CN106754992B - A kind of application, expression vector and its application of grape disease-resistant related gene VvPUB17 - Google Patents
A kind of application, expression vector and its application of grape disease-resistant related gene VvPUB17 Download PDFInfo
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- CN106754992B CN106754992B CN201710051147.7A CN201710051147A CN106754992B CN 106754992 B CN106754992 B CN 106754992B CN 201710051147 A CN201710051147 A CN 201710051147A CN 106754992 B CN106754992 B CN 106754992B
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- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
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Abstract
The invention discloses application, expression vector and its applications of a kind of grape disease-resistant related gene VvPUB17, belong to plant genetic engineering field.The present invention clones and isolates VvPUB17 gene from grape leave, and the plant overexpression carrier containing VvPUB17 gene is transformed into arabidopsis and grape leave, VvPUB17 gene expression amount can significantly increase the disease resistance of the arabidopsis of genetic transformation and the high expression of grape disease-resistant related gene, prove that VvPUB17 gene plays a significant role in enhancing plant disease-resistant, the research of VvPUB17 gene has great importance to disease-resistant plants new varieties are cultivated.
Description
Technical field
The present invention relates to a kind of application of grape disease-resistant related gene VvPUB17 in disease resistance of plant, also relate to
Expression vector and its application comprising grape disease-resistant related gene VvPUB17, belong to plant genetic engineering field.
Background technique
In recent years the study found that protein ubiquitination wide participation plant defense adjust, proteins ubiquitin modification
Key enzyme be ubiquitin ligase E3, it determines the specific recognition of substrate protein, be related to Plant pathogen interaction, including
The defense reaction of early stage, the interaction of gene-for-gene and inductivity are disease-resistant.U-box albumen is a kind of novel E3 albumen, is had
E3 activity.Research finds that U-box albumen is widely present in fungi, plant, in animal, in the eucaryote U- identified at present
In box albumen, the number of plant U-box (plant U-box protein, PUB) albumen is far more than other biological.Exist at present
More than 60 a PUB albumen are had been found that in arabidopsis, and the PUB albumen confirmed is reacted with infecting for plant defense pathogen
It is related, such as the expression of 3 U-box type E3 ligases of PUB22, PUB23 and PUB24 can be by flg22 type PAMP in arabidopsis
(Pathogen-associated molecular pattern) induce (Trujillo M etc., 2008, Current
Biology 18(18):1396-1401).Under the invasion of pathogen, the generation ratio of 22/23/24 Trimutant active oxygen of PUB
Wild type significantly improves, and also can obviously inhibit the growth of pathogen.Consistent with this, active oxygen generates related gene three
It is also obviously induced in mutant material.In Trimutant material, other PAMP, such as elf18 and chitin can also draw
Play oxidative burst.These results indicate that the E3 ligase such as PUB22, PUB23 and PUB24 is PAMPs activation PTI in plant
(PAMP-triggered immunity) downstream signal factor institute is required, it was demonstrated that PUB22, PUB23 and PUB24 are that plant is supported
The disease-resistant related gene of anti-microbial pathogen intrusion.
However, the especially most of novel U-box protein gene of ubiquitin ligase gene are in Plant defense responses at present
Middle effect is still not very clear.VvPUB17 gene of the present invention is grape list subunit U-box family ubiquitinbond enzyme family
One of member.By the way that grape U-box gene family is screened and identified, VvPUB17 gene is demonstrated by a variety of diseases of grape
The inducing expression of opportunistic pathogen.Therefore, it is to study the new way of plant disease-resistant to the exploitation of grape VvPUB17 gene, is turned by heredity
Change related gene improvement Disease Resistance of Grapevine weak kind and other plant varieties, will further enhance plant to the disease-resistant of pathogen
Property, the anti-spectrum of plant is widened, is of great significance for cultivating plant disease-resistant new varieties.
Summary of the invention
The object of the present invention is to provide a kind of grape disease-resistant related gene VvPUB17 answering in enhancing disease resistance of plant
With.
A second object of the present invention is to provide a kind of plant overexpression carriers.
Third object of the present invention is to provide a kind of application of plant overexpression carrier in enhancing disease resistance of plant.
In order to achieve the goal above, the technical scheme adopted by the invention is that:
A kind of application of grape disease-resistant related gene VvPUB17 in enhancing disease resistance of plant, the nucleotides sequence of the gene
Column as shown in SEQ ID NO:5, length 2695bp, open reading frame (ORF) from the 274th~2418 bit base (SEQ ID NO:
1), the amino acid sequence of its encoded protein (terminator codon is not translated) as shown in SEQ ID NO.2, the protein
Molecular weight is 78.2kDa, and isoelectric point 6.82 includes U-box and two ARMrepeat conserved domain.
Specifically, application of the grape disease-resistant related gene VvPUB17 in enhancing disease resistance of plant, comprising the following steps:
1) grape disease-resistant related gene VvPUB17 is connected in expression vector, forms plant overexpression carrier；
2) it takes plant overexpression carrier to transfect Agrobacterium, then is transferred in plant cell.
A kind of plant overexpression carrier, the expression vector contain as grape shown in SEQ ID NO:5 is disease-resistant related
Because of the open reading frame of VvPUB17, as shown in SEQ ID NO:1.
Specifically, the expression vector is pCAMBIA3301-VvPUB17, preparation step is as follows:
1) using grape cDNA as template, P1, P2 are primer, carry out pcr amplification reaction, and amplified production returns after electrophoretic analysis
Receive target fragment；
Forward primer P1:5 '-GGGAGATCTATGGCCTCCGCTGCGATAGTAT-3 ',
Reverse primer P2:5 '-GGGGGTGACCCTACAAAACAGGCACTGAAATGGGC-3'；
It is at restriction enzyme site Bgl II, P2 underscore at P1 underscore is restriction enzyme site BstE II；
2) with target fragment and plant expression vector in Bgl II, BstE II difference double digestion step 1)
PCAMBIA3301 recycles target fragment and linearized vector, screening after connection, conversion, verify to get.
