CN109576303B - Cucumber green mottle mosaic virus induced gene silencing vector and construction method and application thereof - Google Patents
Cucumber green mottle mosaic virus induced gene silencing vector and construction method and application thereof Download PDFInfo
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Abstract
The invention constructs a novel VIGS vector which can be applied to cucurbitaceae crops based on CGMMV, and provides a cucumber green mottle mosaic virus recombinant vector carrying Phytoene Dehydrogenase (PDS) gene segments. The cucurbitaceae plants such as watermelon, melon, cucumber and bottle gourd are natural hosts of CGMMV, and the recombinant vector can effectively reduce the expression level of the PDS gene in the cucurbitaceae crops, so that the plants generate a photobleaching phenotype with the PDS gene silenced. The successful construction of the recombinant vector can be used for researching gene functions, and lays a foundation for excavating cucurbitaceae plant genes with important agronomic characters and cultivating excellent, disease-resistant and characteristic varieties.
Description
Technical Field
The invention particularly relates to a cucumber green mottle mosaic virus induced gene silencing vector and a construction method and application thereof.
Background
According to the latest statistics in 2016, grain growers, the production of cucurbits, such as watermelon, melon and cucumber, has increased year by year in the last decade and plays an important role in fruits and vegetables. With the improvement of agricultural production level and the pursuit of people for high-quality melon vegetables, important agronomic character genes of cucurbitaceae crops are utilized to cultivate excellent, disease-resistant and characteristic varieties, and genome sequencing of cucurbitaceae plants such as cucumbers, watermelons and melons lays a foundation for excellent gene mining and gene function research. The genetic transformation of the cucurbit crops is time-consuming and labor-consuming and has extremely low transformation efficiency, so that the establishment of a virus-induced gene silencing technology platform promotes the research of gene functions, accelerates the research of functional genomics of the cucurbit crops and promotes the sustainable development of the cucurbit crop industry in China. Although the VIGS system has been successfully applied to many crops, there are some limiting factors, and the host range of vector application, selection of effective target gene fragments, stability of silencing efficiency, and the like are still problems to be solved. The development of a vector with high silencing efficiency is an important feature of the present invention, which attempts to construct a vector by CGMMV isolated with cucurbit crops as the main host.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provides a cucumber green mottle mosaic virus induced gene silencing vector and a construction method and application thereof.
The purpose of the invention is realized by the following technical scheme:
a method for constructing a cucumber green mottle mosaic virus-induced gene silencing vector comprises the following steps:
(1) selecting a proper multiple cloning site according to the promoter information of the cucumber green mottle mosaic virus genome and the coat protein subgenome;
(2) constructing a vector containing a BamHI enzyme cutting site based on the full-length infectious clone of cucumber green mottle mosaic virus, and naming the vector as CGMMV-BamHI;
(3) amplifying PDS gene segments to obtain PDS gene segments with lengths of 69bp,150bp,213bp and 300bp respectively;
(4) the amplified PDS gene fragment is connected into a linearized vector CGMMV-BamHI to form recombinant vectors CGMMV-BamHI-PDS69, CGMMV-BamHI-PDS150, CGMMV-BamHI-PDS213 and CGMMV-BamHI-PDS300 respectively. The connection of the PDS gene fragment and the carrier CGMMV-BamHI adopts a homologous recombination method, and the method has the advantages of simple operation, time saving and high efficiency.
The method for constructing the cucumber green mottle mosaic virus-induced gene silencing vector comprises the following steps of (3): total RNA was extracted from leaf tissue of cucurbitaceae plants, then reverse transcription was performed using Oligo dT primer, PrimeScript II reverse transcriptase to synthesize first strand cDNA for RT-PCR, and PCR amplification was performed using cucurbitaceae plant cDNA as template and primer.
