CN106591323A - Wild vitis quinquangularis anti-disease gene and application thereof - Google Patents
Wild vitis quinquangularis anti-disease gene and application thereof Download PDFInfo
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Abstract
The invention discloses a wild vitis. quinquangularis anti-disease gene and application thereof. The full length of a complete open reading frame sequence is 1095bp, and 364 amino acids are encoded. Moreover, a pCAMBIA2300-35S-VqWRKY52 over-expression vector is constructed, a model plant, namely, arabidopsis thaliana is imported by an inflorescence infection method, and over-expression is performed in the arabidopsis thaliana, so that the resistance of the arabidopsis thaliana to powdery mildew and pseudomonas syringae parasitic in living organisms is improved remarkably, and the resistance to saprophytic botrytis cinerea is lowered. A wild vitis. quinquangularis-24 anti-disease gene VqWRKY52 participates in an SA-mediated anti-disease signal path, can enhance the anaphylactic reaction, can be used for enhancing the resistance of plants to the fungus powdery mildew and the pseudomonas syringae parasitic in the living organisms, and plays a role in regulating and controlling the anaphylactic reactions of the plants through regulation and control of the expression of the VqWRKY52 gene, so that the reactions of the plants to different pathogenic bacteria are regulated and controlled.
Description
Technical field
The present invention relates to plant disease resistance genes are identified and gene engineering technology field, more particularly to a kind of wild Vitis quinquangularis resist
Ospc gene and its application.
Background technology
Plant can only rely on each unlike animal has mobile protective cells and posteriori immune system, plant immune system
Plant cell and by infecting the signal that site discharges.Phytopathogen is evolved in the evolutionary process long-term with plant
Diversified life strategy defines a set of oneself distinctive immune system helping oneself to infect plant, its transfer
The record factor plays wherein very important regulating and controlling effect.
WRKY transcription factor is a wherein important class transcription factor, is played in the whole immune system network of plant
Just regulating and controlling or playing a part of negative regulation.WRKY transcription factor be in plant find peculiar transcription regulatory factor, its albumen
Matter N-terminal contains the zinc finger of highly conserved aminoacid sequence WRKYGQK and CX4-5CXNHXH/C (X is arbitrary aminoacid)
(Zinc-finger type) structure, downstream is by special combination target gene promoters region W boxes (T) TGACC (A/T) nucleoside
Acid sequence, so as to regulate and control the expression of corresponding gene, plays its Molecular biological function.The classification of WRKY transcription factor is according to transcription
The number and zinc fingerses of contained domain in the factor, general WRKY transcription factor can be divided into three major types：1st class contains 2
WRKY conserved domains, zinc fingerses type is C2H2 types；2nd class comprises only 1 WRKY conserved domain, its zinc fingers
For C2H2 types；3rd class is identical with the 2nd class, comprises only 1 WRKY conserved domain, but its zinc fingers is C2HC types.
The existing substantial amounts of research in arabidopsiss and Oryza sativa L. shows effect of the WRKY transcription factor in disease-resistant, most
WRKY transcription factor is all inducible expression.General WRKY transcription factor upstream is by the protein kinase MAPK in MAPK signal pathways
Phosphorylation activation, downstream can specificity combined with W-box, and then stimulate the expression of related disease-resistant gene, produce disease resistance anti-
Should；One kind directly can be produced albumen by pathogenic bacterium inducing, and the mostly effector produced by pathogen makes WRKY protein expressions
Amount is improved, and WRKY gene regulates and controls again related disease-resistant gene, so as to improve the disease resistance of plant, if AtWRKY70 and AtWRKY18
It is the resistances against diseases that arabidopsiss are improved by this regulative mode；Phase interaction can also be passed through between multiple WRKY transcription factor
For adjusting resistivity of the plant to pathogen, AtWRKY18, AtWRKY40 compare single prominent with the Trimutant of AtWRKY60
Variant increased to pseudomonas syringae resistance, but graw mold of tomato performance resistance is weakened.Rice Os WRKY23 gene is being intended
Overexpression can improve pathogenesis-related proteins expression in southern mustard, improve the resistance to pseudomonas syringae.