Above-mentioned plant overexpression carrier is (containing the grape disease-resistant related gene VvPUB17 as shown in SEQ ID NO:5
The expression vector of open reading frame) enhancing the application in disease resistance of plant.
Beneficial effects of the present invention:
The present invention provides a kind of new gene VvPUB17 to the resistance of pathogen for enhancing plant.Portugal is cloned by separation
The DNA fragmentation of VvPUB17 gene in grape blade connect with the carrier for capableing of the overexpression target gene, it is thin to be transformed into plant
Born of the same parents can improve plant to the resistance of pathogen by overexpression VvPUB17 gene, control the generation of disease, reduce or keep away
Exempt from loss brought by plant disease.Meanwhile the VvPUB17 gene that the present invention develops also is that plant disease-resistant has widened the anti-of plant
Spectrum overcomes the problem of traditional breeding method is unable to plant inter-species transfer disease-resistant related gene.
Detailed description of the invention
Fig. 1 is agarose gel electrophoretogram after the pCAMBIA3301-VvPUB17 digestion of plant overexpression carrier；
Fig. 2 is the expression quantity variation that grape leave is inoculated with different time VvPUB17 gene after powdery mildew；
Fig. 3 is the identification of VvPUB17 genetic transformation Arabidopsis plant disease resistance；
Fig. 4 is the identification of disease resistance after VvPUB17 gene instantaneous conversion grape leave.
Specific embodiment
Only invention is further described in detail for following embodiments, but does not constitute any limitation of the invention.
Embodiment 1
The separation of grape disease-resistant related gene VvPUB17 is cloned, and the specific method is as follows:
1) grape leave total serum IgE is extracted according to SDS/ phenol method
Prepare extracting solution: be added into 2.0mL centrifuge tube 850 μ L Extraction buffers (140mM LiCl, 10mM EDTA,
10mM Tris, 5% (w/v) SDS, 2% (w/v) PVP) and 30 μ L beta -mercaptoethanols, it mixes well, for use.
Liquid nitrogen precooler mortar takes 0.2g grape young leaflet tablet in mortar, and after adding appropriate liquid nitrogen to be fully ground, packing is to containing
Have in the 2.0mL centrifuge tube for preparing extracting solution in advance, sufficient vortex mixes, and 12000rpm is centrifuged 5min under the conditions of 4 DEG C；In absorption
Clear liquid is added isometric chloroform-isoamyl alcohol (24:1) into another 2.0mL centrifuge tube, and sufficient vortex mixes, under the conditions of 4 DEG C
12000rpm is centrifuged 15min；Aspirate supernatant is added the KAc (pH4.8) of 1/3 volume 5M, fills into another 2.0mL centrifuge tube
Divide to be vortexed and mix, 12000rpm is centrifuged 10min under the conditions of 4 DEG C；Aspirate supernatant is into another 2.0mL centrifuge tube, the bodies such as addition
Long-pending chloroform-isoamyl alcohol (24:1), sufficient vortex mix, and 12000rpm is centrifuged 10min under the conditions of 4 DEG C；Aspirate supernatant is to another
In one 1.5mL centrifuge tube, 1/3 volume 8M LiCl is added, sufficient vortex mixes, -20 DEG C of placement 2h or more, then under the conditions of 4 DEG C
12000rpm is centrifuged 15min；Supernatant is lightly removed, is precipitated twice (under the conditions of 4 DEG C with 800 μ L, 75% ethanol washing
12000rpm is centrifuged 8min)；Air drying 10min waits for that ethyl alcohol sufficiently volatilizees, and 30 μ L DEPC-H are added2O dissolution precipitating, i.e.,
Obtain grape leave total serum IgE.
2) total serum IgE is purified
It is added in 1.5mL centrifuge tube: 100 μ g, DNase I 10U, 10 × DNase buffer of total serum IgE, 10 μ L,
RNasin100U, DEPC-H2O polishing mixes well, 37 DEG C of incubation 30min to 100 μ L；It is different that isometric phenol-chloroform-is added
Amylalcohol (25:24:1), sufficient vortex mix, and 12000rpm is centrifuged 10min under the conditions of 4 DEG C；Aspirate supernatant to another 1.5mL from
It in heart pipe, is added isometric chloroform-isoamyl alcohol (24:1), sufficient vortex mixes, and 12000rpm is centrifuged 10min under the conditions of 4 DEG C；
Aspirate supernatant is into another 2mL centrifuge tube, 1/10 volume 3M NaAC (pH 5.2) of addition, 2.5 times of volume dehydrated alcohols, and -20
DEG C place 2h or more；Then 12000rpm is centrifuged 15min under the conditions of 4 DEG C；Supernatant is lightly removed, with 800 μ L, 75% ethyl alcohol
Washing precipitating is twice (12000rpm is centrifuged 8min under the conditions of 4 DEG C)；Air drying 10min waits for that ethyl alcohol sufficiently volatilizees, and 20 μ are added
L DEPC-H2O dissolution precipitating；RNA purifying is completed.
3) the first chain of cDNA is synthesized by template reverse transcription of RNA
It is added in PCR pipe: Random 6mers (50 μM) 1 μ L, dNTP Mixture (10mM each) 1 μ L, Total
2 μ g, RNase free dH of RNA2O polishing mixes well, brief centrifugation makes solution to PCR pipe bottom to 10 μ L.In PCR instrument
Upper 65 DEG C of reactions 5min, on ice chilling；It is added in PCR pipe:Buffer 4 μ L, RNase
Inhibitor (40U/ μ L) 0.5 μ L,1 μ L, RNase Free dH of RTase (200U/ μ L)2O polishing is to 20
μL.Following reactions: 30 DEG C of 10min are carried out in PCR instrument；42℃60min；95℃5min；4 DEG C of preservations；After the completion of reverse transcription i.e.
Obtain the first chain of cDNA；- 40 DEG C save backup.