The method for constructing the cucumber green mottle mosaic virus-induced gene silencing vector comprises the following steps of selecting four regions with highest homology by comparing PDS gene sequences of watermelons, melons, cucumbers and bottle gourds and designing primers for amplifying the PDS fragments, wherein the primer sequences 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 gene silencing vector induced by cucumber green mottle mosaic virus comprises recombinant vectors CGMMV-BamHI-PDS69, CGMMV-BamHI-PDS150, CGMMV-BamHI-PDS213 and CGMMV-BamHI-PDS 300.
Use of a cucumber green mottle mosaic virus-induced gene silencing vector comprising:
(1) respectively inoculating recombinant vectors CGMMV-BamHI-PDS69, CGMMV-BamHI-PDS150, CGMMV-BamHI-PDS213 and CGMMV-BamHI-PDS300 to cucurbitaceae crops such as watermelon, melon, cucumber and bottle gourd;
(2) observing and recording the phenotype caused by the PDS gene silencing of each vector, measuring and analyzing the silencing efficiency, and detecting the stability of the vector.
The application of the cucumber green mottle mosaic virus induced gene silencing vector comprises the following steps of: the watermelon, melon and bottle gourd are selected from two cotyledons and one true leaf period, and the cucumber is selected from two cotyledons and two true leaf periods.
In summary, due to the adoption of the technical scheme, compared with the prior art, the invention has the following advantages:
the invention constructs a novel VIGS vector which can be applied to cucurbitaceae crops based on CGMMV, and provides a cucumber green mottle mosaic virus recombinant vector carrying Phytoene Dehydrogenase (PDS) gene segments. The cucurbitaceae plants such as watermelon, melon, cucumber and bottle gourd are natural hosts of CGMMV, and the recombinant vector can effectively reduce the expression level of the PDS gene in the cucurbitaceae crops, so that the plants generate a photobleaching phenotype with the PDS gene silenced. The successful construction of the recombinant vector can be used for researching gene functions, and lays a foundation for excavating cucurbitaceae plant genes with important agronomic characters and cultivating excellent, disease-resistant and characteristic varieties.
Drawings
FIG. 1 is an amplification electrophoretogram of DNA fragment 1 and DNA fragment 2;
FIG. 2 is a schematic structural diagram of CGMMV VIGS vector (CGMMV-BamHI);
FIG. 3 is the phenotype of leaf albinism that occurs after infection of watermelons with recombinant vectors carrying different PDS gene segments;
FIG. 4 is the phenotype of albino leaves that occurs after cucumber infection with recombinant vectors carrying different PDS gene segments;
FIG. 5 is the phenotype of leaf whitening following melon infection with recombinant vectors carrying different PDS gene segments;
FIG. 6 shows the phenotype of leaf whitening after bottle gourd is infected with recombinant vectors carrying different PDS gene segments;
FIG. 7 is the photo-bleached phenotype of leaves after 79 days of inoculation on the bottle gourds;
FIG. 8 shows the relative expression level of PDS gene in the whitest leaf after qRT-PCR;
FIG. 9 shows the phenotype of albino leaves at different positions after the bottle gourd is infected with a recombinant vector (CGMMV-BamHI-PDS69) carrying a 69bp PDS gene fragment for 34 days;
FIG. 10 shows the phenotype of albino leaves at different sites after the bottle gourd is infected with a recombinant vector (CGMMV-BamHI-PDS150) carrying a 150bp PDS gene fragment for 34 days and 54 days;
FIG. 11 shows the phenotype of albino leaves at different sites, which appear after the bottle gourd is infected with a recombinant vector (CGMMV-BamHI-PDS213) carrying a 213bp PDS gene fragment for 34 days and 54 days.
FIG. 12 shows the phenotype of albino leaves at different sites, which appear after the bottle gourd is infected with a recombinant vector (CGMMV-BamHI-PDS300) carrying a 300bp PDS gene fragment for 34 days.
FIG. 13 shows the relative quantification of PDS gene in leaves of plants of Lagenaria glauca inoculated with vectors containing different PDS fragments.
