstDC3000 kinds, while be also to understand transcription factor in depthVdWRKY70Biochemical character and physiological function in Vitis davidii Foex body lay the foundation.
Description
Technical field
The invention belongs to agricultural biological technical field, and in particular to Vitis davidii Foex transcription factorVdWRKY70And its planted cultivating
Application in thing resistant variety.
Background technology
Plant is subjected to the biotics such as fungi, bacterium, virus, mycoplasma, nematode in growth and development process, shows as
It is disease-resistant or susceptible.Plant immune system is made up of two immune responses：The immune response that pathogen-associated molecular pattern excites
（PAMP-triggered immunity, PTI）The immune response excited with effect protein（Effector triggered
Immunity, ETI）.PTI and ETI belong to systemic acquired resistance（Systemic acquired resistance, SAR）,
SAR is disease resistance response of the plant to pathogen of SA mediations,WRKYTranscription factor is the important transcription in SA signal transduction process
The factor, very important effect is played in expression of the regulation and control with plant defense response related gene.
Grape is that one of widest fruit is cultivated in China, and grape disease is serious all the more with the development of grape industry, in vain
Maize ear rot is by fungi（Coniothyrium diplodiella）It is caused, it is one of four major diseases of grape most serious.White rot
Harm of the disease to grape even causes total crop failure, the production of serious threat grape including weakening tree vigo(u)r, destroying branches and tendrils, the underproduction.Pierce Portugal
Grape are a kind of Wild ornamental resources of Vitaceae Vitis East Asia population, and it has very strong to anthrachose of grape, white rot, anthracnose etc.
Resistance turn into grape wet-heat resisting, the precious resources of breeding for disease resistance.It is peculiar from China using Modern Molecular Biotechnology means
Germ plasm resource in find resistant gene, identification resistant variety, be grape industrialization production and China's excellent resources protection compel
Being essential will.
Belong in grape full-length genomeWRKYThere are 80 members in family,VdWRKY70CDS sequence 931bp, belong to typical case
'sWRKYThe family member of transcription factor the IIIth.It is anti-that one or a few transcription factor can regulate and control a series of downstream Defense responses
Answer the expression of gene, it is possible to by importing or improveing the gene engineering method of transcription factor come comprehensive improvement disease resistance of plant
Shape.Therefore, seek has the transcription factor gene of positive regulating and controlling effect is disease-resistant to identification to turn base in the reaction of Vitis davidii Foex Defense response
Because Vitis davidii Foex new varieties have very important meaning.
The content of the invention
It is an object of the present invention to provide Vitis davidii Foex transcription factorVdWRKY70And its application in plant resistance to environment stress kind is cultivated,
By Real-Time Fluorescent Quantitative PCR Technique, it is found that transgenic arabidopsis has definitely resistance to Pst DC3000, can cultivate
The anti-Pst DC3000 kinds of arabidopsis, while be also to understand transcription factor in depthVdWRKY70Biochemical character in Vitis davidii Foex body
And physiological function lays the foundation.
To achieve the above object, the present invention uses following technical scheme：
The present invention provides a kind of Vitis davidii Foex transcription factorVdWRKY70, Vitis davidii Foex transcription becauseSub- VdWRKY70Encoding nucleoside
For acid sequence as shown in sequence table SEQ ID NO.1, its length is 931bp.
The present invention also provides a kind of Vitis davidii Foex transcription factorVdWRKY70Application in plant resistance to environment stress kind is cultivated.
Further, the plant resistance to environment stress kind is Genes For Plant TolerancePstDC3000 kinds.
Further, the plant resistance to environment stress kind resists for arabidopsisPstDC3000 kinds.
Further, arabidopsis is cultivated to resistPstThe method of DC3000 kinds is as follows：, will using agricultural biotechnologies method
Vitis davidii Foex transcription factorVdWRKY70Arabidopsis cell is transformed into, is expressedVdWRKY70Transgenic arabidopsis, in table
ReachVdWRKY70Transgenic arabidopsis on be inoculated withPstDC3000, after a few days, analyzed using Real-Time Fluorescent Quantitative PCR Technique
Transcription factorVdWRKY70The relative expression quantity of gene, wildtype Arabidopsis thaliana is contrasted, relative expression quantity is higher to be resisted for arabidopsisPstDC3000 kinds.
Further, the primer used in the Real-Time Fluorescent Quantitative PCR Technique is as follows：
Sense primer VdWRKY70-F：5’-ATGGCAACCAAACAGGTCCAGAT-3’；
Anti-sense primer VdWRKY70-R：5’-GGCGTTGCTATCATCTTGATTGTTT-3’.
Further, the reference gene used in the Real-Time Fluorescent Quantitative PCR Technique is as follows：
Atactin-F：GAAATCACAGCACTTGCACC；
Atactin-R：AAGCCTTTGATCTTGAGAGC.
Compared with prior art, the beneficial effects of the present invention are：
1. transcription factorVdWRKY70Gene can strengthen arabidopsis to whiterot fungi, powdery mildew andPstDC3000 resistance, the present invention
By be inoculated with successively on transgenic arabidopsis and wildtype Arabidopsis thaliana whiterot fungi, powdery mildew orPstDC3000, pass through phenotype
Observation, quantitative character statistics, Trypan Blue observation and Real-Time Fluorescent Quantitative PCR Technique, find transgenic arabidopsis dialogue
Rotten bacterium, powdery mildew orPstDC3000 has definitely resistance, thus can cultivate the anti-whiterot fungi of arabidopsis, powdery mildew andPst
DC3000 kinds.