4) PCR amplification is carried out by template of cDNA
PCR clones VvPUB17 gene PCR reaction system are as follows: 1 μ L of cDNA template, forward and reverse primer P3, P4 each 2 μ L, PCR
5 μ L, dNTP Mix of Buffer, 2.5 μ L, DNA Polymerase, 1.0 μ L, PCR-Grade Water polishing to 50 μ L, PCR
Response procedures are as follows: 94 DEG C of 30sec, 94 DEG C of 30sec, 56 DEG C of 30sec, 72 DEG C of 3min, 30cycles, 72 DEG C of 10min, 4 DEG C
Forever。
Forward primer P3:5 '-AGAGTCTGAGAAACGAGATTCAGAC-3 ' (SEQ ID NO:3)；
Reverse primer P4:5 '-AAGCAGCTTTTCCACATTATACTATTACC-3 ' (SEQ ID NO:4).
5) detection and recycling
The product of PCR amplification is carried out to 1% agarose gel electrophoresis, recycles target fragment, is connected to pMD19-T load
Body, coupled reaction system are as follows: 0.5 μ L, Solution I of pMD19-T carrier 2.5 μ L, 2.0 μ L of target fragment are mixed well,
2h conversion is reacted under the conditions of 16 DEG C, by connection product transformed competence colibacillus TOP10 cell, on additional 100mg/L Amp culture medium
Blue hickie screening is carried out, picking positive colony is seeded to LB liquid medium and shakes bacterium culture, extracts plasmid and obtains positive plasmid
PMD19-T-VvPUB17 send company's sequencing detection, and VvPUB17 gene order is as shown in SEQ ID NO:5.
Embodiment 2
The building of grape disease-resistant related gene VvPUB17 overexpression carrier, the specific method is as follows:
The pMD19-T-VvPUB17 plasmid prepared using embodiment 1 is template, and using P1, P2 as primer, it is anti-to carry out PCR amplification
It answers, PCR product recycles target fragment after agarose gel electrophoresis；It is restriction enzyme site Bgl II, P2 underscore at P1 underscore
Place is restriction enzyme site BstE II.
Forward primer P1:5 '-GGGAGATCTATGGCCTCCGCTGCGATAGTAT-3'(SEQ ID NO:10)；
Reverse primer P2:5 '-GGGGGTGACCCTACAAAACAGGCACTGAAATGGGC-3’(SEQ ID NO:11)。
Double digestion above-mentioned purpose segment and plant expression vector pCAMBIA3301, recycling are distinguished with Bgl II, BstE II
Target fragment is attached with linearized vector；Coupled reaction system are as follows: pCAMBIA3301 linearized vector 5 μ L, Solution
10 μ L of I, 5 μ L of target fragment, mixes well, reacts 2h under the conditions of 16 DEG C；Connection product converts TOP10 competent cell, attached
Add and screened on the LB culture medium of kanamycins, picking positive colony be seeded to LB liquid medium (card containing 100mg/L that
Mycin) in shake bacterium culture, extract plasmid, send company's sequencing detection, the correct plasmid of sequence is named as plant overexpression carrier
pCAMBIA3301-VvPUB17.Correct sequence is subjected to double digestion, endonuclease reaction system are as follows: 4 μ L, Bgl II restriction endonuclease of plasmid
0.5 μ L, BstE II restriction endonuclease 0.5 μ L, ddH215 μ L of O, mixes well, and reacts 2h under the conditions of 37 DEG C；Digestion products are through fine jade
Sepharose electrophoresis releases 2100bp's or so after pCAMBIA3301-VvPUB17 digestion as a result as shown in the band of Fig. 12
Electrophoretic band.M is DNA Marker in Fig. 1, and 1 is before plasmid enzyme restriction, 2 is after plasmid enzyme restriction.
Embodiment 3
Plant overexpression carrier pCAMBIA3301-VvPUB17 converts Agrobacterium, and the specific method is as follows:
Electric shock cup, which is placed under ultraviolet lamp, handles 20min；Agrobacterium GV3101 competent cell is taken out, is placed on ice to melt,
It is transferred to after thawing in electric shock cup, and 10 μ L plasmids is added, mixed well, be placed in 5min on ice；Electric shock cup is transferred to electric shock instrument
Middle carry out shock treatment；The conversion fluid in the cup that shocks by electricity is taken out into the centrifuge tube of 1.5mL after processing, 1mL LB culture is added
Base cultivates 1h under the conditions of 28 DEG C；Brief centrifugation 15s in centrifuge removes part supernatant, is coated on after suspension containing antibiotic
On the plate of (gentamicin of 60mg/L, the kanamycins of 100mg/L), it is placed in 28 DEG C of cultures.Picking monoclonal has been seeded to
In the LB liquid medium of antibiotic (gentamicin of 60mg/L, the kanamycins of 100mg/L), cultivated under the conditions of 28 DEG C for 24 hours,
It is detected through bacterium solution PCR, positive colony for testing in next step.
