4种葡萄类病毒检测及序列分析

Detection of four grapevine virvoids and sequence analysis

采用苹果锈果类病毒属(Apscaviroid)保守区域设计的通用引物和4种葡萄类病毒特异引物,对采自辽宁、山东、新疆、甘肃和宁夏的48个葡萄样品进行RT-PCR检测及序列分析。结果表明,葡萄黄斑类病毒1(Grapevine yellow speckle viroid 1,GYSVd1)、葡萄黄斑类病毒2(Grapevine yellow speckle viroid 2,GYSVd2)、澳大利亚葡萄类病毒(Australian grapevine viroid,AGVd)和啤酒花矮化类病毒(Hop stunt viroid,HSVd)检出率分别为41.7%、22.9%、75%和83.3%。通用引物能够检测出大部分样品中的GYSVd1、GYSVd2、AGVd。序列分析结果显示,中国辽宁兴城GYSVd1分离物(GU170805)属于TypeⅠ,与澳大利亚TypeⅠ分离物同源性为99%,与澳大利亚及日本TypeⅡ分离物同源性为97%,与中国GYSVd3分离物及美国、日本TypeⅢ分离物同源性仅为89%,而与GYSVd2分离物同源性均在80%以下。GYSVd2扩增产物(HM211853)与其余分离物的相似性在97%以上,与两个北京分离物DQ377131和DQ377129最为相似,相似性为99%。辽宁AGVd分离物(HM211854)与其他分离物同源性都在97%以上。山东HSVd分离物(HM357802)与北京、日本葡萄分离物的同源性均在98%以上,与德国葡萄分离物同源性为97%,与马铃薯、啤酒花、柑橘等其他作物上的分离物同源性为97%以下。

RT-PCR was employed to detect four viroids,including Grapevine yellow speckle viroid 1（GYSVd1）,Grapevine yellow speckle viroid 2（GYSVd2）,Australian grapevine viroid（AGVd）,Hop stunt viroid（HSVd）,in the grapevine samples collected from Liaoning,Shandong,Gansu,Xinjiang,Ningxia,in which the primers used for the PCR amplification were those designed against the conserved region of the Apscaviroid group and the specific ones for GYSVd1,GYSVd2,AGVd,HSVd.The results showed that the ratio of samples with GYSVd1,GYSVd2,AGVd and HSVd were 41.7%,22.9%,75% and 83.3%.The common primers designed from the conserved region of the Apscaviroid group could successfully detect the GYSVd1,GYSVd2,AGVd and HSVd in most samples.Sequence alignment analysis indicated that Xingcheng GYSVd1 isolate（GU170805） belong to TypeⅠ,and the sequence identity levels between it and Australia TypeⅠisolate,Australia and Japan TypeⅡ isolates,China GYSVd3 and America and Japan Type Ⅲ isolates were 99%,97%,89%,respectively.It had a lower than 80% identity level with the GYSVd2 isolates.The sequence of amplicon of GYSVd2 isolate（HM211853）showed over 97% identity with those of other GYSVd2 isolates.The sequence identity level of Xingcheng AGVd isolate（HM211854） and other AGVd isolates is over 97%.The sequences of Shandong HSVd isolate（HM357802） and Beijing and Japan grapevine HSVd isolates were similar with an over 98% identity.However,HM357802 was less identical with the sequences of Germany grapevine HSVd isolate and the HSVd isolates from potato,hop and citrus.