Detailed Description
The following describes in further detail embodiments of the present invention.
Example 1 construction of CGMMV-mediated VIGS vector
(1) Taking CGMMV full-length infectious clone (PXT1-CGMMV) as a template, carrying out PCR amplification by using primers CP-TC-F and CP-TC-R to change a CP start codon ATG into ACG, recovering PCR products, then transforming the products into escherichia coli competent Top10, selecting three positive clones for sequencing verification, taking an extracted plasmid with a correct sequencing result and naming the extracted plasmid as PXT 1-CGACG. The primer sequences are as follows:
CP-TC-F:5’-CTGTTTCTTTTGAAGACGGCTTACAAT-3’
CP-TC-R:5’-CGTCTTCAAAAGAAACAGAACTGGACTC-3’。
(2) the primers PXT1-F/78B-99-R and PXT1-R/78B-99-F are respectively used for amplification by taking PXT1-CGACG and PXT1-CGMMV as templates. A DNA fragment 1 containing CGMMV nt1-5840(GenBank accession: KY753929) and a DNA fragment 2 containing CGMMV nt5651-6423 were obtained, respectively, see FIG. 1. The primer sequences are as follows:
PXT1-F:ATGCCTGCAGTCAACATGGTGGAG
PXT1-R:CATGTTGACTGCAGGCATGCAAGC
78B-99-F:ACTTAATGGATCCAGTTATAGGTCTAGGTCGCAG
78B-99-R:CTATAACTGGATCCATTAAGTAAAGTCCTGACGGGA。
(3) the fragment 1 and the fragment 2 are connected by means of homologous recombination and then transferred into Top10, and a plasmid is extracted and named as CGMMV-BamHI. The CGMMV-BamHI vector contains a repeated 190bp CGMMV CP subgenomic promoter and a single restriction enzyme cutting site BamHI, please refer to FIG. 2.
Example 2 cloning and insertion of target Gene fragments
(1) Total RNA was extracted from leaf tissues of cucurbits (watermelon, melon, cucumber and bottle gourd) using an RNAscope total RNA kit (Tiangen Biotech, Beijing, China), and then reverse transcribed using Oligo dT primer, PrimeScript II reverse Transcriptase (TAKARA) to synthesize first strand cDNA for RT-PCR.
(2) The Phytoene Desaturase (PDS) gene, which forms the beta-carotene synthesis pathway, was selected as the target gene. Four pairs of primers are designed by referring to the known sequence information of the cucurbit plant PDS genome in an NCBI database and combining the sequence information of the constructed vector so as to amplify PDS genes of four segments with different lengths. The primer sequences 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。
(3) PCR amplification is carried out by using the primer by using the Cucurbitaceae plant cDNA as a template to obtain PDS gene segments with the lengths of 69bp,150bp and 213bp and 300bp respectively.
(4) Carrying out enzyme digestion linearization on the CGMMV-BamHI vector by using BamHI enzyme, then connecting different PDS gene fragments into the linearization vector in a homologous recombination mode respectively, and recombining to obtain CGMMV-BamHI-PDS69, CGMMV-BamHI-PDS150, CGMMV-BamHI-PDS213 and GMMV-BamHI-PDS300 respectively.
Example 3 Agrobacterium transformation of recombinant vectors and verification of inoculation thereof
(1) Respectively adding 5 μ L vector plasmids (CGMMV-BamHI-PDS69, CGMMV-BamHI-PDS150, CGMMV-BamHI-PDS213 and CGMMV-BamHI-PDS300) into 50 μ L GV3101 competent cells, standing on ice for 10min, quickly freezing for 5min with liquid nitrogen, warm bathing in a water bath kettle at 37 deg.C for 5min, and standing on ice for 2 min. mu.L of LB liquid medium without antibiotics was put on a shaker at 28 ℃ for 2 hours at 200rpm, centrifuged at 12,000rpm for 1min, and the cells were collected and plated on LB solid medium containing antibiotics (50 ng/. mu.l Kan, 50 ng/. mu.l Rif). After 48h, positive clones were selected by picking the cells from the plates and culturing overnight in 200. mu.l LB liquid medium containing antibiotics (50 ng/. mu.l Kan, 50 ng/. mu.l Rif).
