侵染昆明玫瑰的李坏死环斑病毒的鉴定及其分子检测

Identification and molecular detection of Prunus necrotic ringspot virus isolated from Kunming

采集昆明地区种植的花叶症状明显的玫瑰样品,运用现代分子生物学技术与常规技术相结合的方法,初步鉴定引起玫瑰花叶病的主要病毒病原为李坏死环斑病毒。该病毒引起玫瑰植株的系统花叶、畸形和皱缩等症状;电镜下病毒粒体为球形,直径为22～23nm;ELISA检测发现该病毒在植株芽、花粉和顶部叶片的浓度最高;同时,根据外壳蛋白的保守区利用Primer5.0设计该病毒的特异引物,对该病毒进行分子检测,得到450bp的预期DNA片断,并在此基础上,进行了巢式RT-PCR的分子检测,表明巢式RT-PCR的检测能力最强。并通过序列的同源性分析得知该病毒的外壳蛋白与已知PNRSV的同源性为98.0%,进一步证明了该病毒为李坏死环斑病毒。

The virus causing rose mosaic disease in Kunming was identified as Prunus necrotic ringspot virus(PNRSV) through both the molecular biology technology and the traditional technology.And the diameter of this sphere virus is 22-23nm.The rose infected by the virus presents systematically mosaic,abnormality and crimple.The concentration of the virus in the shoot,pollen and top leaves are highest compared with other parts of the plant.And then with the primers,designed based on the conservative region of the PNRSV CP,we can gain a 450bp fragment.While,the nested RT-PCR was also applied to detect the virus with the primers CP1,CP2,P1 and P2 which was proved to be the most sensitive compared with other techniques.And the similarity of the CP sequences between this PNRSV and the known species is 98.0%,which confirmed pathogen of this disease is PNRSV.