Plant resistant pathogen infection relies primarily on two big class Plant hormone signal approach, wherein, SA signal pathways are main
Resistance of the mediated plant to live body parasitical fungi, such as powdery mildew etc.；JA/ET signal pathways are primarily involved in plant to saprophytic
Resistance of bacterium, such as botrytis cinerea etc..Phytohormone regulate and control plant immunization when often with other response elements together with act on, for example
Anaphylaxiss and active oxidative burst.Anaphylaxiss are closely related with plant disease-resistant, in biogenous fungal infection plant, plant
Thing can form large-area cell death site is infected, and to prevent funguses from obtaining nutrition from plant, this is beneficial to limit true
The growth of bacterium, so as to improve the disease resistance of plant.SA signal pathways tend to infecting site or remote organization excites allergy anti-
Should, while activating the expression of disease-resistance-related protein, activation system acquired resistance.Plant responding pathogen reacts, often adjoint
Active oxidative burst, active oxygen plays an important role in anaphylaxiss.
Disease-resistant related WRKY transcription factor research is relatively more in model plant arabidopsiss, on Fructus Vitis viniferae with regard to research also not
It is not also many that WRKY turns the disease-resistant functional study of the green factor.China possesses abundant amur grape resource, some of which kind
Very strong with strain disease resistance, such as business -24 (S-24) but its quality are not so good as European grape kind.By learning from other's strong points to offset one's weaknesses, by wild Portugal
Grape are disease-resistant to be combined with the fine quality of European grape, for research and using these resources carry out Fructus Vitis viniferae breeding for disease resistance have it is important
Meaning.Therefore, the gene of disease-resistant correlation is excavated from Chinese wild grape and its disease-resistant function is studied, its disease resistance response is understood
Regulation Mechanism, its disease resistance response key gene is found, and susceptible variety is improved by genetic transformation, so as to acquired character is excellent
Preferable kind.
The content of the invention
It is an object of the present invention to provide a kind of wild Vitis quinquangularis disease-resistant gene, and it is disease-resistant to demonstrate the wild Vitis quinquangularis
The function and its possible mechanism of action of the disease-resistant aspect of gene.
In order to realize above-mentioned task, the present invention adopts following technical solution：
A kind of wild Vitis quinquangularis disease-resistant gene, it is characterised in that the wild Vitis quinquangularis disease-resistant gene be wild Vitis quinquangularis business-
24 disease-resistant gene VqWRKY52, its coding region sequence is as follows：
Jing applicant further study showed that the wild Vitis quinquangularis disease-resistant gene can be used for improving the anti-live body of plant and post
Raw funguses powdery mildew and pseudomonas syringae ability.And regulation and control plant hypersensitive is played by regulating and controlling the expression of VqWRKY52 genes,
Further reach reaction of the regulation and control plant to various pathogenic bacteria.
The wild Vitis quinquangularis disease-resistant gene that the present invention is given is first from wild Vitis quinquangularis
(Vitis.quinquangularis) one disease-resistant gene of wild Vitis quinquangularis business -24 of the WRKY families being cloned in business -24
VqWRKY52, and further study the disease-resistant aspect of the disease-resistant gene VqWRKY52 of wild Vitis quinquangularis business -24 function and its
Possible mechanism of action, is that Fructus Vitis viniferae and other crop disease-resistant breedings provide theoretical foundation.
Applicant constructs first pCAMBIA2300-35S-VqWRKY52 Overexpression vectors, and by titbit dip method
It is conducted into model plant arabidopsiss.Have studied the transgenic of the disease-resistant gene VqWRKY52 overexpressions of wild Vitis quinquangularis business -24
Strain and wild to impinging upon inoculation powdery mildew, after pseudomonas syringae and botrytis cinerea, the disease resistance response of various strains.
Further study showed that according to applicant, the disease-resistant gene VqWRKY52 of wild Vitis quinquangularis business -24 can be significantly improved
Arabidopsiss increase the susceptibility to botrytis cinerea to powdery mildew and the resistance of pseudomonas syringae.
