香石竹斑驳病毒云南分离物全基因组序列测定和侵染性克隆构建

Sequence and construction of infectious cDNA clones of Carnation mottle virus isolate in Yunnan

香石竹斑驳病毒(Carnation mottle virus,CarMV)是危害香石竹的一种重要病毒,本研究通过构建农杆菌介导的CarMV侵染性克隆来进一步研究该病毒基因功能。首先获得CarMV云南分离物全长序列,与报道的上海分离物相似性达到94.78%;将CarMV全长cDNA基因序列构建到具有35S启动子的双元表达载体pCV-nGFP,通过农杆菌浸润到本氏烟中瞬时表达,本氏烟系统叶片RT-PCR能检测到CarMV。试验结果表明,本研究构建Car-MV侵染性克隆通过农杆菌介导可快速高效侵染植物,可以用于病毒基因功能研究;同时CarMV云南分离物全长序列测定结果表明其属于P164 K331组群。

Carnation mottle virus was a serious constraint to carnation. The objective of this study is to generate the Agrobacterium tumefacien-mediated infectious cDNA clones of Carnation mottle virus to study the gene function of the virus. Firstly, we got full cDNA sequences of CarMV from Yunnan Province, the nucleic acid similarity of which with Shanghai isolate was 94.78%. The full-length cDNAs of CarMV were inserted into pCV-nGFP with a 35S promoter. They were then introduced into A. tumefacien, and infiltrated into Nicotiana benthamiana. RT-PCR detection of systemic leaves of N. benthamiana showed that they were infected with CarMV. The result suggested that the Agrobacterium-mediated infection of N. benthamiana by the infectious cDNA clones of CarMV was successful and efficient, and it could be used to study the gene of virus. The full-length sequences of CarMV which isolated from Yunnan belonged to the group P^164K^331.