Embodiment 4
Plant overexpression carrier pCAMBIA3301-VvPUB17 is transformed into plant
1) plant overexpression carrier pCAMBIA3301-VvPUB17 is transformed into arabidopsis
By the Agrobacterium inoculation containing plant expression vector pCAMBIA3301-VvPUB17 to 10mL LB liquid medium
In (gentamicin containing 60mg/L, the kanamycins of 100mg/L), 28 DEG C of cultures are for 24 hours；5mL bacterium solution is taken to be transferred to 50mL fresh
LB liquid medium (kanamycins of gentamicin and 100mg/L containing 60mg/L), 28 DEG C are continued to cultivate, until bacterium solution OD600
Reach 0.6 or so；Bacterium solution is transferred in centrifugal bottle or centrifuge tube, under room temperature, revolving speed is that 4000rpm is centrifuged 10min, is gone
Except supernatant collection thallus；It is resuspended in permeabilization buffer (0.5 × MS, 5% sucrose, 0.03%Silwet L-77), adjusts OD600Extremely
0.8；Fruit pod existing in arabidopsis floral is removed, inflorescence be completely immersed in 20s in penetrating fluid (10~30s, or with move
Liquid device will directly permeate drop on inflorescence), remove the penetrating fluid in arabidopsis leaf or stalk immediately, plant is lain in into pallet
In, pallet is covered with plastic film, film is removed afterwards for 24 hours, continues to cultivate in greenhouse；To improve transformation efficiency, used after 7 days
Same method infects again；Arabidopsis plant by conversion carries out normal management, harvests seed when fruit pod existing white；It will receive
The transgenic seed obtained impregnates 10min with 0.2% TritonX-100；Again with 10% sodium hypochlorite surface sterilization, 12min；
Sterilizing water washing five times, each 2min；With yellow liquid transfer gun head by seed be layered on the herbicide basta containing 20mg/L MS (0.5 ×
MS, 1% sucrose, 1% agar, pH 5.7) on plate, 4 DEG C dark culturing two days；Incubator is then transferred to be cultivated, if
Set condition of culture: 22 DEG C of temperature, illumination 16h.It still grows fine after herbicide (20mg/L Basta) screening, true leaf blade
And growing point is dark green and root stretches and penetrates the seedling of culture medium and can primarily determine as transgenic plant, transplanting to nutritive cube
In continue to cultivate.
2) plant overexpression carrier pCAMBIA3301-VvPUB17 is transformed into grape
By the Agrobacterium inoculation of conversion plant expression vector pCAMBIA3301-VvPUB17 to 10mL LB liquid medium
In (gentamicin containing 60mg/L, the kanamycins of 100mg/L), 28 DEG C of cultures are for 24 hours；5mL bacterium solution is taken to be transferred to 50mL fresh
LB liquid medium (kanamycins of gentamicin and 100mg/L containing 60mg/L), 28 DEG C are continued to cultivate, until bacterium solution OD600
Reach 0.6 or so；Bacterium solution is transferred in centrifugal bottle or centrifuge tube, under room temperature, revolving speed is that 4000rpm is centrifuged 10min, is gone
Except supernatant collection thallus；Permeabilization buffer (10mM MES pH5.6, the 10mM MgCl of same volume is added2, 2% (w/v)
Sucrose and 150mM acetosyringone) it is resuspended；Grape leave is placed in suitable re-suspension liquid, it is true to be placed in ability of swimming
Vacuumize process is carried out in idle loop pump, starts timing, slow release after 30min when vacuum is pumped when strong pointer is 0.085MPa
Gas；The grape leave handled well is taken out, the bacterium solution of blade surface is removed with sterilized filter paper, blade distal shaft is face-up
It is placed in pallet, petiole absorbent cotton moisturizing, pallet is covered with preservative film, is placed in incubator and cultivates.
Test example
1, expression pattern analysis of the grape disease-resistant related gene VvPUB17 in grape
In order to confirm that VvPUB17 gene participates in the regulation of disease resistance response, using quantitative fluorescent PCR (Real-time PCR)
Technology analyzes expression pattern of the VvPUB17 gene after grape leave is inoculated with powdery mildew (powdery mildew, PM).Inoculation side
Method according to Wang method (Wang Y etc., Vitis 34:159-164), SDS/ phenol method extract inoculation powdery mildew after different time
The total serum IgE of (0,12,24,36,48,60,72h) grape leave, by PrimeScriptTMRT-PCR Kit specification (is purchased from
TAKARA company) reverse transcription is carried out, the reverse transcription product of 7 different times detects grape as Real-time pcr template
VvPUB17 gene pairs grape leave is inoculated with the response after powdery mildew.Real-time PCR uses SYBR Premix Ex TMTaq
II kit (is purchased from TAKARA company), and according to kit specification, in the iCycler iQ5Real-time PCR instrument (U.S.
Bio-Rad company) on carry out real-time quantitative PCR reaction.Reaction system are as follows: 1 μ L, SYBR Mix of template 12.5 μ L, it is forward and reverse
Each 1 μ L of primer, sterile purified water polishing to 25 μ L；Response procedures are as follows: 95 DEG C of 5min, 95 DEG C of 30s, 68 DEG C of 30s, 72 DEG C of 30s, 30
A circulation, 72 DEG C of 5min, 4 DEG C of 10min.P5, P6 are the forward and reverse primer of VvPUB17 gene magnification, and VvActin is internal reference base
Cause, P7, P8 are the forward and reverse primer of VvActin gene magnification.
Forward primer P5:5 '-CTGTAACGGCAATGCTTAACCTAT-3 ' (SEQ ID NO:6)；
Reverse primer P6:5 '-CCTCGTCCGCTATTCTCTTCTTA-3 ' (SEQ ID NO:7).
Forward primer P7:5 '-TCCTGTGGACAATGGATGGA-3 ' (SEQ ID NO:8)；
Reverse primer P8:5 '-CTTGCATCCCTCAGCACCTT-3 ' (SEQ ID NO:9).
Thresh value is defaulted as 30 by PCR instrument, records each reaction fluorescence signal respectively by background and enters exponential increase rank
Then recurring number (threshold cycle, Ct) corresponding to the inflection point of section uses 2- △ △CtMethod is not to be inoculated with the leaf of powdery mildew
Piece is control, carries out normalization to the relative expression quantity of different time points VvPUB17 gene.As a result as shown in Fig. 2, grape leave
Middle VvPUB17 gene 12h expression quantity after being inoculated with powdery mildew sharply increases, for 24 hours when expression quantity peak, expressed in 36h
Amount is declined, and subsequent expression quantity sharply increases again, reaches a peak, subsequent and on a declining curve, explanation again in 60h
VvPUB17 gene is by powdery mildew inducing expression.
