CN109576303A - The gene silencing vector and its construction method of a kind of induction of cucumber green mottle mosaic virus and application - Google Patents
The gene silencing vector and its construction method of a kind of induction of cucumber green mottle mosaic virus and application Download PDFInfo
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Abstract
The present invention is based on CGMMV to construct the new VIGS carrier that can be applied in ground family crop, and provides the cucumber green mottle mosaic virus recombinant vector for carrying phytoene dehydrogenase (PDS) genetic fragment.The cucurbitaceous plants such as watermelon, muskmelon, cucumber and bottle gourd are the natural hosts of CGMMV, and recombinant vector of the invention can effectively reduce the expression of PDS gene in ground family crop, and plant is made to generate the photobleaching phenotype that PDS gene is silenced.The successful building of the recombinant vector can be used for studying gene function, and to excavate the cucurbitaceous plant gene with Main Agronomic Characters, cultivation is excellent, and disease-resistant, the kind of characteristic lays the foundation.
Description
Technical field
Present invention relates particularly to a kind of cucumber green mottle mosaic virus induction gene silencing vector and its construction method with
Using.
Background technique
According to food and agricultural organization's recent statistics data in 2016, the yield of cucurbitaceous plant such as watermelon, muskmelon and cucumber exists
Increased year by year in past 10 years, and has played important function in fruits and vegetables.Raising and people with level of agricultural production
Pursuit to high-quality melon vegetables, cultivate excellent, disease-resistant and characteristic product using ground family crop Main Agronomic Characters gene
Kind is imperative, and the gene order-checking of the cucurbitaceous plants such as cucumber, watermelon and muskmelon is excellent gene excavating and gene function
It can study and lay a good foundation.The genetic transformation of ground family crop takes time and effort and transformation efficiency is extremely low, therefore, the base of virus induction
Because the foundation of silent technology platform promotes the research of gene function, accelerate the research of ground family crop functional genomics, promotees
Into the sustainable development of China's ground family crop industry.Although VIGS system has been successfully applied in many crops, deposit
In some restrictive factors, for the host range of carrier application, the selection of effective target fragment and silence efficiency it is steady
Qualitative etc. is still problem to be solved.Developing the high carrier of silence efficiency is important feature of the invention, and the present invention attempts
By the CGMMV that is separated using ground family crop as main host come carrier construction.
Summary of the invention
It is an object of the present invention to solve the deficiency of the existing technology and provide a kind of inductions of cucumber green mottle mosaic virus
Gene silencing vector and its construction method and application.
The purpose of the present invention is what is be achieved through the following technical solutions:
A kind of construction method of the gene silencing vector of cucumber green mottle mosaic virus induction, comprising the following steps:
(1) it is suitable to be selected according to cucumber green mottle mosaic virus genome and coat protein sub-genomic promoter information
Multiple cloning sites；
(2) carrier of overall length infectious clone building restriction enzyme site containing BamHI based on cucumber green mottle mosaic virus, and
It is named as CGMMV-BamHI；
(3) PDS genetic fragment is expanded, obtains the PDS genetic fragment of 69bp, 150bp, 213bp and 300bp length respectively；
(4) the PDS genetic fragment of above-mentioned amplification is connected into linearized vector CGMMV-BamHI, is respectively formed recombination and carries
Body CGMMV-BamHI-PDS69, CGMMV-BamHI-PDS150, CGMMV-BamHI-PDS213 and CGMMV-BamHI-
PDS300.The method that the connection of PDS genetic fragment and support C GMMV-BamHI use homologous recombination, this method is easy to operate, saves
When and efficiency it is very high.
A kind of construction method of the gene silencing vector of above-mentioned cucumber green mottle mosaic virus induction, wherein step (3)
In, further includes: total serum IgE is extracted from cucurbitaceous plant leaf texture, it is then inverse using Oligo dT primer, PrimeScript II
Transcriptase carry out reverse transcription synthesis be used for RT-PCR the first chain cDNA, using cucurbitaceous plant cDNA as template, using primer into
Row PCR amplification.
