半夏3种花叶病毒的mRT-PCR鉴定及遗传进化分析  

mRT-PCR identification and genetic evolution analysis of mosaic viruses in three Pinellia ternata

作　　者：李锦超 董俊美 孟义江[2] 杨太新[1] 葛淑俊[1] LI Jinchao;DONG Junmei;MENG Yijiang;YANG Taixin;GE Shujun(College of Agronomy,Hebei Agricultural University/Key Laboratory of Crop Gennplasm Resources research and Utilization in North China,Ministry of Education,Baoding 071000,China;College of Life Science,Hebei Agricultural University/Key Laboratory of Crop Germplasm Resources research and Utilization in North China,Ministry of Education,Baoding 071000,China)

机构地区：[1]河北农业大学农学院/教育部华北作物种质资源研究与利用重点实验室,河北保定071000 [2]河北农业大学生命科学学院/教育部华北作物种质资源研究与利用重点实验室,河北保定071000

出　　处：《河北农业大学学报》2023年第1期80-87,共8页Journal of Hebei Agricultural University

基　　金：国家现代农业产业技术体系(CARS-21);河北省现代农业产业技术体系中药材产业创新团队(HBCT201806201,HBCT201806202)。

摘　　要：半夏(Pinellia ternata)因长期无性繁殖导致病毒病高发。本研究以河北安国疑似病毒感染的半夏为材料,通过优化扩增反应体系及反应程序,建立了可以同时检测黄瓜花叶病毒(Cucumber mosaic virus, CMV)、大豆花叶病毒(Soybean mosaic virus, SMV)和芋花叶病毒(Dasheen mosaic virus, DsMV)的多重RT-PCR体系,RNA稀释106倍时亦能同时检测3种病毒。对侵染病毒外壳蛋白进行基因克隆和遗传进化分析,结果表明CMV的外壳蛋白编码区序列为657 bp,与浙江侵染半夏的CMV(DQ399550.1)序列相似性高达96.16%,与感染其他寄主的病毒序列差异较大;SMV和DsMV外壳蛋白序列分别为843和828 bp,与同种株系遗传变异度分别达到0.38和0.37以上,独立为1个进化支。生物信息学统计分析发现,病毒外壳蛋白序列高度保守,具有较为丰富的变异位点,碱基转换率大于颠换率,CMV AT偏倚度大于GC,SMV、DsMV与之相反。研究结果为半夏病毒检测和脱毒提供了技术和理论基础。The incidence of viral disease is high in Pinellia ternata due to long-term asexual reproduction. In this study, a multiplex RT-PCR system was established to detect Cucumber mosaic virus(CMV), Soybean mosaic virus(SMV) and Dasheen mosaic virus(DsMV) simultaneously in pinellia that was suspected of virus infection in Anguo,Hebei Province. After optimizing the amplification reaction system and reaction procedure, three viruses could be detected at the same time when the RNA was diluted 10 million times. The genes encoding the viral coat protein were cloned and their genetic evolution were analyzed. The results showed that the CMV coat protein(CP) was encoded by a sequence of 657 bp that was 96.16% similar to the CMV(DQ399550.1) sequence isolated from virus infected Pinaceae from Zhejiang Province and significantly different from virus infected other hosts. The sequences encoding the coat proteins of SMV and DsMV were 843 bp and 828 bp. Their genetic variation of other SMV and DsMV was above 0.38 and 0.37, and they were in an independent clade in the phylogenetic tree. Statistical analysis of the CP sequences showed that the virus coat protein sequences included highly conserved domains and regions with abundant mutation sites in which base conversion rate was higher than inversion rate, and the AT skew was greater than GC for CMV that was opposite in SMV and DsMV. The results will provide a technical and theoretical basis for virus detection and detoxification of Pinellia ternata.

关 键 词：半夏 花叶病毒 多重RT-PCR 遗传进化 

分 类 号：S432.11[农业科学—植物病理学]