侵染广西甘蔗的高粱花叶病毒分子鉴定初报

Preliminary Report on Identification of Sorghum Mosaic Virus Infecting Sugarcane in Guangxi

在南宁市郊采到1份表现褪绿条纹、花叶的糖蔗(桂糖95-53)样品(编号为SC-NN1),经差速离心和PEG二次沉淀法提取病毒粗汁液,电镜观察,该病毒为略弯曲的线状,ELISA检测显示该病毒与甘蔗花叶病毒(Sugarcane mosaic virus,SCMV)、高粱花叶病毒(Sorghum mosaic virus,SrMV)多克隆抗血清呈阳性反应,与SCMV单克隆抗血清呈阴性反应,初步判断分离物为SrMV。针对SrMV CP基因设计特异引物,利用RT-PCR和免疫捕获反转录PCR(Immunocapture reverse transcript PCR,IC-RT-PCR)对SC-NN1进行分子检测鉴定,两种方法均扩增出1条长约1.1kb的片段,而健康对照未扩增到任何片段。扩增片段经克隆后测序,序列包含1010个核苷酸,测序结果在NCBI上用BLAST进行同源性分析,该序列与SrMV浙江、云南分离物对应的核苷酸序列同源性大于95.32%,氨基酸序列同源性大于97%,根据马铃薯Y病毒科(Potyviridae)病毒种和株系的划分标准,将SC-NN1鉴定为SrMV。

A sampl e of sugarcane leaves showing chlorotic stripe and mosaic symptoms was collected in the Nanning suburb, and purified preparation from the infected sugarcane leaves by one cycle of differential centrifugation and PEG double precipitation. Filamentous particles were observed under electron microscope. ELISA test indicated that the virus isolate had positive reaction with SCMV and SrMV muhi-clonal antiserum and negative reaction with SCMV monoclonal antiserum. The isolate was pre-identified as SrMV. A specific primer designed according to the coat protein gene of SrMV was used in the RT-PCR and IC-RT-PCR to test the sample. A 1.1kb segment was obtained in both methods and no segment in healthy control. The segment nucleotide sequence analysis indicated that the segment contain 1010 nucleotides. Sequence similarity searches were performed using the BLAST program （http：//www.ncbi.nlm.nih.gov）. The result showed that the isolate had more than 95.32% nucleotide sequence and 97% amino acid sequence similarities with SrMV from Zhejiang and Yunnan. So the isolate was identified as SrMV according to the classify standard of Potyviridae.