The construction method of the gene silencing vector of above-mentioned a kind of cucumber green mottle mosaic virus induction, wherein by PDS piece
The primer of Duan Jinhang amplification is the PDS gene order by comparing watermelon, muskmelon, cucumber and bottle gourd, and selection homology is most
High region design, the primer sequence are as follows:
78-69P-X:GAGTGGATGAGACTCTTGCACAGTTAAATTATCTTGAGCCTCCAGTTATAGGTCTAGGTC
78-69P-S:GAGTCTCATCCACTCTTGCACAGTTAAATTATCTTGAGCCTCCATTAAGTAAAGTCCTG
78-213-F:CCCGTCAGGACTTTACTTAATGGATCCATGCTTACTTGGCCAGAG
78-213-R:CGACCTAGACCTATAACTGGATCCAATGCATTGCATAGAAAGTTC
78-150-F:cccGTCAGGACTTTACTTAATGGATCCGGATATGGGCTATTTTAAGGA
78-150-R:CGACCTAGACCTATAACTGGATCCCCCGACTTCTCATCCACT
78-300-F:CCCGTCAGGACTTTACTTAATGGATCCTTTGGGGCTTATCCCAA
78-300-R:CGACCTAGACCTATAACTGGATCCTCTCATCCACTCTTGC。
A kind of gene silencing vector of cucumber green mottle mosaic virus induction, including recombinant vector CGMMV-BamHI-
PDS69, CGMMV-BamHI-PDS150, CGMMV-BamHI-PDS213 and CGMMV-BamHI-PDS300.
A kind of application of the gene silencing vector of cucumber green mottle mosaic virus induction, comprising:
(1) by recombinant vector CGMMV-BamHI-PDS69, CGMMV-BamHI-PDS150, CGMMV-BamHI-PDS213
It is inoculated on ground family crop watermelon, muskmelon, cucumber and bottle gourd respectively with CGMMV-BamHI-PDS300；
(2) observing and recording each carrier makes phenotype caused by PDS gene silencing, and measurement analysis silence efficiency detects carrier
Stability.
A kind of application of the gene silencing vector of above-mentioned cucumber green mottle mosaic virus induction, wherein ground family crop
Inoculating date is respectively as follows: watermelon, muskmelon and bottle gourd selection two panels cotyledon, a piece of leaf period, cucumber and selects two panels cotyledon two panels true
Ye Qi.
In conclusion by adopting the above-described technical solution, compared with prior art, the present invention having the advantage that
The present invention is based on CGMMV to construct the new VIGS carrier that can be applied in ground family crop, and provides and take
Cucumber green mottle mosaic virus recombinant vector with phytoene dehydrogenase (PDS) genetic fragment.Watermelon, muskmelon, cucumber
It is the natural host of CGMMV with cucurbitaceous plants such as bottle gourds, recombinant vector of the invention can effectively reduce PDS in ground family crop
The expression of gene makes plant generate the photobleaching phenotype that PDS gene is silenced.The successful building of the recombinant vector can be used for
Gene function is studied, to excavate the cucurbitaceous plant gene with Main Agronomic Characters, cultivates excellent, disease-resistant, the kind of characteristic
It lays the foundation.
Detailed description of the invention
Fig. 1 is that DNA fragmentation 1 and DNA fragmentation 2 expand electrophoretogram；
Fig. 2 is the structural schematic diagram of CGMMV VIGS carrier (CGMMV-BamHI)；
Fig. 3 is to carry the recombinant vectors of different PDS genetic fragments to infect the phenotype of the blade albefaction occurred after watermelon；
Fig. 4 is to carry the recombinant vectors of different PDS genetic fragments to infect the phenotype of the blade albefaction occurred after cucumber；
Fig. 5 is to carry the recombinant vectors of different PDS genetic fragments to infect the phenotype of the blade albefaction occurred after muskmelon；
Fig. 6 is to carry the recombinant vectors of different PDS genetic fragments to infect the phenotype of the blade albefaction occurred after bottle gourd；
Fig. 7 is the photobleaching phenotype that 79 days rear blades are inoculated on bottle gourd；
Fig. 8 is the PDS gene relative expression quantity of most white blade after qRT-PCR detection inoculation；
Fig. 9 is after the recombinant vector (CGMMV-BamHI-PDS69) of carrying 69bp PDS genetic fragment infects bottle gourd 34 days
The phenotype of the Different sites of leaf albefaction of appearance；
Figure 10 is that the recombinant vector (CGMMV-BamHI-PDS150) of carrying 150bp PDS genetic fragment infects 34 He of bottle gourd
The phenotype of the Different sites of leaf albefaction occurred after 54 days；
Figure 11 is that the recombinant vector (CGMMV-BamHI-PDS213) of carrying 213bp PDS genetic fragment infects 34 He of bottle gourd
The phenotype of the Different sites of leaf albefaction occurred after 54 days.