Under the conditions of three kinds of pathogen infections, the accumulation of the active oxygen (ROS) in transgenic line is apparently higher than wild right
According to, and there are strong anaphylaxiss in transgenic line, for live body parasitism powdery mildew and half live body parasitism Flos Caryophylli vacation unit cell
For bacillus, strong anaphylaxiss limit growth of pathogenic bacteria, can be to their disease resistance but grey for saprophytic bacteria so as to strengthen
For mycete, strong anaphylaxiss are conducive on the contrary it to infect, so transgenic line is reduced to its resistance.Result above is all
Show that overexpressions of the adversity gene VqWRKY52 of wild Vitis quinquangularis business -24 in arabidopsiss enhances plant hypersensitive response, carry
The high ability of the anti-live body parasitical fungi of plant, reduce anti-saprophytic bacteria ability.
Description of the drawings
Fig. 1 is responses of the disease-resistant gene VqWRKY52 of wild Vitis quinquangularis business -24 to SA and JA.Wherein, A figures are to use qRT-
PCR method sees the relative expression quantity of the disease-resistant gene VqWRKY52 of wild Vitis quinquangularis business -24, and figure B is that wild Vitis quinquangularis business -24 is disease-resistant
Gene VqWRKY52 Assay of promoter activity, * represents that compared with the control there were significant differences, and * * are represented and have compared with the control extremely notable
Difference (* 0.01<P<0.05, * * P<0.01).
Fig. 2 is the different growths in arabidopsiss T3 is for plant of the disease-resistant gene VqWRKY52 promoteres of wild Vitis quinquangularis business -24
The expression pattern situation in stage；Wherein, A figures are to be grown in the MS culture medium mature embryo of 24 hours, and scale is 200 μm；B figures are 5
The seedling in its age, scale is 500 μm；C figures are the young plant of 2 week old, and scale is 2mm；D figures are 3 week old young plants；E figures are titbit, mark
Chi is 2mm；F figures are flower, and scale is 1mm；G figures are the style and flower pesticide of flower, and scale is 200 μm；H figures are silique, and scale is
1mm。
Fig. 3 is wild control, pad4 mutants, transgenic line VqWRKY52 (#28), VqWRKY52 (#30) and
The Potted orchard of VqWRKY52 (#33) is inoculated with the reaction of powdery mildew.Wherein, A figures are the morphology photograph of 7 days after each strain inoculation white lead
Piece；B figures are the plant cell death that platform expects blue dyeing observation powdery mildew induction, and scale represents 200 μm on figure.
Fig. 4 is wild control, pad4 mutants, transgenic line VqWRKY52 (#28), VqWRKY52 (#30) and
The accumulation of colony growth and active oxygen after the Potted orchard inoculation powdery mildew of VqWRKY52 (#33).Wherein, A figures be inoculation seven days after
Platform expects blue dyeing observation powdery mildew thalli growth and plant cell death, and arrow is pointed out that the plant cell that powdery mildew is induced is dead
Die, scale represents 100 μm on figure；B figures are to O with NBT2 -Dyeing sees 0 hour after inoculation, 24 hours, 48 hours, and 72 hours
Accumulation afterwards, scale represents 2cm on figure；C figures be inoculation seven days after conidium number statistics, * is represented to be had compared with wild control
Significant difference, * * are represented pole significant difference (* 0.01 compared with wild control<P<0.05, * * P<0.01).
Fig. 5 be after powdery mildew is processed disease-resistant related gene in wild control, transgenic line VqWRKY52 (#28),
Expression in VqWRKY52 (#30) and VqWRKY52 (#33).Reference gene is Actin2.* represent compared with wild control
There were significant differences, and * * are represented pole significant difference (* 0.01 compared with wild control<P<0.05, * * P<0.01).
Fig. 6 is wild control, pad4 mutants, transgenic line VqWRKY52 (#28), VqWRKY52 (#30) and
The Potted orchard of VqWRKY52 (#33) is inoculated with the reaction of pseudomonas syringae.Wherein, A figures are 5 days after each strain inoculation pathogen
Morphology photo；B figures be inoculation three days after bacterial growth amount statistics；C figures are that 24 hours, 48 is little 0 hour after inoculation pathogen
When, and expect the plant cell death that blue dyeing observation bacterium induces with platform in 72 hours, scale represents 200 μm on figure；D figures are H2O2With
O2 -The accumulation of 0 hour and 48 hours, H after inoculation pathogen2O2And O2 -Dyeed with NBT and DAB respectively.Each experiment repeats three
Secondary, each independent experiment at least processes 6 blades, and scale represents 200 μm on figure.* to represent and have significance difference compared with wild control
Different (* 0.01<P<0.05).