2, the identification of grape disease-resistant related gene VvPUB17 disease resistance
1) grape disease-resistant related gene VvPUB17 Disease Resistance Identification in arabidopsis
The arabidopsis T3 of conversion carries out Disease Resistance Identification after growing 60 days for transgenic plant.Arabidopsis powdery mildew
(Golovinomyces cichoracearum UCSC1) is bred and is saved on pad4 Arabidopsis Mutants.Selection life
Long consistent transgenic plant, according to method (Wang W etc., 2007, Molecular Plant-Microbe of Wang
Interactions 20:966-976) inoculation powdery mildew.Transgenic plant and WT lines are detected after inoculation 6 days to white powder
The disease resistance of disease detects apoptosis using Trypan blue decoration method, as a result as (WT is wild to Fig. 3 in Fig. 3
Type, #1, #2, #4 are followed successively by transgenosis system 1, transgenosis system 2 and transgenosis system 3) shown in, 3 transgenosis systems are relative to wild type
It is more disease-resistant, show lighter susceptible symptom；3 transgenosis system plant leafs generate hypersensitivity, WT lines without or
Generate slight hypersensitivity；These results suggest that overexpression VvPUB17 gene causes Arabidopsis plant disease resistance to enhance.
2) grape disease-resistant related gene VvPUB17 Disease Resistance Identification in grape
It verifies grape disease-resistant related gene VvSTS, VvPR1, VvPR10, VvNPR1 and enters grape in VvPUB17 genetic transformation
Expression after blade.VvPUB17 gene instantaneous conversion grape leave for 24 hours after, according to Wang method (Wang Y etc., 1995,
Vitis 34:159-164) inoculation powdery mildew, continues culture for 24 hours after the completion of inoculation.Using to grape leave water spray processing as pair
According to, conversion VvPUB17 genome and control group grape leave total serum IgE are extracted, it is disease-resistant with Real-time round pcr detection grape
The expression of related gene VvSTS, VvPR1, VvPR10 and VvNPR1.It is that the forward and reverse of VvSTS gene magnification draws with P9, P10
Object, P11, P12 are the forward and reverse primer of VvPR1 gene magnification, and P13, P14 are the forward and reverse primer of VvPR10 gene magnification,
P15, P16 are the forward and reverse primer of VvNPR1 gene magnification, primer and the phase in test example 1 of internal reference VvActin gene magnification
Together.
Forward primer P9:5 '-GAAACGCTCAACGTGCCAAGG-3 ' (SEQ ID NO:12)；
Reverse primer P10:5 '-GTAACCATAGGAATGCTATGTAGC-3'(SEQ ID NO:13).
Forward primer P11:5 '-GGAGTCCATTAGCACTCCTTTG-3 ' (SEQ ID NO:14)；
Reverse primer P12:5 '-CATAATTCTGGGCGTAGGCAG-3 ' (SEQ ID NO:15).
Forward primer P13:5 '-CCAACCAATCCTCCTCCTCTTC-3 ' (SEQ ID NO:16)；
Reverse primer P14:5 '-CATCTCCGTCAACCACAGTGTA-3 ' (SEQ ID NO:17).
Forward primer P15:5 '-TCTCCGATTCCAACGACTTCAG-3 ' (SEQ ID NO:18)；
Reverse primer P16:5 '-CATCATCAACGCACGCACAA-3 ' (SEQ ID NO:19).
PCR reaction carries out in iCycler iQ5Real-time PCR instrument (Bio-Rad company, the U.S.).With 2- △ △Ct
The relative expression quantity of method calculating gene.As a result as shown in figure 4, after being inoculated with powdery mildew, the grape leave of VvPUB17 gene is converted
Its expression quantity significantly increases relative to unconverted grape leave by middle disease-resistant related gene VvSTS, VvPR1, VvPR10, VvNPR1
Add, show to convert after VvPUB17 gene that each disease-resistant related gene expression quantity rises in grape leave, enhances the anti-of transformed plant
Characteristic of disease.
Sequence table
SEQUENCE LISTING
<110>University Of Science and Technology Of He'nan
<120>a kind of application, expression vector and its application of grape disease-resistant related gene VvPUB17
<170> PatentIn version 3.5
<211> 2145
<212> DNA
<213>sequence
<221>open reading frame of grape disease-resistant related gene VvPUB17