Figure 12 is that the recombinant vector (CGMMV-BamHI-PDS300) of carrying 300bp PDS genetic fragment infects bottle gourd 34 days
The phenotype of the Different sites of leaf albefaction occurred afterwards.
Figure 13 is the relative quantification of PDS gene in Different sites of leaf of the bottle gourd plant inoculation containing different PDS fragment vectors
As a result.
Specific embodiment
A specific embodiment of the invention is further described in detail below.
The building for the VIGS carrier that embodiment 1.CGMMV is mediated
(1) it is carried out for template with primer CP-TC-F and CP-TC-R with CGMMV overall length infectious clone (PXT1-CGMMV)
PCR amplification, so that CP initiation codon ATG changes into ACG, PCR product is recycled, and is then transformed into E. coli competent
In Top10, selects three positive colonies to carry out sequence verification, take sequencing result correctly to extract plasmid and be named as PXT1-
CGACG.Primer sequence is as follows:
CP-TC-F:5’-CTGTTTCTTTTGAAGACGGCTTACAAT-3’
CP-TC-R:5’-CGTCTTCAAAAGAAACAGAACTGGACTC-3’。
(2) primer PXT1-F/78B-99-R and PXT1-R/78B-99-F are used, respectively with PXT1-CGACG and PXT1-
CGMMV is that template is expanded.It is obtained respectively comprising CGMMV nt1-5840 (GenBank accession:KY753929)
DNA fragmentation 1 and DNA fragmentation 2 comprising CGMMV nt5651-6423, referring to Figure 1.Primer sequence is as follows:
PXT1-F:ATGCCTGCAGTCAACATGGTGGAG
PXT1-R:CATGTTGACTGCAGGCATGCAAGC
78B-99-F:ACTTAATGGATCCAGTTATAGGTCTAGGTCGCAG
78B-99-R:CTATAACTGGATCCATTAAGTAAAGTCCTGACGGGA。
(3) segment 1 and segment 2 are attached by way of homologous recombination and are then gone in Top10, extract plasmid simultaneously
It is named as CGMMV-BamHI.The CGMMV-BamHI carrier contain repetition 190bp CGMMV CP sub-genomic promoter and
One single restriction enzyme site BamHI, refers to Fig. 2.
The clone of 2. target fragment of embodiment and insertion
(1) it is planted using RNAsimple total serum IgE kit (Tiangen Biotech, Beijing, China) from Curcurbitaceae
Total serum IgE is extracted in object (watermelon, muskmelon, cucumber and bottle gourd) leaf texture, then uses Oligo dT primer, PrimeScript II
Reverse transcriptase (TAKARA) carries out the first chain cDNA that reverse transcription synthesis is used for RT-PCR.
(2) select to be formed the phytoene desaturase of beta carotene route of synthesis (PDS) gene as target base
Cause.With reference to cucurbitaceous plant PDS Genomic sequence information known in ncbi database, and combine the sequence letter of the carrier of building
Breath four pairs of primers of design, to expand the PDS gene of four different length segments.Primer sequence is as follows:
78-69P-X:GAGTGGATGAGACTCTTGCACAGTTAAATTATCTTGAGCCTCCAGTTATAGGTCTAGGTC
78-69P-S:GAGTCTCATCCACTCTTGCACAGTTAAATTATCTTGAGCCTCCATTAAGTAAAGTCCTG
78-213-F:CCCGTCAGGACTTTACTTAATGGATCCATGCTTACTTGGCCAGAG
78-213-R:CGACCTAGACCTATAACTGGATCCAATGCATTGCATAGAAAGTTC
78-150-F:cccGTCAGGACTTTACTTAATGGATCCGGATATGGGCTATTTTAAGGA
78-150-R:CGACCTAGACCTATAACTGGATCCCCCGACTTCTCATCCACT
78-300-F:CCCGTCAGGACTTTACTTAATGGATCCTTTGGGGCTTATCCCAA
78-300-R:CGACCTAGACCTATAACTGGATCCTCTCATCCACTCTTGC。
(3) using cucurbitaceous plant cDNA as template, PCR amplification is carried out with above-mentioned primer, obtains 69bp, 150bp respectively,
The PDS genetic fragment of 213bpand 300bp length.
(4) CGMMV-BamHI carrier is subjected to linearization for enzyme restriction with BamHI enzyme, then by different PDS genetic fragment point
It is not connected in linearized vector in a manner of homologous recombination, recombination obtains CGMMV-BamHI-PDS69, CGMMV- respectively
BamHI-PDS150, CGMMV-BamHI-PDS213 and GMMV-BamHI-PDS300.