Fig. 7 be inoculation pseudomonas syringae after disease-resistant related gene in wild control, transgenic line VqWRKY52 (#
28), the expression in VqWRKY52 (#30) and VqWRKY52 (#33).Reference gene is Actin2.* represent and compareed with wild
Compare that there were significant differences, * * are represented pole significant difference (* 0.01 compared with wild control<P<0.05, * * P<0.01).
Fig. 8 is wild control, transgenic line VqWRKY52 (#28), VqWRKY52 (#30) and VqWRKY52's (#33)
Potted orchard is inoculated with the reaction of botrytis cinerea.Wherein, A figures are the morphology photograph of 3 days after the in vitro lotus throne blade inoculation pathogen of each strain
Piece；B figures are to be inoculated with 0 hour, 24 hours, 48 hours and 72 hours after pathogen to expect that the plant of blue dyeing observation bacterium induction is thin with platform
Born of the same parents are dead, and scale represents 2cm on figure；C figures be inoculation three days after lesion diameter statistics；D figures are H2O2And O2 -In inoculation pathogen
The accumulation of 0 and 48 hour afterwards, H2O2And O2 -Dyeed with NBT and DAB respectively.In triplicate, each independent experiment is extremely for each experiment
6 blades are processed less, and scale represents 2cm on figure.* represent compared with wild control that there were significant differences (* 0.01<P<0.05).
Fig. 9 be after inoculation botrytis cinerea disease-resistant related gene in wild control, transgenic line VqWRKY52 (#28),
Expression in VqWRKY52 (#30) and VqWRKY52 (#33).Reference gene is Actin2.* represent compared with wild control
There were significant differences, and * * are represented pole significant difference (* 0.01 compared with wild control<P<0.05, * * P<0.01).
The present invention is described in further detail below in conjunction with drawings and Examples.
Specific embodiment
Application inventor utilizes reverse transcriptional PCR (Reverse Transcription-Polymerase Chain
Reaction, RT-PCR), the chains of cDNA first are synthesized as template with the blade total serum IgE reverse transcription of wild Vitis quinquangularis business -24, using same
Source clone technology, according to European grape PINOT NOIR genome sequence design primer is adopted, and amplification has obtained wild Vitis quinquangularis business -24 and resisted
Ospc gene VqWRKY52, the wild Vitis quinquangularis disease-resistant gene be the disease-resistant gene VqWRKY52 of wild Vitis quinquangularis business -24, Complete Open
Reading frame sequence total length 1095bp, encodes 364 aminoacid.
In order to the disease-resistant gene VqWRKY52 of a step research wild Vitis quinquangularis business -24 resists the concrete work(of biotic in plant
Can, the inventor of applicant constructs pCAMBIA2300-35S-VqWRKY52 Overexpression vectors, by it in wild arabidopsiss
Overexpression in plant.It was found that there is the overexpression strain of the disease-resistant gene VqWRKY52 of wild Vitis quinquangularis business -24 can significantly improve
Arabidopsiss increase the susceptibility to botrytis cinerea to powdery mildew and the resistance of pseudomonas syringae.VqWRKY52 is in arabidopsiss
Overexpression enhance plant hypersensitive response, improve the ability of the anti-live body parasitical fungi of plant, reduce anti-saprophytic bacteria
Ability.
Under study for action, applicant has carried out the side of body of three kinds of different type pathogen to seedling transfer-gen plant and wild control
Compel to process, and determine cell death situation, as a result the accumulation of active oxygen and the expression of disease-resistant related gene show wild
The disease-resistant gene VqWRKY52 of downy grape business -24 receives SA abduction deliverings, and important work is played in the process of cell death of pathogenic bacterium inducing
With.
The following is the coding region sequence and biotic function reality of the disease-resistant gene VqWRKY52 of wild Vitis quinquangularis business -24
The concrete steps of checking.
