Construction Of A VIGS Vector Based On Cucumber Green Mottle Mosaic Virus

Cucurbits are important fruits and vegetables in China.Breeding new cultivars needs better understanding the elite genes for agronomic traits.The genome sequencing of cucumber,watermelon and melon and other cucurbit crops laid the foundation for mining elite gene and researching gene function.The genetic transformation of cucurbit crops is time-consuming and labor-intensive,and the transformation efficiency is extremely low.The establishment of the virus-induced gene silencing(VIGS)technology platform facilitates the use of VIGS technology for the research of functional genomics in cucurbit crops.In this study,Cucumber green mottle mosaic virus(CGMMV),which can infect the cucurbit plants under natural conditions,was selected as the material.Based on the infectious clone of the virus,we created a VIGS vector that can be used to study the gene function of cucurbit plants by exploring the insertion site,identifying the subgenomic promoter(SGP)of the coat protein(CP)and evaluation of silencing effects.The major results of this study are as follows:(1)The insertion sites between the CGMMV CP gene stop codon and the 3' non-coding region are not suitable for constructing theVIGS vector.Based on the CGMMV infectious clone(pXT1-CGMMV),the viral vector(1a23)containing the single restriction site HindIII was obtained by point mutation and homologous recombination.The vector proved to be invasive by observing the symptom of the host plants and conducting DAS-ELISA and RT-PCR analysis.A VIGS vector containing PDS gene fragments of different lengths(114 bp,213 bp and 300 bp)was constructed on the basic of the 1a23,and demonstrate that only the vector included gene fragments of 114 bp in length could systematically infect.However,due to the strong pathogenicity of the vector,the silent phenotype of the host plant is coveredby viral symptoms.(2)Identification of the SGP of the CGMMV CP.pXT1-CGMMV was mutated by point mutation and homologous recombination,and 7 partial MP deletion mutants and 10 CP partial deletion vectors for transient expression of EGFP were constructed.To determine the SGP active region of CGMMV CP,the accumulation of CP subgenome RNA and the relative expression of EGFP were detected by NorthernBlot and qRT-PCR,and these results were further verified by bioinformatics.(3)A VIGS vector containing the 190 bp CGMMV CP subgenomic promoter and a single restriction enzyme site(BamHI)was successfully constructed: CGMMV-BamHI.Based on the information of identification the SGP of the CGMMV CP,an insertion site for constructing the CGMMV VIGS vector was confirmed.It was also found the inserts with length of 100~300 bp can produce silencing phenotype.When the insert was a 69 bp hairpin structure,the time when inoculated plants produced silent phenotypes was earlier,and the silencing efficiency was between the inserts with length of 150 bp and 213 bp.In summary,these results reveal the insertion sites of the foreign gene fragment in constructing the VIGS vector and mapping of CP SGP of CGMMV,then a CGMMV VIGS vector for rapid and efficient cucurbit functional genomic research was developed.The vector has the advantages of mild mosaicsymptoms in cucurbit plants,high silencing efficiency and easy operation,which will contribute to identify genes involved in disease-resistance and development,etc.