<222> (1)..(2145)
<400> 1
ATGGCCTCCG CTGCGATAGT ATCGTCTCTG CGGCGGCGGA GATCGCCGTC TTTGGAGGCT 60
TTCTTGGCGC CGGTGGATCT AAACGAGGTG GCTCTTGTAC GGACACTGGC CACCATCTCC 120
ATGGAGCTTA TATTTGCGTT TTCCGATAGG CCTTGGCCGT TTCAGCGCAA GAATTCGAGG 180
TCCTTGATTC GGAAAATTGA GGTCTTTCTG GTGCTGTTAG AGTTCCTGAG GGATTGCAAT 240
TTGAGTCTGC CTTCGGCGGC GGTGCTTTGC TTCAAGGAGC TTTACCTGCT TCTGTATCGG 300
TCGAAGATAC TTCTCGATTA TTGCTTGCAA TCAAGTAAAT TGTGGCTTCT GTTGCAGAAC 360
CAGTCGATTT CGGGGCATTT TCATGATCTT AATCAGGAAA TTTCGACGCT GTTGGATGTA 420
TTTCCGATGG AGGAACTTGA ATTGACTGAA GATATAAGGG AGCAGCTTGA GCTTTTGCAG 480
AAACAGGTGA GGAGGGCGAA GTTGTTTCTT GATAAGAATG ATGAGGGGTT GAGGCTGAGG 540
TTGTATTCTT TTCTTGATGA CTTTGGGAGT GGGCGGATTC CTGATCCAGT GGAGCTGCGG 600
TTGTTTTTTG TGGATAGATT AGGGATTCGG GATGCGAAGA GTTGTAGGGC TGAAATTGAG 660
TTTTTGGAGG AGCAGATTTA TAGTCACGAG GGAGATGTTG AGCCGAATGT TGCTGTGCTT 720
AATGGGTTCG TTGCGCTTAC TCGATATTGC AGATTCTTGC TATTTGGATT TGAGGAGAGC 780
GAGGTAGAAA TGAGTTTTGG GATTAAGAAG CCGAGGAAAG GGTTGATTAC TCAAGAAATT 840
GGGGATACCT TCATAACAGT CCCCAAGGAC TTTTGTTGCC CCATATCTTT GGATGTGATG 900
CGAGACCCAG TAATAATTTC AACAGGACAG ACATATGATC GAACTTCAAT ATCAAGGTGG 960
ATGGAAGAAG GGCATTGTAG TTGCCCAAAG ACAGGGCAGA TGCTTGCTCA CCCTCGCCTT 1020
GTTCCCAATC GAGCTCTCAG GAATTTGATC ACACAATGGT GCACTGCTTA TGGAATCACC 1080
TTGGATCCAC CAGACAGCCC AGATAGTGTT GTAGAAACTT TTGCAGCAGC TTTGCCAACC 1140
AAAGCTGCTA TTGAAGCCAA CAAAGCCACA GCAGCGCTTC TCGTCCAACA GCTTGCAAGT 1200
GGTTCACAGG GTGCAAAGAC TGTAGCTGCC CGTGAGATAC GTTTATTAGC AAAAACAGGG 1260
AAGGAAAACC GTGCGTATAT AGCAGAAGCT GGGGCAATCC CCCATCTTCT CAAGCTACTC 1320
TCATCTCCAA ATTCTGTCGC ACAAGAGAAT TCTGTAACGG CAATGCTTAA CCTATCAATA 1380
TATGACAAGA ACAAAAGCCG AATTATGGAT GAGGATGGGT GTTTAGGTTT GATTGTTGAA 1440
GTTCTGATAT TTGGGCACAC AACGGAAGCA AGGGAAAATG CAGCTGCGAC ATTGTTCAGC 1500
CTTTCTGCTG TTCATGATTA TAAGAAGAGA ATAGCGGACG AGGGTGGGGC AGTTGAAGCC 1560
CTGGCAGGGC TGTTGAGAGA GGGAACCCCA AGAGGAAGGA AAGATGCTGT AACGGCTCTA 1620
TTTAATCTAT CAACCCACAC AGATAATTGT GCCAGAATGG TAGCGTCAGG GGCTGTAACT 1680
GCATTAGTAG CGGCCTTGGG AACTGAGGGG GTTGCAGAGG AAGCAGCGGG AGCATTGGCC 1740
TTGATTGTTA GGCGGCCAAT TGGGGCTGAA GCAGTAGGGA GGGAGGAAAT GGCAGTGGCT 1800
GGGTTATTAG GAATGATGCG GTGTGGGACT CCAAGGGGGA AAGAGAATGC AGTTGCAGCA 1860
TTGCTTGAAC TGTGCAGAAG TGGCGGGACA GCTGCAACAG AAAGGGTCCT TAAGGCTCCA 1920
GCGCTGGCTG GTTTGCTTCA GACTCTGTTG TTTACAGGTA CAAAGCGCGC TAGGAGAAAG 1980
GCTGCATCCC TTGCCAGAGT TTTCCAGAGG TGTGAGAATG CAGCCTTGCA TTTTGGTGGA 2040
CTTGGTGTAG GGTATGCATT TGCCAGAAAC TCATCTGCAA ATACCGATGC AAGCTTTTCT 2100
AGTGAGGTTT CCATGCAAAT GCCCATTTCA GTGCCTGTTT TGTAG 2145
<211> 714
<212> PRT
<213>sequence
<221>protein of grape disease-resistant related gene VvPUB17 coding
<222> (1)..(714)
<400> 2
MASAAIVSSL RRRRSPSLEA FLAPVDLNEV ALVRTLATIS MELIFAFSDR PWPFQRKNSR 60
SLIRKIEVFL VLLEFLRDCN LSLPSAAVLC FKELYLLLYR SKILLDYCLQ SSKLWLLLQN 120
QSISGHFHDL NQEISTLLDV FPMEELELTE DIREQLELLQ KQVRRAKLFL DKNDEGLRLR 180
LYSFLDDFGS GRIPDPVELR LFFVDRLGIR DAKSCRAEIE FLEEQIYSHE GDVEPNVAVL 240
NGFVALTRYC RFLLFGFEES EVEMSFGIKK PRKGLITQEI GDTFITVPKD FCCPISLDVM 300
RDPVIISTGQ TYDRTSISRW MEEGHCSCPK TGQMLAHPRL VPNRALRNLI TQWCTAYGIT 360
LDPPDSPDSV VETFAAALPT KAAIEANKAT AALLVQQLAS GSQGAKTVAA REIRLLAKTG 420
KENRAXIAEA GAIPHLLKLL SSPNSVAQEN SVTAMLNLSI YDKNKSRIMD EDGCLGLIVE 480
VLIFGHTTEA RENAAATLFS LSAVHDYKKR IADEGGAVEA LAGLLREGTP RGRKDAVTAL 540
FNLSTHTDNC ARMVASGAVT ALVAALGTEG VAEEAAGALA LIVRRPIGAE AVGREEMAVA 600
GLLGMMRCGT PRGKENAVAA LLELCRSGGT AATERVLKAP ALAGLLQTLL FTGTKRARRK 660
AASLARVFQR CENAALHFGG LGVGYAFARN SSANTDASFS SEVSMQMPIS VPVL 714
<211> 25
<212> DNA
<213>sequence
<221>forward primer P3
<222> (1)..(25)
<400> 3
AGAGTCTGAG AAACGAGATT CAGAC 25
<211> 29
<212> DNA
<213>sequence
<221>reverse primer P4
<222> (1)..(29)
<400> 4
AAGCAGCTTT TCCACATTAT ACTATTACC 29
<211> 2695
<212> DNA
<213>sequence
<221>grape disease-resistant related gene VvPUB17
<222> (1)..(2695)
<400> 5
AGAGTCTGAG AAACGAGATT CAGACAAGTA GTAGAATCAC ACACAAAAAT AAACAATAAG 60
