Construction Of Full-length CDNA Clone And Virus-induced Gene Silencing Vector Of Papaya Mosaic Virus In HaiNan

Papaya mosaic virus （PaMV） is one of the plant viruses that limit papaya production, belonging to the genus Potexvirus family. The complete genomic sequence of PaMV of Hainan isolate （PaMV-HN, GenBank:JX524226） was determined, which shared with a99.6%nucleotide sequence identity with the PaMV isolate from USA （GenBank:NC₀01748）. The complete genomic sequence of PaMV-HN was6656nucleotides（without the3’ploy A） long and the genome contained five typically conserved open reading frames （ORF）, flanked with5’and3’non coding regions of99and119nucleotides, respectively.Two infectious clones containing the full-length of a Hainan isolate of PaMV cDNA were constructed. Plasmid PaMV-HN-3is an Agrobacterium-mediated system for PaMV-HN that insert a27nucleotides ployA at the3’end of the full-length cDNA of PaMV-HN and the CaMV35S promoter and NOS terminator were inserted from the HBT95-SGFP （S65T）-NOS vector. Plasmid PaMV-HN-4is an in vitro transcription system that the the full-length cDNA of PaMV-HN to the T-easy vector was ligated by in-fusion PCR in order to insert the T7promoter. Infectivity of PaMV-HN-3（63.3%） and PaMV-HN-4（33.3%） on papaya plant by Agrobacterium infiltration and the Friction inoculation, respectively. The agrobacterium-mediated system has a higher infectivity and handier operation, is more appropriate for the papaya infection.Virus-induced gene silencing （VIGS） is a recently developed gene transcript suppression technique for characterizing the function of plant genes. We have developed a VIGS vector derived from PaMV-HN. A subunit gene promoter and a lethal gene ccdB were inserted before the CP gene by overlapping long distance PCR （OE-LD PCR）, it is convenient to substitute target genes for the gene ccdB by one-step, ligation-independent cloning （OZ-LIC）.In this study, we substitute the papaya phytoene desaturase（PDS） gene fragment for the gene ccdB as the silencing fragment by OZ-LIC. As expected, PDS mRNA levels were decended in leaves exhibiting a phenomenon of photobleaching after the agrobacterium infiltration.The gene silencing efficiency on papaya plant is56.7%and29.4%of the Silencing phenotype were not systemic gene silencing.The complete nucleotide sequences of PaMV is the first time to report in China which provide not only a new clues for the PaMV disease protection, but also at the molecular level for the further function study of PaMV. With the method of OZ-LIC, the construction of PaMV VIGS vector allows the high-throughput cloning of silencing fragments without the use of costly enzymes required for recombination. It has a vital significance for the high-throughput analysis of papaya genome gene function.