A, early-stage Study analysis in, Fructus Vitis viniferae WRKY family genes pathogen process and HORMONE TREATMENT after expression base
On plinth, using Homology-based cloning, the chains of cDNA first are synthesized as template with the blade total serum IgE reverse transcription of wild Vitis quinquangularis business -24, expanded
Increasing has obtained the disease-resistant gene VqWRKY52 sequences of wild Vitis quinquangularis business -24, the disease-resistant gene VqWRKY52 of wild Vitis quinquangularis business -24
Coding region sequence it is as follows：
B, the entire open reading frame of the adversity gene VqWRKY52 sequences of wild Vitis quinquangularis business -24 insertion CaMV35S is opened
Mover downstream, constructs plant Overexpression vector and is conducted into wild type by agriculture bacillus mediated titbit dip method
Arabidopsiss Colombia C0.Screening obtains the good VqWRKY52 transgenic lines (#28, #30, #33) of Phenotype.
C, referring to Fig. 1-9, by identifying VqWRKY52 transgenic lines (#28, #30, #33), arabidopsiss can be significantly improved
To powdery mildew and the resistance of pseudomonas syringae, increase the susceptibility to botrytis cinerea.Additionally, after pathogen inoculation, turning base
Because the accumulation of the active oxygen (ROS) in strain be also significantly more than in wild control, and than control have higher anaphylaxiss.With
Upper result all shows that the disease-resistant gene VqWRKY52 of wild Vitis quinquangularis business -24 plays an important role in plant disease-resistant signal pathway.
The specific embodiment that inventor provides is the following is, all methods are if no special instructions conventional side in following enforcement
Method.Below in an example the disease-resistant gene WRKY52 of wild Vitis quinquangularis business -24 is abbreviated as VqWRKY52.
Embodiment 1：VqWRKY52 is to the response of hormone and the tissue specificity in arabidopsiss and Space-time speciality table
Reach
Applicant has found that VqWRKY52 receives grape powdery mildew abduction delivering, in order to determine it to SA in the research of early stage
With the response of JA, expressions of the VqWRKY52 under both HORMONE TREATMENT is analyzed, as a result found little in pathogen process 1
When and 12 hours, VqWRKY52 can be expressed by SA induced strongs, but can not by MeJA induce (Figure 1A).
In order to further verify this result, the promoter region of the 2107bp of VqWRKY52 is cloned to be fused to
Before gus reporter gene, Pro is formedVqWRKY52:GUS report structures, the work that instantaneous conversion Tobacco Analysis promoter is responded to hormone
Property, use Pro35S:GUS is used as control.As a result show that VqWRKY52 promoteres can receive SA abduction deliverings, it is impossible to by JA induction (figures
1B), this is consistent with the result of Figure 1A.
In order to have a look tissue specificity and Space-time speciality expression, Pro is usedVqWRKY52:GUS stable conversion arabidopsiss, obtain
T3 is obtained for plant, using the plant of different development stage GUS staining analysiss are carried out.Sprouting stage early stage does not have obvious GUS to live
Property be detected (Fig. 2A), as the very low activity of growth of plant is found (Fig. 2 B) in cotyledon tip and root system.Growth
In MS culture medium in two weeks big plant in a organized way in have active with strong GUS, especially in blade (Fig. 2 C).
In Seedling of growing up, old blade has with strong GUS activity (Fig. 2 D) than young blade.In whole arabidopsiss titbit and Fruit pod
Find in dyeing in addition to the seed in flower pesticide and Fruit pod, remaining tissue has very strong GUS activity (Fig. 2 E-H).