2. the present invention is by by transcription factorVdWRKY70With other known plantsWRKY70And the race of arabidopsis the IIIth ownsWRKYThe cluster analysis of gene, drawsVdWRKY70WithFcWRKY70、CaWRKY70、AtWRKY70、AtWRKY54、ClWRKY70
Functionally there is certain similitude, also illustrate thatVdWRKY70Effect in biotic and abiotic stress is several with this
Individual gene has certain similitude., will additionally by Subcellular Localization technologyVdWRKY70Albumen is positioned at transgenic plan
In the nucleus of southern mustard, therefore the present invention is to understand transcription factor in depthVdWRKY70Biochemical character and life in Vitis davidii Foex body
Reason function lays the foundation.
Brief description of the drawings
Fig. 1 is that transgenic arabidopsis and wildtype Arabidopsis thaliana are inoculated with the phenotype sight after whiterot fungi in the embodiment of the present invention three
Examine comparison diagram.
Fig. 2 is quantitative after transgenic arabidopsis in the embodiment of the present invention three and wildtype Arabidopsis thaliana inoculation whiterot fungi
Shape Statistical Comparison figure.
Fig. 3 expects to carry out platform after transgenic arabidopsis in the embodiment of the present invention three and wildtype Arabidopsis thaliana inoculation whiterot fungi
The disease-resistant situation schematic diagram of indigo plant dyeing.
Fig. 4 is that transgenic arabidopsis and wildtype Arabidopsis thaliana are inoculated with after whiterot fungi when different in the embodiment of the present invention three
The delayed early transcription factorVdWRKY70Expression situation schematic diagram.
Fig. 5 is that transgenic arabidopsis and wildtype Arabidopsis thaliana are inoculated with the phenotype sight after powdery mildew in the embodiment of the present invention four
Examine comparison diagram.
Fig. 6 is quantitative after transgenic arabidopsis in the embodiment of the present invention four and wildtype Arabidopsis thaliana inoculation powdery mildew
Shape Statistical Comparison figure.
Fig. 7 expects to carry out platform after transgenic arabidopsis in the embodiment of the present invention four and wildtype Arabidopsis thaliana inoculation powdery mildew
The disease-resistant situation schematic diagram of indigo plant dyeing.
Fig. 8 is that transgenic arabidopsis and wildtype Arabidopsis thaliana are inoculated with after powdery mildew when different in the embodiment of the present invention four
The delayed early transcription factorVdWRKY70Expression situation schematic diagram.
Fig. 9 is transgenic arabidopsis and wildtype Arabidopsis thaliana inoculation in the embodiment of the present invention fivePstTable after DC3000
Type observes comparison diagram.
Figure 10 is transgenic arabidopsis and wildtype Arabidopsis thaliana inoculation in the embodiment of the present invention fivePstAfter DC3000
Quantitative character Statistical Comparison figure.
Figure 11 is transgenic arabidopsis and wildtype Arabidopsis thaliana inoculation in the embodiment of the present invention fivePstDC3000 is laggard
The disease-resistant situation schematic diagram of row Trypan Blue.
Figure 12 is transgenic arabidopsis and wildtype Arabidopsis thaliana inoculation in the embodiment of the present invention fivePstAfter DC3000
Different times transcription factorVdWRKY70Expression situation schematic diagram.
Figure 13 is transcription factor of the present inventionVdWRKY70With other known plantsWRKY70And the race of arabidopsis the IIIth ownsWRKYThe cluster analysis figure of gene.
Figure 14 is transcription factor of the present inventionVdWRKY70Subcellular Localization figure in arabidopsis.
Embodiment
Following examples are used to illustrate the present invention, but are not used to limit protection scope of the present invention.If specializing, in fact
Apply the conventional meanses that technological means used is well known to those skilled in the art in example.
LB culture mediums（In units of 1L）Prepare：In 10g Tryptone（Tryptone）, 5g Yeast Extract
（Yeast extract）, 10g NaCl in plus 800mL deionized waters, be sufficiently stirred dissolving, with KOH tune pH to 7.0, add deionization
Culture medium is settled to the agar that 1L is added to 15g, sterilizing by water.
The preparation of PDA culture medium（In units of 1L）：200g potato decortications are taken, stripping and slicing youngster, with eight layers of filtered through gauze, are added
20g glucose is settled to 1L, pours into the conical flask for adding 15g agar, sterilizing.
The transcription factor of embodiment oneVdWRKY70Clone
1.1 design of primers
Using homologous clone method, according in NCBIVvWRKY70Gene, sequence use the primer in VectorNTI 11
Find design primer clone's mRNA sequences, sequence are：
VdWRKY70F：ATGCACTGCACCTGGTCGGAAA
VdWRKY70R：ACACTCAAACCGATAAAACATCATC
1.2RNA extraction
Choose and ' young leaflet tablet of Vitis davidii Foex 0943 ', plant total serum IgE is extracted using Bioteke plant RNA extractions kit.Use
1000 micro ultra-violet and visible spectrophotometers of Thermo Scientific Nanodrop measure concentration and through Ago-Gels
Electrophoresis determines RNA integrality.CDNA first chain is obtained using the RR047 reverse transcription reagent box of TakaRa companies.
