黄瓜绿斑驳花叶病毒辽宁营口分离物基因组测定及分段基因克隆

Genome sequencing and segmental gene cloning of Cucumber green mottle mosaic virus isolate from Yingkou city, Liaoning province

黄瓜绿斑驳花叶病毒Cucumber green mottle mosaic virus(CGMMV)是葫芦科作物的重要检疫性病毒。本研究以田间自然感病的西瓜叶片为试材,采用RT-PCR获得该病毒(CGMMV-LNYK)基因组全长(登录号MG745849),以pBluescriptⅡSK(-)为载体,分别克隆其所编码的4段基因,并采用人工接种验证它们的体外侵染活性。结果表明:该病毒与韩国分离物KW(登录号AF417242)亲缘关系最近,相似度达99.8%,并成功克隆了CGMMV-LNYK编码的4段基因,分别记为RNA1～RNA4。经人工接种,发现RNA1和RNA4能侵染葫芦,表现明显花叶症状,与该病毒接种葫芦症状一致,且接种RNA1后的症状较RNA4明显,4种RNA混合接种症状最为明显,经RT-PCR检测,发病植株可扩增到与接种的RNA大小相等的片段。

Cucumber green mottle mosaic virus (CGMMV) is an important quarantine virus of cucurbits. In this study, the complete nucleotide sequence of the virus (GenBank accession no. MG745849) was obtained by RT-PCR in the naturally infected watermelon leaves. The four gene fragments encoded by the CGMMV were cloned into pBluescript Ⅱ SK(-) containing T7 promoter, respectively, and the invasive activity in vitro was verified by artificial inoculation. The results showed that the CGMMV was most closely related to the KW (GenBank accession no. AF417242) isolated from Korea, with an identity of 99.8%. The 4 infectious gene fragments of the CGMMV were successfully constructed and the segments RNA1 and RNA4 could infect cucurbits and cause obvious systemic mosaic in leaves. Moreover, the symptoms caused by the segment RNA1 were more severe than those by RNA4 after artificial inoculation. The symptoms were most obvious when the four segments of RNA inoculated at the same time. The RNA fragment amplified in diseased plants was equal in length to the inoculated viral RNA by RT-PCR.