Embodiment 2：Responses of the VqWRKY52 transgenic arabidopsis T3 for seedling to powdery mildew
In order to further determine effects of the VqWRKY52 in plant defense, by 3 T3 for transgenic line (#28, #
30, #33), arabidopsiss wild type (WT) and pad4 mutants inoculation arabidopsiss powdery mildew, see its response to pathogen, invent
People will grow all big adult Seedling inoculation powdery mildews of 3-4, the different strain active oxygens of observation, dead cell accumulation and growth of pathogenic bacteria shape
Condition, counted the conidium quantity of powdery mildew single bacterium colony, quantitative analyses relative expression's situation of disease-resistant related gene.As a result
Show that three transgenic lines (#28, #30, #33) are light in the wilder control (WT) of powdery mildew inoculation sequela situation, pad4
The onset state most heavy (Fig. 3 A) of mutant.There is strong powdery mildew induction in three transgenic lines (#28, #30, #33)
Cell death, and wild control (WT) cell death is lighter, although pad4 mutants morbidity most serious, is barely perceivable bright
Aobvious cell death (Fig. 3 B).In order to be further characterized by result above, we observe powdery mildew in each morbidity strain blade
Thalli growth situation.It was found that powdery mildew be grown in significantly suppression is received in 3 transgenic lines, and infecting site
There is obvious cell death (Fig. 4 A).Conidium statistical magnitude in transgenic line (Fig. 4 C) substantially few than WT plant.
O2 -Accumulation it is often relevant with plant cell death, so it is white in inoculation further to determine different genotype
Powder 0 hour, 24 hours, 48 hours, and be O in 72 hours2 -Accumulation.In the genotype of all detections, do not have within 0 hour and 24 hours
Obvious difference has been found, large-area active oxidative burst has all been occurred in that.At 48 hours and 72 hours, transgenic line was compared
There is substantial amounts of O in WT and pad42 -Aggregation, accumulation in 72 hours is especially more, and the accumulation of active oxygen is presented mottled.(Fig. 4 B)
Verified VqWRKY52 receives SA abduction deliverings to previous experiments, but is not induced by JA.In order to further appreciate that
Response of the VqWRKY52 genes in plant responding powdery mildew stress, inventor have detected SA signals way by real-time quantitative PCR
Expression (Fig. 5) of the disease-resistant related gene in footpath in wild control (WT) and transgenic line (#28, #30, #33).
AtICS1 genes participate in the biosynthesiss of SA and affect the accumulation of SA, and 24 hours expressions are raised after pathogen is processed, and 48
Hour reaches peak value, starts within 72 hours to reduce, and the expression in its 3 transgenic lines all compares wild type in this 3 time points
It is high.AtEDS1 also assists in the related signal pathways of SA, plays an important role in the biosynthetic upstreams of SA.Its expression is little in 24 and 48
When it is similar to AtICS1, but at 72 hours, its expression was lower than wild type in overexpression strain.Other AtPR1 and AtPR5 bases
Because after treatment expression in 24,48 and 72 hours is raised, and the expression of overexpression strain is higher than wild type.Experiment above is said
Bright VqWRKY52 works in the signal pathway that SA is mediated, and SA related gene expresses enhancing in overexpression strain.
These results all show that overexpressions of the disease-resistant gene VqWRKY52 of wild Vitis quinquangularis business -24 in arabidopsiss can be bright
It is aobvious to improve resistance of the arabidopsiss for powdery mildew.
Embodiment 3：Responses of the VqWRKY52 transgenic arabidopsis T3 for seedling to pseudomonas syringae
In order to determine effects of the VqWRKY52 in Genes For Plant Tolerance pseudomonas syringae, by 3 T3 for transgenic line (#
28, #30, #33) and arabidopsiss wild type (WT) inoculation pseudomonas syringae, its response to pathogen is seen, 3-4 will be grown
All big adult Seedling inoculation pathogen, the different strain active oxygens of observation, dead cell accumulation and growth of pathogenic bacteria amount, have counted inoculation
The quantity of pathogen in 3 days rear blades, and quantitative analyses relative expression's situation of disease-resistant related gene (Fig. 6,7).As a result
Show three transgenic lines (#28, #30, #33) in the light (figure of the wilder control (WT) of inoculation pathogen sequela situation
6A).Meanwhile, the increment of antibacterial in different genotype has been counted after being inoculated with 3 days, find the antibacterial life in overexpression plant
Length is inhibited (Fig. 6 B).At 0 hour, all genotype did not found cell death, at 24 hours, started in transgenic line
There is cell death, and WT does not occur, a large amount of cell deaths occur in 48h and continue to strengthen in 72h in transgenic line.WT exists
72h is suitable (Fig. 6 C) when the cell death amount for occurring ties up to 24h with transgenic line.The a large amount of cell deaths occurred in 72h can
Can be relevant with active oxidative burst.So determining H2O2And O2 -In the status of accumulating nutrient of 72h.As a result find, in 72h, overexpression strain
System accumulates more active oxygens (Fig. 6 D) than wild type.