1.3 PCR are expanded
PCR amplifications use the NEB super fidelity enzymatic amplifications of Phusion TM, and reaction system is：HF buffer 10 μ L, 2.5
The mmol/L μ L of d NTPs 2.5, the μ L of template 2, upstream and downstream primer（10mmol/L）The super fidelity enzymes of each 2.5 μ L, Phusion TM
0.5 μ L, distilled water are mended to 50 μ L.PCR response procedures are：98 DEG C of min of pre-degeneration 3；98 DEG C of denaturation 10s, 58 DEG C of 10 s of annealing,
72 DEG C of extension 30s, totally 31 circulations；Last 72 DEG C of extensions 10min, 4 DEG C of preservations.
1.4 glue reclaim
PCR primer runs electrophoresis with 2% agarose gel, and glue reclaim is carried out with the agarose gel QIAquick Gel Extraction Kit of Tiangeng.Afterwards will be pure
The EXTaq of product TaKaRa after changeTMPolymerase adds A, reaction system：10x buffer 2 μ L, 2.5mmol Mg2+ 2μ
L, 2.5 mmol/L the μ L of dNTPs 2, the μ L of template 14.Response procedures：72 DEG C of 10 min of reaction.
1.5 Ta clones publish in instalments body
With reference to pGEMT-easy TM vector constructions specification structure VdWRKY70-T carriers, Transformed E .coli DH5 α Escherichia coli
Blue hickie screening is carried out, monoclonal is chosen and send raw work bioengineering（Shanghai）Limited company is sequenced, and sequencing result uses
Vector NTI splicings compare, and obtain transcription factorVdWRKY70Coding nucleotide sequence such as sequence table SEQ ID NO.1 institutes
Show, its length is 931bp.
The structure of the arabidopsis overexpression vector of embodiment two
Arabidopsis chooses Colombia's type arabidopsis；The T plasmids that PCR amplifications template is crossed using sequence verification；Plant expression vector
From pCAMBIA1303；Agrobacterium strains are LBA4404；NEB NCO I and Bst E II restriction endonuclease, Axygen mini-scale plasmids
Extracts kit is purchased from Zhengzhou Bo Mei companies；1/2MS, hygromycin B, kanamycins etc. are purchased from Zhengzhou Bao Sai biotech firms；
PEG4000, Cellulase R10, Mecerozym R10, mannitol, potassium chloride, MES, BSA, 0.45 μm of filter etc. are purchased from
Zhengzhou Chao Yan biotech firms.
Protoplasts of Arabidopsis thaliana broken by ultrasonic prepares method (Maas et al, 1995) of the conversion with reference to Maas C, is copolymerized using laser
Focusing microscope observation converts cultured protoplasts of Arabidopsis thaliana broken by ultrasonic.Nuclear localization signal analysis prediction uses Nuc Pre（http://
www.sbc.su.se/~maccallr/nucpred/cgi-bin/single.cgi）.
It is prepared by Agrobacterium LBA4404 competence：Plate, picking are drawn on the LB solid mediums containing 50 mg/L rifampins
Unit cell shakes bacterium 48h to bacterium solution muddiness in the LB fluid nutrient mediums containing 50 mg/L rifampins, by 1：100 ratios are inoculated into
Bacterium 5h is shaken on the fresh LB fluid nutrient mediums containing 50mg/L rifampins of 50ml.Bacterium solution is placed in ice bath 30min on ice, 4 DEG C,
5000g centrifuges 5min；The calcium chloride suspension that supernatant adds 1ml 0.1% is abandoned, ice bath 5min, 4 DEG C, 5000g centrifuges 5 min；Abandon
Reset and add the calcium chloride resuspension into 800 μ L0.02%, often the μ L ice baths of pipe 100 are standby for the pipe of packing 8.
The plasmid that 2 μ g need to convert is added in the Agrobacterium competence prepared, is mixed, liquid nitrogen flash freezer 1min, 37 DEG C of water
5min is bathed, 1ml LB fluid nutrient mediums is added, 28 DEG C, 200rpm, shakes bacterium 5h.Centrifugal concentrating be coated onto containing 50mg/L rifampins and
On the LB flat boards of 50mg/L kanamycins, 28 DEG C of culture 48h, the checking of picking single bacterium colony.
Arabidopsis planting conditions are 22 DEG C, and 16h illumination, 8h is dark, and water is a few days ago poured in conversion.The Agrobacterium of conversion shakes bacterium
To OD values be 2.0 or so, 5000g room temperatures concentration centrifugation 5min, abandon reset and add 10% sucrose solution resuspension, 5000g room temperatures from
Heart 5min adds 10% sucrose solution to be resuspended floating to OD1.0, addition Sillwet L-77 to final concentration 0.02%.Select thaliana flower
Converted when just showing money or valuables one carries unintentionally, inflorescence is dipped into 1min in Agrobacterium bacterium solution, taking-up is stained with filter paper and inhales bacterium solution unnecessary on stem.22 DEG C black
Placed two days under dark condition, pay attention to moisturizing, converted again once every 7 days.Three weeks or so after conversion, treat that fruit folder maturation collects seed.