(2) The positive clones were cultured overnight in LB liquid medium containing antibiotics (50 ng/. mu.l Kan, 50 ng/. mu.l Rif) at a ratio of 1:100 in a shaker at 28 ℃ and 200rpm, and then centrifuged at 6,000rpm for 5min to collect the cells, which were then inoculated with an inoculation buffer (10mM MgCl. sub.g/L)210mM MES and 100. mu.M AS), the cells were suspended, allowed to stand at room temperature for 2 to 3 hours, inoculated by injection with a 1mL syringe from the back of cotyledon leaves of cucurbits (watermelon, melon, cucumber and bottle gourd), and then placed in a 16 hour/8 hour (light/dark) culture room at 28 ℃ at 25 ℃ for further culture.
Example 4 application of Gene silencing in Cucurbitaceae plants
The recombinant viruses (CGMMV-BamHI-PDS69, CGMMV-BamHI-PDS150, CGMMV-BamHI-PDS213 and CGMMV-BamHI-PDS300) were inoculated into watermelon, melon, cucumber and bottle gourd. Photobleaching can occur on uninoculated leaves 9-17 days after inoculation (dpi), see FIGS. 3-6; and maintained for a period of more than 2 months, see fig. 7.
Example 5 determination of silencing efficiency of CGMMV VIGS vectors
Total RNA was extracted from leaf samples inoculated with the most distinct vector-containing phenotypes and then passed through PrimeScriptTMRT reagent Kit with gDNA Eraser (TAKARA) reverse transcription Kit from 1ug RNA first strand cDNA relative expression measurements of the PDS gene were performed by qRT-PCR using SYBR Green I Master Mix. SelectingSequences outside the targeted silencing region were selected to design primers for determining PDS gene expression in cucumber, melon, bottle gourd and watermelon (CuPDS-679F/CuPDS-906R and water-q-F/water-q-R). In cucumber, melon and bottle gourd, actin gene is used as internal reference, and primers are cumactin-F/cumactin-R. The Cla016178 gene is used as an internal reference in watermelon, and the primer is Cla016178-F/Cla 016178-R. qRT-PCR was performed using a total of 20 μ Ι _ of reaction mixture: mu.L of diluted cDNA, 10. mu.L of LSYBRGreen I Master, 0.5. mu.M of each forward and reverse primer and 6. mu.L of double distilled water. The qRT-PCR procedure was as follows: 95 ℃ for 5 minutes, then 95 ℃ for 10 seconds, 58 ℃ for 20 seconds, 72 ℃ for 30 seconds, 45 cycles. Expression of the PDS gene was calculated using the 2-. DELTA.CT method. The expression level of the PDS gene in the negative control (CGMMV-BamHI) was set to an arbitrary value (1.0) to calculate the relative expression levels of the other samples, each of which was made in 3 replicates, see FIG. 8. The primer sequences are 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。
example 6 identification of the stability of the VIGS vector constructed based on CGMMV
It was observed that the photobleached phenotype of plants infected with the PDS fragment-containing recombinant vector could be maintained for a long time, see fig. 7. Referring to FIGS. 9-12, FIG. 9 shows the phenotype of leaf whitening at different parts of bottle gourd after 34 days of infection with a recombinant vector (CGMMV-BamHI-PDS69) carrying a 69bp PDS gene fragment, wherein L6, L7 and L9 represent the 6 th, 7 th and 9 th true leaves above the inoculated cotyledon, respectively; FIG. 10 shows the phenotype of leaf whitening at different sites after the infection of bottle gourd with recombinant vector (CGMMV-BamHI-PDS150) carrying 150bp PDS gene fragment (CGMMV-BamHI-PDS150) for 34 and 54 days, L4 and L11 respectively represent the 4 th and 11 th true leaves above the inoculated cotyledon; FIG. 11 shows the phenotype of leaf whitening at different sites after the infection of bottle gourd with recombinant vector (CGMMV-BamHI-PDS213) carrying 213bp PDS gene fragment (CGMMV-BamHI-PDS213) for 34 and 54 days, L4 and L12 respectively represent the 4 th and 12 th true leaves above the inoculated cotyledon; FIG. 12 shows the phenotype of leaf whitening at different sites after the bottle gourd is infected with a recombinant vector (CGMMV-BamHI-PDS300) carrying a 300bp PDS gene fragment for 34 days, and L7, L8, L9 and L10 represent the 7 th, 8 th, 9 th and 10 th true leaves above the inoculated cotyledon, respectively. At 54 days after inoculation, a photobleaching phenotype caused by PDS gene silencing was still observed in the upper, fresh leaves of the bottle gourd plants.