In order to further appreciate that response of the VqWRKY52 genes in Genes For Plant Tolerance pseudomonas syringae, by real-time quantitative
PCR have detected 0h after inoculation pathogen, and 6h, 12h and 24h disease-resistant related gene is in wild control (WT) and transgenic line (#
28, #30, #33) in expression change (Fig. 7).As a result AtPR1, the AtPR5 and AtEDS1 abduction delivering in wild type is shown, but
It is but to be suppressed in transgenic line, this response with overexpression strain to powdery mildew is contrary.For JA approach related genes
It is also such for AtPDF1.2.The cell of the bacterium induction that above description of test causes in arabidopsiss overexpression VqWRKY52 is dead
Dying, to strengthen the expression of the gene related to SA be unmatched.
These results show that overexpressions of the disease-resistant gene VqWRKY52 of wild Vitis quinquangularis business -24 in arabidopsiss is substantially carried
High resistances of the adult plants to pseudomonas syringae, it is relevant with the cell death of bacterium induction, and with the table of SA related genes
It is little up to relation.
Embodiment 4：Responses of the VqWRKY52 transgenic arabidopsis T3 for seedling to botrytis cinerea
The cell death of bacterium induction plays an important role in the biogenous pathogen of plant resistant, and applicant wants to have a look
Overexpression strain for saprophytic bacteria response.By 3 T3 for transgenic line (#28, #30, #33) and arabidopsiss wild type (WT)
Inoculation botrytis cinerea, sees its response to pathogen；The adult Seedling inoculation pathogen that growth 3-4 weeks are big, the different strains of observation are lived
Property oxygen, dead cell accumulation and lesion diameter, and quantitative analyses relative expression's situation of disease-resistant related gene (Fig. 8, Fig. 9).
As a result show that three transgenic lines (#28, #30, #33) will be in the wilder control (WT) of inoculation pathogen sequela situation again
(Fig. 8 A, C).Meanwhile, cell death situation is observed when botrytis cinerea 0h, 24h, 48h and 72h is inoculated with, observe in 0h and 48h
The accumulation (Fig. 8 B, D) of active oxygen.As a result show that the anaphylaxiss after inoculation botrytis cinerea of overexpression strain are eager to excel than wild type, and
Have accumulated more active oxygens.But overexpression VqWRKY52 enhances the susceptibility to botrytis cinerea.
In order to further appreciate that effect of the VqWRKY52 genes in regulation and control botrytis cinerea resistance, examined by real-time quantitative PCR
0h after inoculation pathogen is surveyed, 12h, 24h and 48h disease-resistant related gene is in wild control (WT) and transgenic line (#28, #
30, #33) expression change (Fig. 9) in.As a result show that JA approach related gene AtPDF1.2 is in inoculation in all genotype
It is induced expression after 12h, and the expression in 3 transgenic lines is higher than wild type.24h after inoculation, in transgenic line
Expression starts to reduce, and wild type continues to raise, and now wild type is higher than transgenic line.In addition, relative to control 0h,
AtPR2 and AtPR5 12h after inoculation botrytis cinerea, 24h and 48h expressions are suppressed, but expression will in transgenic line
Wild type is significantly higher than, the expression inhibiting of these genes in transgenic line is which imply lighter than wild type.These results
Imply that SA signal pathways are enhanced considerably in transgenic line, and SA signal pathways and JA signal pathways are antagonisms, SA letters
The enhancing of number approach may make JA approach die down, this be unfavorable for plant to saprophytic bacteria opposing, susceptibility will be increased.
Result above shows that overexpressions of the disease-resistant gene VqWRKY52 of wild Vitis quinquangularis business -24 in arabidopsiss is substantially carried
High susceptibilities of the adult plants to botrytis cinerea.