Dry seed, which is placed on, to be added 900 μ L and shakes 9min containing 0.2% 70% ethanol of polysorbas20 in 2ml EP pipes.Abandon
The ethanol reset and added into 90% is washed 3 times, is finally poured on the filter paper of sterilizing and dried with 100% ethanol suspension.1/2MS culture mediums are configured,
Hygromycin B is added before a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices to final concentration 25mg/L.The arabidopsis seed of sterilizing is uniformly sprinkled upon on 1/2MS culture mediums, it is close
Envelope is put to return again in 22 DEG C of incubators for two days in 4 DEG C of refrigerators and cultivated, and the selection result is observed after one week, can be using normal growth to turn base
Because of type arabidopsis positive plant.
Transcription factor in the arabidopsis of embodiment threeVdWRKY70The reaction of gene pairs whiterot fungi
3.1 Phenotypic Observation
Upgrowth situation good transgenic arabidopsis and wildtype Arabidopsis thaliana are chosen, whiterot fungi is connect using needle point method（white
rot）, its phenotype is observed respectively after seven days, as a result as shown in figure 1, wherein a1, which is wildtype Arabidopsis thaliana, infects the disease-resistant of whiterot fungi
Situation, a2 are the disease-resistant situation that transgenic arabidopsis infects whiterot fungi.From figure 1 it appears that wildtype Arabidopsis thaliana is than turning
Genotype arabidopsis infects more serious.
After seven days, according to four kinds of degree situations of Infectikon whiterot fungi in transgenic arabidopsis and wildtype Arabidopsis thaliana
Quantitative character statistics is carried out, as a result as shown in Figure 2.It can also be seen that transgenic arabidopsis compares wildtype Arabidopsis thaliana from Fig. 2
Anti- whiterot fungi ability it is stronger.
3.2 Trypan Blues are observed
Trypan Blue is carried out to the blade for being inoculated with whiterot fungi 0h, 24h, 48h, 72h, 96h, be put into 50mL added with dyeing liquor from
In heart pipe, 95 DEG C of water-bath 3min, it is stored at room temperature several hours or stays overnight.Washed with chloraldurate, low speed concussion, make its colour fading,
Change liquid 2 ~ 3 times, wait control（Acellular dead blade）Decolourize complete, preserved with 60% glycerine.Transgenosis is observed under the microscope
The disease-resistant situation of type arabidopsis and wildtype Arabidopsis thaliana, as a result as shown in figure 3, wherein b1, which is wildtype Arabidopsis thaliana blade, carries out platform
Expect the disease-resistant situation of blue dyeing, b2 is the disease-resistant situation that transgenic Arabidopsis leaf carries out Trypan Blue.Can be with from Fig. 3
Find out, connect bacterium 48h, the situation of a small amount of meronecrosis of wildtype Arabidopsis thaliana；Bacterium 72h is met, wildtype Arabidopsis thaliana has big face
Long-pending meronecrosis situation, and the meronecrosis area of transgenic arabidopsis is substantially much smaller, explanationVdWRKY70Gene has
There is the ability of definitely anti-whiterot fungi.
Transcription factor in 3.3 arabidopsisVdWRKY70The reaction of gene pairs powdery mildew
3.3.1 Total RNAs extraction and purifying
Divide 0h, 24h, 48h, 72h, 96h picking leaves piece, after liquid nitrogen frozen, -80 DEG C of preservations.It is broken by grinding using CTAB methods
Transgenic Arabidopsis leaf is added in EP pipes of the 2ml without RNase, is rapidly added 1mLCTAB extract solutions, 20 μ L β-sulfydryl second
Alcohol, concussion mix, and 65 DEG C of water-bath 15min, during which overturn 1-2 times.Add 200 μ L chloroform-isoamyl alcohols（Chloroform：Isoamyl alcohol=24：1）,
Turn upside down, mix 5min, 4 DEG C of condition 12000rpm centrifuge 10min, suct clearly in new centrifuge tube, add isometric chloroform, shake
Swing 5min.Under the conditions of 4 DEG C, 12000rpm centrifugation 10min, supernatant is drawn in new centrifuge tube, adds 1/3 to 1/4 volume 1mol/L's
LiCl, -20 DEG C of precipitates overnights.Under the conditions of 4 DEG C, 12000rpm centrifugation 30min, supernatant, precipitation 500-700 μ L 75% second are abandoned
Alcohol washs, and is repeated 2 times.Precipitation 5-8min is dried, with 50 μ L without RNase（DEPC water）Dissolving precipitation.RNA is purified, reference
The operating instructions of TAKTRA companies Dnase I.The 30min under the conditions of 37 DEG C in PCR instrument afterwards.Add 540 μ LDEPC water, add isometric
600 μ L chloroforms, concussion shake up 3-5min, 12000rpm centrifugation 10min, take supernatant（About 500 μ L）, add the anhydrous second of two volumes
Alcohol, -70 DEG C of precipitation 30-60min.12000rpm centrifuges 10min（4℃）, supernatant is abandoned, precipitation is washed with 500 μ L 75% ethanol,
It is repeated 2 times.Precipitation 5-8min is dried, ethanol is volatilized completely, dissolves RNA, -80 DEG C of preservations with the DEPC water without RNase in right amount.
3.3.2 cDNA is synthesized
To extract, purify to obtain total serum IgE as template, with reference to the PrimeScript RT reagent Kit of TAKARA companies
With gDNA Eraser（Perfect Real Time）Application method carry out the synthesis of grape cDNA the first chains.
