滇重楼辣椒轻斑驳病毒分离物的鉴定及全基因组序列分析.docxVIP

滇重楼辣椒轻斑驳病毒分离物的鉴定及全基因组序列分析 杨林毅 陈潞 陈泽历 摘要：滇重楼（Paris polyphylla var. yunnanensis）是多年生草本植物，其药用部位为地下根茎，是一种重要的中药材。为明确侵染滇重楼的病毒病原，对采自云南省曲靖市的滇重楼进行透射电镜观察、DAS-ELISA和RT-PCR技术检测。在透射电镜下观察到杆状病毒粒子，大小为（200～300） nm×（20～36） nm。对采集到的7份病样进行DAS-ELISA检测，结果表明，其中5份病样呈阳性。通过RT-PCR技术扩增、克隆获得1个全长为6 357 bp的曲靖分离物PMMoV-QJ的全基因组序列。将PMMoV-QJ的全基因组序列与国内外已经报道的10个PMMoV分离物进行同源性分析比对，其同源性为99.42%～99.81%。基于全基因序列的系统发育分析表明，PMMoV-QJ与韩国分离物亲缘关系密切。本研究明确了该分离物的亲缘关系，从分子水平鉴定该分离物，为后续滇重楼病毒病的预防提供理论依据。 关键词：滇重楼（Paris polyphylla var. yunnanensis）;辣椒轻斑驳病毒（PMMoV）;鉴定;序列分析 Abstract： Paris polyphylla var. yunnanensis is a perennial herb， and its medicinal part is underground rhizomes. It is an important chinese medicinal material. In order to clarify the viral pathogens infecting the Paris polyphylla， the transmission electron microscopy， DAS-ELISA and RT-PCR techniques were performed on the Paris polyphylla collected from Qujing， Yunnan province. Baculovirus particles were observed under transmission electron microscopy and were（200～300）nm ×（20～36）nm in size. The results of DAS-ELISA on 7 samples collected showed that 5 of them were positive. A whole genome sequence of a 6 357 bp Qujing isolate （PMMoV-QJ， accession number MK784568） was cloned by RT-PCR amplification. The homologous sequence of PMMoV-QJ were analyzed by homology with 10 PMMoV isolates reported at home and abroad， and the homology was between 99.42% and 99.81%. Phylogenetic analysis based on the whole gene sequence showed that PMMoV-QJ was closely related to Korean isolates. This study clarifies the genetic relationship of the isolate， and identifies the isolate from the molecular level， which provides a theoretical basis for the prevention of the subsequent viral disease. Key words： Paris polyphylla var. yunnanensis; pepper mild mottle virus（PMMoV）; identification; sequence analysis 滇重樓（Paris polyphylla var.yunnanensis）又称独角莲，属延龄草科重楼属（Paris）多年生草本植物，其药用部位为地下根状茎，分布于亚欧大陆温带和热带地区，在中国主要集中在西南地区，其中以云南省滇中、滇东和滇东北、滇西和滇西北为主[1]。滇重楼宜阴畏晒，喜湿忌燥，通常生长在