The Agrobacterium-mediated Transformation and its inoculation verifying of 3. recombinant vector of embodiment
(1) 5 μ L vector plasmid (CGMMV-BamHI-PDS69, CGMMV-BamHI-PDS150, CGMMV- are taken respectively
BamHI-PDS213 and CGMMV-BamHI-PDS300) it is added in the GV3101 competent cell of 50 μ L, it stands on ice
10min is placed in after standing 2min on ice through warm bath 5min in liquid nitrogen flash freezer 5min, 37 DEG C of water-baths.Antibiosis is not added in 500 μ L
The LB liquid medium of element is placed in 28 DEG C, carries out recovery culture 2h in the shaking table under the conditions of 200rpm, then 12,000rpm from
Heart 1min collects thallus, is coated on the LB solid medium containing antibiotic (50ng/ μ l Kan, 50ng/ μ lRif).
Unit cell is picked from the plate after 48h into LB liquid medium of the 200 μ l containing antibiotic (50ng/ μ l Kan, 50ng/ μ lRif)
Overnight incubation, screening positive clone.
(2) by the positive colony screened and the LB Liquid Culture for containing antibiotic (50ng/ μ l Kan, 50ng/ μ l Rif)
Base with the ratio of 1:100 as being incubated overnight in the shaking table under the conditions of 28 DEG C, 200rpm, then through 6,000rpm 5min
Thalline were collected by centrifugation, with inoculation buffer (10mM MgCl2, 10mM MES and 100 μM of AS) thallus is suspended, room temperature is quiet
2-3h is set, injection inoculation is carried out from cucurbitaceous plant (watermelon, muskmelon, cucumber and bottle gourd) cotyledon leaf back with 1mL syringe, connects
28 DEG C/25 DEG C are placed it in after kind, 16h/8h (illumination/dark) culturing room continues to cultivate.
Application of 4. gene silencing of embodiment in cucurbitaceous plant
By recombinant virus (CGMMV-BamHI-PDS69, CGMMV-BamHI-PDS150, CGMMV-BamHI-PDS213 and
CGMMV-BamHI-PDS300 it) is seeded in watermelon, muskmelon, cucumber and bottle gourd.It 9 to 17 days after inoculation (dpi), can be not
Photobleaching occurs on the leaf of inoculation, refers to Fig. 3~6；And 2 months time is remained above, refer to Fig. 7.
The measurement of embodiment 5.CGMMV VIGS carrier silence efficiency
From inoculation containing total serum IgE is extracted in the most apparent leaf sample of different carriers phenotype, then pass through
PrimeScriptTMRT reagent Kit with gDNA Eraser (TAKARA) reverse transcription reagent box is closed from 1ug RNA
At the first chain cDNA.PDS gene relative expression quantity measurement by qRT-PCR using SYBR Green I Master Mix into
Row.Sequence design outside selection targeted silent region is for measuring cucumber, muskmelon, and PDS gene expression draws in bottle gourd and watermelon
Object (CuPDS-679F/CuPDS-906R and wate-q-F/wate-q-R).In cucumber, muskmelon and bottle gourd, actin is used
Gene is as internal reference, primer cumsactin-F/cumsactin-R.Cla016178 gene is used as the internal reference in watermelon, primer
For Cla016178-F/Cla016178-R.The diluted cDNA of qRT-PCR:2 μ L is carried out using the reaction mixture of 20 μ L in total,
10 μ LSYBRGreen I Master, positive anti-primer each 0.5 μM and 6 μ L distilled waters.QRT-PCR program is as follows: 95 DEG C 5 minutes,
Then 95 DEG C 10 seconds, 58 DEG C 20 seconds, 72 DEG C 30 seconds, carry out 45 circulation.The table of PDS gene is calculated using 2- Δ Δ CT method
It reaches.The expression of PDS gene in negative control (CGMMV-BamHI) is set as arbitrary value (1.0) to calculate other samples
Relative expression levels, each sample does 3 repetitions, refers to Fig. 8.Primer sequence is as follows:
Wate-q-F:TGCTTTAGCGTTTTGGGGGA
Wate-q-R:GACGTGCAGAAGCACGAAAA
CuPDS-679F:TGTGTGGATTACCCTAGACC
CuPDS-906R:CCAAGCTGCTACCTTTCCAC
Cumsactin-F:ATGGTCAAGGCTGGATTTGC
Cumsactin-R:TGAGCTTCATCACCAACATAGGC
Cla016178-F:GAACTTGGCACCTGTCCTGT
Cla016178-R:GAACAGTGCAACAGCCTCAA。