The relative expression of the PDS gene in the leaves at different positions is detected by qRT-PCR, and the expression of the PDS gene in the latest leaves can still be reduced, please refer to FIG. 13, which shows that the vector has good stability and can be used for identifying the function of the gene in cucurbit crops.
The above-mentioned embodiments are only for illustrating the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and to implement the same, and the present invention is not limited to the embodiments, i.e. all equivalent changes or modifications made according to the spirit of the present invention are still within the scope of the present invention.
Claims (4)
1. A method for constructing a cucumber green mottle mosaic virus-induced gene silencing vector is characterized by comprising the following steps:
(1) selecting a proper multiple cloning site according to the promoter information of the cucumber green mottle mosaic virus genome and the coat protein subgenome;
(2) constructing a vector containing a BamHI enzyme cutting site based on the full-length infectious clone of cucumber green mottle mosaic virus, and naming the vector as CGMMV-BamHI;
(3) amplifying the PDS gene segments to obtain PDS gene segments with lengths of 69bp,150bp,213bp and 300bp respectively, and further comprising the following steps: extracting total RNA from leaf tissue of cucurbitaceae plants, performing reverse transcription by using Oligo dT primer and PrimeScript II reverse transcriptase to synthesize first strand cDNA for RT-PCR, and performing PCR amplification by using the cucurbitaceae plant cDNA as a template and using the primer; the primer sequences 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；
(4) the amplified PDS gene fragment is connected into a linearized vector CGMMV-BamHI to form recombinant vectors CGMMV-BamHI-PDS69, CGMMV-BamHI-PDS150, CGMMV-BamHI-PDS213 and CGMMV-BamHI-PDS300 respectively.
2. A cucumber green mottle mosaic virus-induced gene silencing vector obtained by the construction method of claim 1, which is characterized by comprising a recombinant vector CGMMV-BamHI-PDS69, CGMMV-BamHI-PDS150, CGMMV-BamHI-PDS213 or CGMMV-BamHI-PDS 300.
3. Use of a cucumber green mottle mosaic virus-induced gene silencing vector according to claim 2, characterized in that it comprises:
(1) respectively inoculating recombinant vectors CGMMV-BamHI-PDS69, CGMMV-BamHI-PDS150, CGMMV-BamHI-PDS213 and CGMMV-BamHI-PDS300 to cucurbitaceae crops such as watermelon, melon, cucumber and bottle gourd;
(2) observing and recording the phenotype caused by the PDS gene silencing of each vector, measuring and analyzing the silencing efficiency, and detecting the stability of the vector.
4. The use of the cucumber green mottle mosaic virus-induced gene silencing vector according to claim 3, characterized in that the cucurbitaceae crop is inoculated at the following stages: the watermelon, melon and bottle gourd are selected from two cotyledons and one true leaf period, and the cucumber is selected from two cotyledons and two true leaf periods.
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