TGTAAGAGAG TTGCACTGTT TTCCTTGTTT TTGTTTTTAT TGTTTTGTTT TTTGTTTCTG 120
GGTTTGTTTA TTAGAGAGAG AGAGAGATTT TGGTGCTGCA GAGGTCTTGA TTTTCCAAGA 180
GATTTGTGCA TCTTCAAAAT TTGTTTGCAG GTAAATTGAG GGGAAATATT TCGCAGTGTT 240
TTTTTTAGTA ACCCTAGATA ACGTTCGAAT TCCATGGCCT CCGCTGCGAT AGTATCGTCT 300
CTGCGGCGGC GGAGATCGCC GTCTTTGGAG GCTTTCTTGG CGCCGGTGGA TCTAAACGAG 360
GTGGCTCTTG TACGGACACT GGCCACCATC TCCATGGAGC TTATATTTGC GTTTTCCGAT 420
AGGCCTTGGC CGTTTCAGCG CAAGAATTCG AGGTCCTTGA TTCGGAAAAT TGAGGTCTTT 480
CTGGTGCTGT TAGAGTTCCT GAGGGATTGC AATTTGAGTC TGCCTTCGGC GGCGGTGCTT 540
TGCTTCAAGG AGCTTTACCT GCTTCTGTAT CGGTCGAAGA TACTTCTCGA TTATTGCTTG 600
CAATCAAGTA AATTGTGGCT TCTGTTGCAG AACCAGTCGA TTTCGGGGCA TTTTCATGAT 660
CTTAATCAGG AAATTTCGAC GCTGTTGGAT GTATTTCCGA TGGAGGAACT TGAATTGACT 720
GAAGATATAA GGGAGCAGCT TGAGCTTTTG CAGAAACAGG TGAGGAGGGC GAAGTTGTTT 780
CTTGATAAGA ATGATGAGGG GTTGAGGCTG AGGTTGTATT CTTTTCTTGA TGACTTTGGG 840
AGTGGGCGGA TTCCTGATCC AGTGGAGCTG CGGTTGTTTT TTGTGGATAG ATTAGGGATT 900
CGGGATGCGA AGAGTTGTAG GGCTGAAATT GAGTTTTTGG AGGAGCAGAT TTATAGTCAC 960
GAGGGAGATG TTGAGCCGAA TGTTGCTGTG CTTAATGGGT TCGTTGCGCT TACTCGATAT 1020
TGCAGATTCT TGCTATTTGG ATTTGAGGAG AGCGAGGTAG AAATGAGTTT TGGGATTAAG 1080
AAGCCGAGGA AAGGGTTGAT TACTCAAGAA ATTGGGGATA CCTTCATAAC AGTCCCCAAG 1140
GACTTTTGTT GCCCCATATC TTTGGATGTG ATGCGAGACC CAGTAATAAT TTCAACAGGA 1200
CAGACATATG ATCGAACTTC AATATCAAGG TGGATGGAAG AAGGGCATTG TAGTTGCCCA 1260
AAGACAGGGC AGATGCTTGC TCACCCTCGC CTTGTTCCCA ATCGAGCTCT CAGGAATTTG 1320
ATCACACAAT GGTGCACTGC TTATGGAATC ACCTTGGATC CACCAGACAG CCCAGATAGT 1380
GTTGTAGAAA CTTTTGCAGC AGCTTTGCCA ACCAAAGCTG CTATTGAAGC CAACAAAGCC 1440
ACAGCAGCGC TTCTCGTCCA ACAGCTTGCA AGTGGTTCAC AGGGTGCAAA GACTGTAGCT 1500
GCCCGTGAGA TACGTTTATT AGCAAAAACA GGGAAGGAAA ACCGTGCGTA TATAGCAGAA 1560
GCTGGGGCAA TCCCCCATCT TCTCAAGCTA CTCTCATCTC CAAATTCTGT CGCACAAGAG 1620
AATTCTGTAA CGGCAATGCT TAACCTATCA ATATATGACA AGAACAAAAG CCGAATTATG 1680
GATGAGGATG GGTGTTTAGG TTTGATTGTT GAAGTTCTGA TATTTGGGCA CACAACGGAA 1740
GCAAGGGAAA ATGCAGCTGC GACATTGTTC AGCCTTTCTG CTGTTCATGA TTATAAGAAG 1800
AGAATAGCGG ACGAGGGTGG GGCAGTTGAA GCCCTGGCAG GGCTGTTGAG AGAGGGAACC 1860
CCAAGAGGAA GGAAAGATGC TGTAACGGCT CTATTTAATC TATCAACCCA CACAGATAAT 1920
TGTGCCAGAA TGGTAGCGTC AGGGGCTGTA ACTGCATTAG TAGCGGCCTT GGGAACTGAG 1980
GGGGTTGCAG AGGAAGCAGC GGGAGCATTG GCCTTGATTG TTAGGCGGCC AATTGGGGCT 2040
GAAGCAGTAG GGAGGGAGGA AATGGCAGTG GCTGGGTTAT TAGGAATGAT GCGGTGTGGG 2100
ACTCCAAGGG GGAAAGAGAA TGCAGTTGCA GCATTGCTTG AACTGTGCAG AAGTGGCGGG 2160
ACAGCTGCAA CAGAAAGGGT CCTTAAGGCT CCAGCGCTGG CTGGTTTGCT TCAGACTCTG 2220
TTGTTTACAG GTACAAAGCG CGCTAGGAGA AAGGCTGCAT CCCTTGCCAG AGTTTTCCAG 2280
AGGTGTGAGA ATGCAGCCTT GCATTTTGGT GGACTTGGTG TAGGGTATGC ATTTGCCAGA 2340
AACTCATCTG CAAATACCGA TGCAAGCTTT TCTAGTGAGG TTTCCATGCA AATGCCCATT 2400
TCAGTGCCTG TTTTGTAGTG TTAACATCTG CCTTGTGTTA CTGTGTTCCC TGGTTATTTA 2460
TGCACCATTT TTGTTGGTAA TGTTCACAAA TTCTTTCATT GAAATGTTAA GAGGAGGTGG 2520
GAAAAGAAAG TTATAGGAAT ACCATATTTG ATGTTTTAGA AAGATTGATG AATGTGTTTA 2580
TCATTTAGTT TCATTTTCTG CTATATTTTG TAATGCCCAA TTACTTGACA AATCAGAATG 2640
AGGAATTCTC TAAAAATGGT CAAGTTGGTA ATAGTATAAT GTGGAAAAGC TGCTT 2695
<211> 24
<212> DNA
<213>sequence
<221>forward primer P5
<222> (1)..(24)
<400> 6
CTGTAACGGC AATGCTTAAC CTAT 24
<211> 23
<212> DNA
<213>sequence
<221>reverse primer P6
<222> (1)..(23)
<400> 7
CCTCGTCCGC TATTCTCTTC TTA 23
<211> 20
<212> DNA
<213>sequence
<221>forward primer P7
<222> (1)..(20)
<400> 8
TCCTGTGGAC AATGGATGGA 20
<211> 20
<212> DNA
<213>sequence
<221>reverse primer P8
<222> (1)..(20)
<400> 9
CTTGCATCCC TCAGCACCTT 20
<211> 31
<212> DNA
<213>sequence
<221>forward primer P1
<222> (1)..(31)
<400> 10
GGGAGATCTA TGGCCTCCGC TGCGATAGTA T 31
<211> 35
<212> DNA
<213>sequence
<221>reverse primer P2
<222> (1)..(35)
<400> 11
GGGGGTGACC CTACAAAACA GGCACTGAAA TGGGC 35
<211> 21
<212> DNA
<213>sequence
<221>forward primer P9
<222> (1)..(21)
<400> 12
GAAACGCTCA ACGTGCCAAG G 21
<211> 24