3.3.3 primer sequence
According toVdWRKY70Gene order uses the primer find design primers in Vector NTI 11：
Sense primer VdWRKY70-F：5’-ATGGCAACCAAACAGGTCCAGAT-3’；
Anti-sense primer VdWRKY70-R：5’-GGCGTTGCTATCATCTTGATTGTTT-3’；
Reference gene：
Atactin-F：GAAATCACAGCACTTGCACC；
Atactin-R：AAGCCTTTGATCTTGAGAGC.
3.3.4 real-time quantitative PCR
Operated with the SYBR Green I Master kits of LightCycler 480 of Roche companies, reaction system
For：LightCycler®480SYBR GreenⅠ:10 μ L, sense primer and each μ L of 0.5 μ L, cDNA template 2 of anti-sense primer, nothing
The μ L of bacterium water 4.Reaction condition is 95 DEG C of pre-degeneration 5min；95 DEG C of denaturation 20s, 60 DEG C of 20s, 72 DEG C of extension 20s, totally 45 are followed
Ring.Each three biology of processing repeat, and the fluorescent value at each time point has three technologies to repeat, and takes average.Data analysis is adopted
With the method for relative quantification.Gene relative expression quantity is finally analyzed using 2- △ △ CT methods, as a result as shown in Figure 4.
From fig. 4, it can be seen that transcription factor after wildtype Arabidopsis thaliana inoculation whiterot fungiVdWRKY70Hardly express, turn base
Because type arabidopsis expression quantity gradually rises, 72h reaches top, then reduces, and illustrates transcription factor VdWRKY70 genes by white
The induction of rotten bacterium.
Transcription factor in example IV arabidopsisVdWRKY70The reaction of gene pairs powdery mildew
4.1 Phenotypic Observation
Upgrowth situation good transgenic arabidopsis and wildtype Arabidopsis thaliana are chosen, powdery mildew is connect using needle point method（powdery
mildew）, its phenotype is observed respectively after eight days, as a result as shown in figure 5, wherein c1, which is wildtype Arabidopsis thaliana, infects the anti-of powdery mildew
State of an illness condition, c2 are the disease-resistant situation that transgenic arabidopsis infects powdery mildew.From figure 5 it can be seen that wildtype Arabidopsis thaliana ratio
Transgenic arabidopsis infects more serious.
After eight days, according to four kinds of degree situations of Infectikon whiterot fungi in transgenic arabidopsis and wildtype Arabidopsis thaliana
Quantitative character statistics is carried out, as a result as shown in Figure 6.It can also be seen that transgenic arabidopsis compares wildtype Arabidopsis thaliana from Fig. 6
Anti- powdery mildew ability it is stronger.
4.2 Trypan Blues are observed
Trypan Blue is carried out to inoculation powdery mildew 0d, 1d, 2d, 3d, 4d blade, is put into the 50mL centrifuge tubes added with dyeing liquor
In, 95 DEG C of water-bath 3min, it is stored at room temperature several hours or stays overnight.Washed with chloraldurate, low speed concussion, make its colour fading, change liquid 2
~ 3 times, wait control（Acellular dead blade）Decolourize complete, preserved with 60% glycerine.Transgenic is observed under the microscope to intend
Southern mustard and the disease-resistant situation of wildtype Arabidopsis thaliana, as a result as shown in fig. 7, wherein d1, which is wildtype Arabidopsis thaliana blade, carries out trypan blue
The disease-resistant situation of dyeing, d2 are the disease-resistant situation that transgenic Arabidopsis leaf carries out Trypan Blue.From figure 7 it can be seen that
Bacterium 1d is met, the situation of a small amount of meronecrosis of wildtype Arabidopsis thaliana meets bacterium 3d, and wildtype Arabidopsis thaliana has the thin of large area
Born of the same parents' necrosis situation, and the meronecrosis area of transgenic arabidopsis is substantially much smaller, explanationVdWRKY70Gene has certain
The ability of the anti-powdery mildew in ground.
Transcription factor in 4.3 arabidopsisVdWRKY70The reaction of gene pairs powdery mildew
1.3.1 in the step of Total RNAs extraction and purifying, cDNA synthesis, primer sequence, real-time quantitative PCR and embodiment one ~
1.3.4 identical, the primer is identical with 1.3.3 in embodiment one, will not be repeated here, as a result as shown in Figure 8.
From figure 8, it is seen that transcription factor after wildtype Arabidopsis thaliana inoculation powdery mildewVdWRKY70Hardly express, turn base
Because type arabidopsis expression quantity gradually rises, 3d reaches top, then gradually reduces, illustrates transcription factorVdWRKY70Gene by
The induction of powdery mildew.
Transcription factor in the arabidopsis of embodiment fiveVdWRKY70Gene pairs Pst DC3000 reaction
5.1 Phenotypic Observation
First by preservationPstDC3000 is chosen in right amount with adding rif LB culture mediums to activate with pipette tipsPstDC3000 bacterial strains
（P.syringae pv. Tomato DC3000））, directly squeeze into it is sterilized shake in tube, be put into shaking table（Ultra-clean work
Platform operates）Shake 14h or so.Afterwards, tube will be shaken to be put into centrifuge, thalline is collected by centrifugation in centrifugal force 2500G, 10min.Repeat
Last step.Use 10mMMgCl2100 times of dilution injects vacuum side of blade to OD600 ≈ 0.002 with syringe nozzle（Make whole blade
Soak）, with injection Mgcl2Blade do blank control, blade incidence is observed, as a result as shown in figure 9, wherein e1 is wild
Type arabidopsis infectsPstDC3000 disease-resistant situation, e2 are that transgenic arabidopsis infectsPstDC3000 disease-resistant situation.From
Fig. 9 can be seen that transgenic arabidopsis and wildtype Arabidopsis thaliana is inoculated with respectivelyPstDC3000, wildtype Arabidopsis thaliana ratio turn base
Because type arabidopsis infect it is more serious.
