青海省辣椒隐症病毒1的鉴定与全基因组序列克隆

Identification and whole genome sequence cloning of pepper cryptic virus-1 in Qinghai Province

【目的】明确青海省辣椒病毒病病原种类,获得乐都长辣椒的辣椒隐症病毒1(Pepper cryptic virus 1,PCV1)分离物的全基因组序列。【方法】2021年6月对青海省辣椒病毒病进行调查并采集疑似病样,对采集的样品分别用通用引物和特异性引物进行RT-PCR检测,后续通过克隆的方法得到PCV1的全基因组序列,用NCBI进行序列比对后用MEGA6.0软件最大似然法(maximum likelihood)、自导复制(bootstrap)1000次构建系统进化树,并运用同源性分析和聚类分析方法对PCV1的2条RNA链进行分析。【结果】采自乐都区的15份辣椒病样中,确认检出10份感染PCV1,其RNA1和RNA2的全长分别为1563 bp和1495 bp。NCBI网站序列比对分析结果表明克隆得到的PCV1青海分离物序列与已报道的PCV1其他分离物的核苷酸序列一致性与氨基酸一致性高于95%。系统发育分析表明RNA1和RNA2分别与其他PCV1分离物聚在一支上,与其同属的辣椒隐症病毒2(pepper cryptic virus 2,PCV2)分离物聚在不同的分支上。【结论】青海省辣椒上检测得到的病毒为PCV1,并获其全基因组序列,对今后保护青海省当地特色辣椒品种资源、不同地区的PCV1基因组遗传方式等研究的相关工作奠定理论基础。

【Objective】The aim of this study was to identify the pathogenic species of pepper virus in Qinghai Province and obtain the whole genome sequence of pepper cryptic virus 1(PCV1)isolate from Ledu Long Pepper.【Method】Suspected disease samples of Capsicum were investigated in June 2021 in Qinghai Province.The samples were detected using RT-PCR with universal and specific primers.The whole genome sequence of PCV1 was obtained using the cloning method,and a phylogenetic tree was constructed using MEGA6.0 software Maximum Likelihood and Bootstrap 1000 times after sequence comparison with NCBI.The two RNA strands of PCV1 were analyzed using homology and cluster analysis methods.【Result】The results showed that of the 15 pepper disease samples collected from Ledu District,10 samples were confirmed to be infected with PCV1,and the total lengths of RNA1 and RNA2 were 1563 bp and 1495 bp,respectively.the nucleotide sequence consistency and amino acid consistency between the cloned PCV1 Qinghai isolate and other reported PCV1 isolates were higher than 95%according to the NCBI website sequence comparison analysis.Phylogenetic analysis showed that RNA1 and RNA2 were clustered on one branch with the other PCV1 isolates,respectively,and the same genus of Pepper cryptic virus 2(PCV2)isolates were clustered on different branches.【Conclusion】The virus detected on pepper in Qinghai Province was identified as PCV1,and its whole genome sequence was obtained.This study lays a theoretical foundation for future research on the protection of local pepper varieties and the genetic mode of PCV1 genome in different regions in Qinghai Province.