Embodiment 6. identifies the stability based on the CGMMV VIGS carrier constructed
It is obtained by observation, can keep very long by the photobleaching phenotype for the plant that the recombinant vector of the segment containing PDS infects
Time refers to Fig. 7.Fig. 9~12 is referred to, Fig. 9 is the recombinant vector (CGMMV-BamHI- for carrying 69bp PDS genetic fragment
PDS69 the phenotype of the Different sites of leaf albefaction occurred after) infecting bottle gourd 34 days, L6, L7 and L9 are respectively indicated on inoculation cotyledon
6th, 7 and 9 true leaf of side；Figure 10 is the recombinant vector (CGMMV-BamHI-PDS150) for carrying 150bp PDS genetic fragment
The phenotype of the Different sites of leaf albefaction occurred after bottle gourd 34 and 54 day is infected, L4 and L11 are respectively indicated above inoculation cotyledon
4th and 11 true leaf；Figure 11 is that the recombinant vector (CGMMV-BamHI-PDS213) of carrying 213bp PDS genetic fragment infects an a kind of edible gourd
The phenotype of the Different sites of leaf albefaction occurred after melon 34 and 54 day, L4 and L12 respectively indicate the 4th and 12 above inoculation cotyledon
Piece true leaf；Figure 12 is that the recombinant vector (CGMMV-BamHI-PDS300) of carrying 300bp PDS genetic fragment infects bottle gourd 34 days
The phenotype of the Different sites of leaf albefaction occurred afterwards, L7, L8, L9 and L10 respectively indicate the 7th, 8,9 and the above inoculation cotyledon
10 true leaves.After inoculation 54 days, the photobleaching table as caused by PDS gene silencing still can be observed in the top of bottle gourd plant most young leaves
Type.
The relative expression quantity of PDS gene in Different sites of leaf is had detected by qRT-PCR, finds PDS base in most young leaves
Because expression quantity can still be lowered, referring to Figure 13, illustrate that the carrier has good stability and can be used for identifying ground family crop
The function of middle gene.
Embodiment described above is merely to illustrate technical idea and feature of the invention, in the art its object is to make
Technical staff can understand the content of the present invention and implement it accordingly, patent model of the invention only cannot be limited with the present embodiment
It encloses, i.e., it is all according to same changes or modifications made by disclosed spirit, it still falls in the scope of the patents of the invention.
Claims (6)
1. a kind of construction method of the gene silencing vector of cucumber green mottle mosaic virus induction, which is characterized in that including following
Step:
(1) more grams suitable according to cucumber green mottle mosaic virus genome and the selection of coat protein sub-genomic promoter information
Grand site；
(2) carrier of overall length infectious clone building restriction enzyme site containing BamHI based on cucumber green mottle mosaic virus, and name
For CGMMV-BamHI；
(3) PDS genetic fragment is expanded, obtains the PDS genetic fragment of 69bp, 150bp, 213bp and 300bp length respectively；
(4) the PDS genetic fragment of above-mentioned amplification is connected into linearized vector CGMMV-BamHI, is respectively formed recombinant vector
CGMMV-BamHI-PDS69, CGMMV-BamHI-PDS150, CGMMV-BamHI-PDS213 and CGMMV-BamHI-PDS300.
2. a kind of construction method of the gene silencing vector of cucumber green mottle mosaic virus induction according to claim 1,
It is characterized in that, in step (3), further includes: extract total serum IgE from cucurbitaceous plant leaf texture, then drawn using Oligo dT
Object, PrimeScript II reverse transcriptase carry out the first chain cDNA that reverse transcription synthesis is used for RT-PCR, with cucurbitaceous plant
CDNA is template, carries out PCR amplification using primer.