After five days, according to five kinds of degree situations of Infectikon whiterot fungi in transgenic arabidopsis and wildtype Arabidopsis thaliana
Quantitative character statistics is carried out, shown in result figure 10.From fig. 10 it can be seen that transgenic arabidopsis resists than wildtype Arabidopsis thaliana
Pst DC3000 abilities are stronger.
5.2 Trypan Blues are observed
To inoculationPstDC3000 blade carries out Trypan Blue, it is put into the 50mL centrifuge tubes added with dyeing liquor, 95 DEG C of water
3min is bathed, several hours is stored at room temperature or stays overnight.Washed with chloraldurate, low speed concussion, make its colour fading, change liquid 2 ~ 3 times, wait pair
According to（Acellular dead blade）Decolourize complete, preserved with 60% glycerine.Transgenic arabidopsis and wild is observed under the microscope
The disease-resistant situation of type arabidopsis, as a result as shown in figure 11, wherein f1 are that wildtype Arabidopsis thaliana blade carries out the anti-of Trypan Blue
State of an illness condition, f2 are the disease-resistant situation that transgenic Arabidopsis leaf carries out Trypan Blue.It can be seen from figure 11 that bacterium 6h is met,
Wildtype Arabidopsis thaliana and transgenic arabidopsis have the situation of a small amount of meronecrosis, meet bacterium 12h, wildtype Arabidopsis thaliana it is thin
Born of the same parents' necrosis situation is more more serious than transgenic arabidopsis, explanationVdWRKY70Gene, which has, definitely to be resistedPstDC3000 energy
Power.
Transcription factor in 5.3 arabidopsisVdWRKY70Gene pairsPstDC3000 reaction
Divide 0d（Before injection）、0d（After injection）, 1d, 2d, 3d picking leaves piece, with liquid nitrogen quick freeze, -80 DEG C of preservations.Total RNAs extraction
It is used to draw and the step of purifying, cDNA synthesis, primer sequence, real-time quantitative PCR is identical with 1.3.1 ~ 1.3.4 in embodiment one
Thing is identical with 1.3.3 in embodiment one, will not be repeated here, as a result as shown in figure 12.
It can be recognized from fig. 12 that transcription factor after wildtype Arabidopsis thaliana inoculation powdery mildewVdWRKY70Hardly express, turn
Genotype arabidopsis expression quantity gradually rises, and 12h reaches top, then gradually reduces, illustrates transcription factorVdWRKY70Base
Because byPstDC3000 induction.
The transcription factor of embodiment sixVdWRKY70With other known plantsWRKY70And the race of arabidopsis the IIIth ownsWRKYBase
The cluster analysis of cause
Utilize ' in Vitis davidii Foex 0943 'VdWRKY70, model plant arabidopsis, golden mandarin orange（Fortunella crassifolia）VdWRKY70, paprike（Capsicum annuum）CaWRKY70, watermelon（Citrullus lanatus）ClWRKY70, wheat
（Triticum aestivum）TaWRKY70, upland cotton（Gossypium hirsutum）GhWRKY70, corn（Zea mays）ZmWRKY70, rice（Oryza sativa）OsWRKY70, model plant arabidopsis（Arabidopsis thaliana）AtWRKY70And the race of arabidopsis the IIIth ownsWRKYGene protein sequence construct phylogenetic tree, with analysisWRKYGene family
The evolutionary relationship of member.Amino acid sequence progress multiple sequence connection is matched somebody with somebody first by the Clustal programs of MEGA7.0 softwares,
The result matched somebody with somebody of Clustal multisequencing connection is output in the softwares of MEGA 7.0, the adjacent tree using the software buildings of MEGA 7.0, and
Examined 1000 times using Boot strap method and Confidence Analysis is carried out to these chadograms, as a result as shown in figure 13.
Referring to Figure 13, the chadogram is divided into four major classes, whereinVdWRKY70WithFcWRKY70、CaWRKY70、AtWRKY70、AtWRKY54、ClWRKY70Get together.And be overexpressed in tobacco and lemonFcWRKY70Make both drought-resistant stress
Ability strengthens.In addition,FcWRKY70By adjusting arginine decarboxylase（ADC）Expression promote putrescine generation.CaWRKY70
In bacterial wilt（R. Solanacearun）, Phytophthora capsici disease（P. capsici）And tobacco mosaic virus (TMV)（TMV）In the presence of lure
Lead expression.It is overexpressedAtWRKY70Cause the expression of SA induction PR genes, so as to increase resisting pseudomonas aeruginosa, soft rot, Huang
Melon powdery mildew ability, but suppress the ability of the anti-alternaria of SA mediations.AtWRKY70WithAtWRKY54Collective effect, in non-life
Strengthen the tolerance of arabidopsis osmotic stress in thing stress.Silence slwrky70 mitigates the anti-Potato Aphid of mi-1- mediations, root
The ability of Root-knot.As can be seen from Figure 13,VdWRKY70WithFcWRKY70、CaWRKY70、AtWRKY70、AtWRKY54、ClWRKY70Functionally there is certain similitude, explanationVdWRKY70Effect in biotic and abiotic stress with
These genes have certain similitude.