<212> DNA
<213>sequence
<221>reverse primer P10
<222> (1)..(24)
<400> 13
GTAACCATAG GAATGCTATG TAGC 24
<211> 22
<212> DNA
<213>sequence
<221>forward primer P11
<222> (1)..(22)
<400> 14
GGAGTCCATT AGCACTCCTT TG 22
<211> 21
<212> DNA
<213>sequence
<221>reverse primer P12
<222> (1)..(21)
<400> 15
CATAATTCTG GGCGTAGGCA G 21
<211> 22
<212> DNA
<213>sequence
<221>forward primer P13
<222> (1)..(22)
<400> 16
CCAACCAATC CTCCTCCTCT TC 22
<211> 22
<212> DNA
<213>sequence
<221>reverse primer P14
<222> (1)..(22)
<400> 17
CATCTCCGTC AACCACAGTG TA 22
<211> 22
<212> DNA
<213>sequence
<221>forward primer P15
<222> (1)..(22)
<400> 18
TCTCCGATTC CAACGACTTC AG 22
<211> 20
<212> DNA
<213>sequence
<221>reverse primer P16
<222> (1)..(20)
<400> 19
CATCATCAAC GCACGCACAA 20
Claims (3)
1. a kind of application of grape disease-resistant related gene VvPUB17 in enhancing Genes For Plant Tolerance powdery mildew resistance, it is characterised in that: should
The nucleotide sequence of gene is as shown in SEQ ID NO:5, and open reading frame is as shown in SEQ ID NO:1, its encoded albumen
The amino acid sequence of matter is as shown in SEQ ID NO.2.
2. application of the plant overexpression carrier in enhancing Genes For Plant Tolerance powdery mildew resistance, it is characterised in that: the plant excess
Expression vector contains the open reading frame of the grape disease-resistant related gene VvPUB17 as shown in SEQ ID NO:5, open reading frame
As shown in SEQ ID NO:1.
3. application of the plant overexpression carrier according to claim 2 in enhancing Genes For Plant Tolerance powdery mildew resistance, special
Sign is: the plant overexpression carrier is pCAMBIA3301-VvPUB17, and preparation step is as follows:
1) using grape cDNA as template, P1, P2 are primer, carry out pcr amplification reaction, and amplified production recycles mesh after electrophoretic analysis
Segment；
Forward primer P1:5 '-GGGAGATCTATGGCCTCCGCTGCGATAGTAT-3 ',
Reverse primer P2:5 '-GGGGGTGACCCTACAAAACAGGCACTGAAATGGGC-3'；
2) it with target fragment and plant expression vector pCAMBIA3301 in Bgl II, BstE II difference double digestion step 1), returns
Receive target fragment and linearized vector, screening after connection, conversion, verify to get.
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| CN201710051147.7A CN106754992B (en) | 2017-01-23 | 2017-01-23 | A kind of application, expression vector and its application of grape disease-resistant related gene VvPUB17 |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102121005A (en) * | 2010-12-03 | 2011-07-13 | 西北农林科技大学 | Grapevine powdery mildew resistance transcription factor gene VpRFP1 promoter sequence and application thereof |
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2017
- 2017-01-23 CN CN201710051147.7A patent/CN106754992B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102121005A (en) * | 2010-12-03 | 2011-07-13 | 西北农林科技大学 | Grapevine powdery mildew resistance transcription factor gene VpRFP1 promoter sequence and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| GenBank登录号：XM_019222704.1;NCBI;《NCBI GENBANK》;20161122;第448-2592位 |
| Overexpression of a stress-responsive U-box protein gene VaPUB affects the accumulation of resistance related proteins in Vitis vinifera ‘Thompson Seedless’;Li Jiao等;《Plant Physiology and Biochemistry》;20161225;第112卷;第53-63页 |
| 拟南芥U-box蛋白的研究进展;鲁玉清等;《分子植物育种》;20081231;第6卷(第5期);第941-948页 |
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Application publication date: 20170531 Assignee: LUOYANG JIENTE BIOTECHNOLOGY CO.,LTD. Assignor: Henan University of Science and Technology Contract record no.: X2019980000341 Denomination of invention: Application of anti-disease related gene VvPUB17 of grape, expression vector and application thereof Granted publication date: 20190614 License type: Common License Record date: 20191111 |
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