3. a kind of construction method of the gene silencing vector of cucumber green mottle mosaic virus induction according to claim 2,
It is characterized in that, the primer sequence is as follows:
78-69P-X:GAGTGGATGAGACTCTTGCACAGTTAAATTATCTTGAGCCTCCAGTTATAGGTCTAGGTC；
78-69P-S:GAGTCTCATCCACTCTTGCACAGTTAAATTATCTTGAGCCTCCATTAAGTAAAGTCCTG；
78-213-F:CCCGTCAGGACTTTACTTAATGGATCCATGCTTACTTGGCCAGAG；
78-213-R:CGACCTAGACCTATAACTGGATCCAATGCATTGCATAGAAAGTTC；
78-150-F:cccGTCAGGACTTTACTTAATGGATCCGGATATGGGCTATTTTAAGGA；
78-150-R:CGACCTAGACCTATAACTGGATCCCCCGACTTCTCATCCACT；
78-300-F:CCCGTCAGGACTTTACTTAATGGATCCTTTGGGGCTTATCCCAA；
78-300-R:CGACCTAGACCTATAACTGGATCCTCTCATCCACTCTTGC。
4. a kind of gene silencing vector of cucumber green mottle mosaic virus induction, which is characterized in that including recombinant vector CGMMV-
BamHI-PDS69, CGMMV-BamHI-PDS150, CGMMV-BamHI-PDS213 and CGMMV-BamHI-PDS300.
5. a kind of application of the gene silencing vector of cucumber green mottle mosaic virus induction characterized by comprising
(1) by recombinant vector CGMMV-BamHI-PDS69, CGMMV-BamHI-PDS150, CGMMV-BamHI-PDS213 and
CGMMV-BamHI-PDS300 is inoculated into respectively on ground family crop watermelon, muskmelon, cucumber and bottle gourd；
(2) observing and recording each carrier makes phenotype caused by PDS gene silencing, and measurement analysis silence efficiency detects the steady of carrier
It is qualitative.
6. a kind of application of the gene silencing vector of cucumber green mottle mosaic virus induction according to claim 5, special
Sign is that ground family crop Inoculating date is respectively as follows: watermelon, muskmelon and bottle gourd selection two panels cotyledon, a piece of leaf period, cucumber choosing
Select two panels cotyledon two panels leaf period.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110423762A (en) * | 2019-08-16 | 2019-11-08 | 海南大学 | A kind of construction method of PVMV full length infectious cDNA |
| CN114107371A (en) * | 2021-12-06 | 2022-03-01 | 中国农业科学院郑州果树研究所 | Cucumber green mottle mosaic virus gene mediated transgenic tobacco method |
| CN114150003A (en) * | 2021-12-06 | 2022-03-08 | 中国农业科学院郑州果树研究所 | Application of cucumber green mottle mosaic virus gene spacer region in resistant tobacco cultivation |
| CN114568439A (en) * | 2022-03-30 | 2022-06-03 | 河南农业大学 | Application of melatonin in preventing and treating cucumber green mottle mosaic virus and method for enhancing prevention and treatment effect of melatonin in cooperation with melatonin |
| CN115725598A (en) * | 2022-07-20 | 2023-03-03 | 沈阳农业大学 | Target gene set related to cucumber green mottle mosaic virus infection and application thereof |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104498521A (en) * | 2014-11-27 | 2015-04-08 | 浙江省农业科学院 | Infectious clone vector of cucumber green mottle mosaic virus (CGMMV), agrobacterium strain and preparation method and application of infectious clone vector of cucumber green mottle mosaic virus (CGMMV) |
| CN109022449A (en) * | 2018-07-25 | 2018-12-18 | 沈阳农业大学 | Cucumber CsMLO1 gene and its silencing expression vector establishment method, application |
-
2018
- 2018-12-29 CN CN201811637184.7A patent/CN109576303B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104498521A (en) * | 2014-11-27 | 2015-04-08 | 浙江省农业科学院 | Infectious clone vector of cucumber green mottle mosaic virus (CGMMV), agrobacterium strain and preparation method and application of infectious clone vector of cucumber green mottle mosaic virus (CGMMV) |
| CN109022449A (en) * | 2018-07-25 | 2018-12-18 | 沈阳农业大学 | Cucumber CsMLO1 gene and its silencing expression vector establishment method, application |
Non-Patent Citations (5)
| Title |
|---|
| AKI IGARASHI ET AL: "Apple latent spherical virus vectors for reliable and effective virus-induced gene silencing among a broad range of plants including tobacco, tomato, Arabidopsis thaliana, cucurbits, and legumes", 《VIROLOGY》 * |
| ZHAO F ET AL: "Development of tobacco rihgspot virus-based vectors for foreign gene expression and virus-induced gene silencing in a variety of plants", 《VIROLOGY》 * |
| 刘美等: "VIGS技术的研究进展及其在葫芦科作物中的应用", 《果树学报》 * |
| 徐幼平 等: "病毒诱导的基因沉默", 《浙江大学学报》 * |
| 郑棚峻 等: "葫芦科作物重要种传病毒研究进展", 《江苏农业科学》 * |
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