The transcription factor of embodiment sevenVdWRKY70Subcellular Localization
Protein is the direct agent of biological function, and the distribution and regulation and control of protein and growth, the development of plant individual are close
It is related.Therefore, the proteins subcellular location understood during this is very necessary to the life process for understanding plant.VdWRKY70
Be to the disease-resistant related gene of grape, therefore understandVdWRKY70Expressive site in plant, for understanding the gene in Portugal
Anti-disease mechanism in grape has a very important role.
By Wuhan Biorun Bio-Tech. Co., Ltd., pass through cloneVdWRKY70Open reading frame sequence, it is glimmering using green
Aequorin（GFP）For reporter gene, the cellular localization for building amalgamation and expression detects carrier pBWA (V) HS-wrky70-
GLosgfp, above-mentioned carrier is subjected to protoplasts of Arabidopsis thaliana broken by ultrasonic conversion, comprised the following steps that：25 DEG C or so cultures are taken to turn for 7 ~ 15 days
Genotype arabidopsis Arabidopsis thaliana Seedlings are some, add 5 ~ 10ml enzymolysis liquids（1.5% Cellulase R10,0.75%
Macerozyme R10,0.6M mannitol, 10mM MES pH=5.7,10mM CaCl2and 0.1%BSA）.All immersions
Tissue is advisable, 28 DEG C of slowly concussions（100rpm）Digest 5 ~ 6h.50g centrifuges 5 min after 40 μm of strainer filterings, removes supernatant, with pre-
Cold W5（154mM NaCl, 125mM CaCl2, 5mM KCl and 2mM MES pH 5.7）Solution 10ml wash 2 times, 50g from
Heart 5min, 4 ~ 25 degree of centrifuging temperature.Add 500 μ L MMG（0.4M mannitol, 15mM MgCl2 and 4mM MES
pH=5.7）Solution suspension.200 μ L protoplast suspensions are taken to add 10 μ L DNAs, 210 μ L PEG solution [40%（W/V）
PEG4000,2M mannitol and 0.1M CaCl2, uniformly mixing.It is stored at room temperature 30 min.Diluted with 1mL W5 solution
Terminating reaction.50g centrifugations 5min collects protoplast.1mL W5 solution is added to wash 1 ~ 2 time.It is eventually adding 1mLW5 solution, 28 DEG C
24 ~ 48h of light culture, is observed by confocal laser scanning microscope, CLSMVdWRKY70Expressive site in arabidopsis, sees simultaneously
Examine the protoplast of wildtype Arabidopsis thaliana, as a result as shown in figure 14, wherein g1 be wildtype Arabidopsis thaliana Subcellular Localization figure, g2
For the Subcellular Localization figure of transgenic arabidopsis.It is seen from figure 14 that the wildtype Arabidopsis thaliana of target gene is not imported
Protoplast, there is green fluorescence in whole protoplast, and import the transgenic protoplasts of Arabidopsis thaliana broken by ultrasonic of target gene, only
Have has green fluorescence in nucleus, explanationVdWRKY70Albumen is positioned in the nucleus of transgenic arabidopsis, and this measure is
Understand transcription factor in depthVdWRKY70Biochemical character and physiological function in Vitis davidii Foex body lay the foundation.
Embodiment described above, simply presently preferred embodiments of the present invention, only to explain the present invention, is not limited
The scope of the present invention processed, to those of ordinary skill in the art, certainly can be according to skill disclosed in this specification
Art content, make other embodiments easily by way of replacing or changing, thus it is all in principle and technique bar of the invention
Changes and improvements that part is done etc., it all should be included in scope of the present invention patent.
