Genetic Diversity And Construction Of An Infectious CDNA Clone Of Cucumber Green Mottle Mosaic Virus

Cucumber green mottle mosaic virus(CGMMV)can infect cucumber, watermelon, melon, gourd and other plants. It causes a serious disease on cucurbit crops with significant economic losses in many countries. In China, it is listed as Quarantine plant pathogen, and has been reported in several places. To know the molecular evolution and mechanisms of pathogenic plays an important role in controlling plant disease. Here, we determined the complete genomes of two isolates obtained from Jinan and Qingdao, respectively; studied its phylogenetic relationship with other CGMMV isolates and constructed an CGMMV infectious clone successfully. The followings are the main results obtained in this study:(1) Two complete genomes of CGMMV isolates were determined. Two CGMMV isolates obtained from Jinan and Qingdao city of Shandong province(named CGMMV-JN and CGMMV-QD, respectively), were sequenced. Sequence analysis showed that the full length genome is 6424 nucleotides(nt) for both isolates. The 5?-UTR and 3?-UTR has 60 nts and 176 nts, respectively. The complete genome of CGMMV-JN contains four open reading frames(ORFs) encoding 129 k Da and 186 k Da RNA-dependent RNA polymerase(RdRp), 29 kDa movement protein(MP), 17.4 kDa coat protein(CP), respectively.(2) The complete genome of CGMMV-JN showed 90.0%-99.7% sequence similarity with other CGMMV isolates at nucleotide levels. Recombination events were detected in RdRp-encoding sequences from isolates from Korea(AF417243), Israel(KF155231), India(DQ767631) and China(KJ658958). Purifying selection were found in Rd Rp and MP genes. In contrast, positive selection was found in CP gene.(3) Based on phylogenetic analysis, all CGMMV isolates can be divided into three groups. Isolates CGMMV-JN and CGMMV-QD belong to group I. Isolates from Liaoning and Taiwan including CGMMV-JN were more closed to that from Japan. Moreover, isolates from Shandong and Jiangsu including CGMMV-QD were much closed to that from Korea. These results suggested that isolates CGMMV-JN and CGMMV-QD were possibly originated from Japan and Korea, respectively.(4) The complete genome of CGMMV-JN were inserted to binary vector pCambia 0390 under 35 S promoter. To make the clone much stable in E.coli, an intron obtained from watermelon were inserted within Rd Rp-encoding sequence. The infectious of obtained clone pCamCGMMV was tested both on Nicotiana benthamiana and watermelon. The N.benthamiana inoculated by p CamCGMMV showed similar symptoms with that infected by wild type CGMMV. The infectious clone pCamCGMMV showed 100% infection efficiency under normal condition in all experiments by RT-PCR.The results obtained in this study will assist future work on gene function and pathogenesis of the virus.