Nucleotide or amino acid sequence table
<110>Xibei Univ. of Agricultural ＆ Forest Science ＆ Technology
<120>Wild Vitis quinquangularis disease-resistant gene and its application
<160>
<210> 1
<211>1095
<212>The coding region sequence of the disease-resistant gene VqWRKY52 of wild Vitis quinquangularis business -24
<213>
<220>
<400> 1
1 ATGGAGAACA TGGGAAGTTG GGAACAAAAG AATCTGATAA ATGAGCTTAC GAATGGGAGA
61 GAGCTAGCCA AACAGCTACA AATCCATCTC AGCGTGTCGT CTTCTTCACA TGACGCCCGT
121 GAATCCTTGG TCCAAAAGAT CCTAACTTCA TACGAGAAGG CCCTTTCATT GCTGAGGTGT
181 AGCGGCCCAG TGGGCGAGCC ACATGCCGCC GCAGCAGGCG GAGGAGGAGC AGTTGGAATA
241 GGGATGTCTG AGTCTCCACG CTCGCTCAGT GGGAGTCCTA GGAGCGAAGA TTCTGATAAG
301 GAGCAGGAGC ACAAAGATGG GTCTAGGAAG AGAAAGACAT TACCAAGGTG GACAAAGCAA
361 GTGCAGATGT CTCTAGGGCC AGGGCCAGAA GGCCCTCTTG ATGATGGGTT TAGTTGGAGG
421 AAGTATGGGC AGAAGGACAT TCTCGGAGCC AAATATCCAA GGAGCTATTA CAAATGCACT
481 CATAGAAATG CCCAAGGCTG TCTGGCTACA AAGCAAGTTC AAAGATCAGA CGATGACCCA
541 ACCATCTTTG AAATCACCTA CCGTGGAAGA CACACTTGCA CCCAAGTCTC CCAACAAATT
601 CCACCGCCGG CAGCACTGCC GGGAAACCAA GGACTGATGG ACAGCAAAGA TCATCAGCAA
661 TTCAACCTCC AACCCCAACA AAACCAGCAA CAAGCACAAG ACATGCTCTT GAGCTTCCAA
721 AGTGGCCTCA GAGTCGTAAC AGAAGGCTTG GACACACCAA GCGACCAAGC ATTTCCCCCC
781 TTTTGCTTCA CTTCAACGTC TAATATTAAG GTCGAAGAAC CCGGTTTCTC GCCTTCCATG
841 ATGGACAACA ATATCGTTGG TAATTTTCCT ACCTCATTTG TATCTCCTGC AGCATCTGGA
901 TCAAACTATT TTTCCATGTC GTCTGATGAA ATGAACAGCC TTGGAGGAAA CCAAAACCTG
961 CAGGCCCCCG AAGCCAATCT CAATGAGATA ATCTCAGCTG CTGCTTCAAC CACAAACCCT
1021 CAGTTTGAGC AGTTCCCGTT TGGTTCCATG GAATTCGATC CGAACTTTAA CTTCGACCAC
1081 CTGGGATTCT TTTAA
Claims (3)
1. wild Vitis quinquangularis disease-resistant gene, it is characterised in that the wild Vitis quinquangularis disease-resistant gene is that wild Vitis quinquangularis business -24 is disease-resistant
Gene VqWRKY52, its coding region sequence is as follows：
2. the wild Vitis quinquangularis disease-resistant gene described in claim 1 is used to improve the anti-live body parasitical fungi powdery mildew and fourth of plant
The application of fragrant pseudomonass ability.
3. application as claimed in claim 2, it is characterised in that play regulation and control plant by regulating and controlling the expression of VqWRKY52 genes
Allergy, and then reach reaction of the regulation and control plant to various pathogenic bacteria.
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| CN116479015A (en) * | 2023-03-30 | 2023-07-25 | 西北农林科技大学 | Grape powdery mildew effector, interaction protein and application thereof |
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| CN116479015A (en) * | 2023-03-30 | 2023-07-25 | 西北农林科技大学 | Grape powdery mildew effector, interaction protein and application thereof |
| CN116479015B (en) * | 2023-03-30 | 2024-04-26 | 西北农林科技大学 | Grape powdery mildew effector, interaction protein and application thereof |
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