SEQUENCE LISTING
<110>Zhengzhou Fruit-tree Inst., Chinese Agriculture Science Academy
<120>Vitis davidii Foex transcription factor VdWRKY70 and its application in plant resistance to environment stress kind is cultivated
<130> 2017.9.30
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 931
<212> DNA
<213>Artificial sequence（VdWRKY70）
<400> 1
atggagtgca cctggtcgga aaagatctcc ggcgatcgga aaagagccat cgacgagctg 60
cttcgaggtc gctccatcac caagcagctt cgcagcgtcc tcgtcggagg ggatcaacag 120
gagtcgaccc aggatcttct agcgaaaatc ttgaggtcat tcaccgacac tctttctata 180
ttgaattccg gcgagtccga cgaggccgtc tcacagattc cggcgagtgg tcagctgtat 240
tcgccgggtt ggcatggccg gaggtcggag gaatcttgcg agagtggtaa gagttccacc 300
aaggatcgca ggggctgtta caagcgaagg aagaattcgc aatcttggat cagaatcacc 360
cctaattttc acgatgacgg ttatgcctgg cggaagtacg gacaaaaagt catactcaat 420
gctaaacatc aaagaagcta ctataggtgc acccacaagc atgaccaggg ctgcatggca 480
accaaacagg tccagatgac tgaagaggag cccccaatgt acaaaaccac ataccacggc 540
cagcacacat gcaaaagcat gctgaagtca tctcagatta tggtggaaaa ttctactgca 600
agagattctt ccattctgct cagctttgaa tcaaacaatc aagatgatag caacgccttt 660
ttctcatcat tccccttgat aaaacaagag gaagagatcc caaatgatga tcaggaggtg 720
acctacaata accataccaa taacaacaac aagaacaaca actcctcatc ctctgattat 780
cttctgtcac tggagctcac cacatttgaa tccaatctgg ggtctgatca tggcgatgtc 840
ctttctgggg ttaactcttc ttgtacggac agcactcaca gtttggatga tatgatgatg 900
gactttgatg atgtttttat cggttttgag t 931
<210> 2
<211> 22
<212> DNA
<213>Artificial sequence（VdWRKY70F）
<400> 2
atgcactgca cctggtcgga aa 22
<210> 3
<211> 25
<212> DNA
<213>Artificial sequence（VdWRKY70R）
<400> 3
acactcaaac cgataaaaca tcatc 25
<210> 4
<211> 23
<212> DNA
<213>Artificial sequence（Sense primer VdWRKY70-F）
<400> 4
atggcaacca aacaggtcca gat 23
<210> 5
<211> 25
<212> DNA
<213>Artificial sequence（Anti-sense primer VdWRKY70-R）
<400> 5
ggcgttgcta tcatcttgat tgttt 25
<210> 6
<211> 20
<212> DNA
<213>Artificial sequence（Atactin-F）
<400> 6
gaaatcacag cacttgcacc 20
<210> 7
<211> 20
<212> DNA
<213>Artificial sequence（Atactin-R）
<400> 7
aagcctttga tcttgagagc 20
Claims (7)
1. Vitis davidii Foex transcription factorVdWRKY70, it is characterised in that the Vitis davidii Foex transcription factorVdWRKY70Encoding nucleoside
Acid sequence is as shown in sequence table SEQ ID NO.1.
2. Vitis davidii Foex transcription factorVdWRKY70Application in plant resistance to environment stress kind is cultivated.
3. Vitis davidii Foex transcription factor according to claim 2VdWRKY70Application in plant resistance to environment stress kind is cultivated, it is special
Sign is that the plant resistance to environment stress kind is Genes For Plant TolerancePstDC3000 kinds.
4. Vitis davidii Foex transcription factor according to claim 2VdWRKY70Application in plant resistance to environment stress kind is cultivated, it is special
Sign is that the plant resistance to environment stress kind resists for arabidopsisPstDC3000 kinds.
5. Vitis davidii Foex transcription factor according to claim 4VdWRKY70Application in plant resistance to environment stress kind is cultivated, its
It is characterised by, cultivates arabidopsis and resistPstThe method of DC3000 kinds is as follows：Using agricultural biotechnologies method, Vitis davidii Foex is turned
Record the factorVdWRKY70Arabidopsis cell is transformed into, is expressedVdWRKY70Transgenic arabidopsis, expressingVdWRKY70Transgenic arabidopsis on be inoculated withPstDC3000, after a few days, turned using Real-Time Fluorescent Quantitative PCR Technique analysis
Record the factorVdWRKY70The relative expression quantity of gene, wildtype Arabidopsis thaliana is contrasted, relative expression quantity is higher to be resisted for arabidopsisPst
DC3000 kinds.
6. Vitis davidii Foex transcription factor according to claim 5VdWRKY70Application in plant resistance to environment stress kind is cultivated, its
It is characterised by, the primer used in the Real-Time Fluorescent Quantitative PCR Technique is as follows：
Sense primer VdWRKY70-F：5’-ATGGCAACCAAACAGGTCCAGAT-3’；
Anti-sense primer VdWRKY70-R：5’-GGCGTTGCTATCATCTTGATTGTTT-3’.
7. Vitis davidii Foex transcription factor according to claim 6VdWRKY70Application in plant resistance to environment stress kind is cultivated, its
It is characterised by, the reference gene used in the Real-Time Fluorescent Quantitative PCR Technique is as follows：
Atactin-F：GAAATCACAGCACTTGCACC；
Atactin-R：AAGCCTTTGATCTTGAGAGC.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110408648A (en) * | 2019-07-05 | 2019-11-05 | 中国农业科学院郑州果树研究所 | A kind of method of quick detection plant disease resistance genes |
| CN112725351A (en) * | 2021-03-23 | 2021-04-30 | 上海师范大学 | Application of gene OsWRKY43 in resisting bacterial blight of rice |
| CN113293169A (en) * | 2021-05-26 | 2021-08-24 | 扬州大学 | Paeonia lactiflora PlMYB108 gene and application thereof in drought resistance of plants |
-
2017
- 2017-10-10 CN CN201710934742.5A patent/CN107501399A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| 冯虎: "刺葡萄0943抗白腐病WRKY调控因子功能分析", 《中国农业科学院学位论文》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110408648A (en) * | 2019-07-05 | 2019-11-05 | 中国农业科学院郑州果树研究所 | A kind of method of quick detection plant disease resistance genes |
| WO2021004098A1 (en) * | 2019-07-05 | 2021-01-14 | 中国农业科学院郑州果树研究所 | Method for rapidly detecting plant disease-resistant genes |
| 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 |
| CN113293169A (en) * | 2021-05-26 | 2021-08-24 | 扬州大学 | Paeonia lactiflora PlMYB108 gene and application thereof in drought resistance of plants |
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