大麦黄矮病毒运动蛋白在烟草中的瞬时表达

Transient Expression of BYDV-MP in Nicotiana benthamiana

[目的]快速鉴定运动蛋白基因在烟草中的瞬时表达,进一步研究该外源基因的功能。[方法]将大麦黄矮病毒的运动蛋白基因定向克隆到马铃薯X病毒载体上,得到重组的马铃薯X病毒,电击转化农杆菌后,利用农杆菌渗透注射技术注射到本生烟草的叶片中,观察病毒对烟草的侵染状况。[结果]渗透注射后第7天观察,重组病毒载体侵染的烟草系统叶有病毒侵染症状,而对照组未有此现象。对2组试验进行逐日跟踪观察,重组病毒载体侵染的烟草有较严重的病毒侵染症状和叶片卷曲现象,后期引起注射叶及系统叶坏死。而空病毒载体侵染的烟草只有轻微的病毒侵染症状,并能够恢复健康。对侵染的烟草进行RT-PCR检测,结果表明外源基因BYDV-MP在烟草体内进行了正常的转录和表达。[结论]BYDV-MP蛋白促进了PVX的系统侵染速度,加重了系统侵染的病毒症状,是病毒病症的决定因子;利用PVX表达载体表达异源MP的方法是可行的。

[Objective] The aim was to identify transient expression of movement protein（MP） gene in Nicotiana benthamiana rapidly and study functions of the exogenous gene.[Method] The MP gene of Barley yellow dwarf virus（BYDV） was cloned into potato virus X（PVX） viral vector,pGR107,and the recombinant PVX vector,PVX-MP,was obtained,then electrotransformed into Agrobacterium tumefaciens.The infection phenomenon on the upper leaves in tobacco was observed after inoculating PVX using Agrobacterium tumefaciens infiltration approach.[Result] Plants which were infected by recombinant virus vector showed the virus infection symptoms when observed at the seventh day after infection.While the control group of PVX inoculation displayed non viral phenomenon.Observations in the time-course revealed different symptoms between two groups.Tobacco which infected by recombinant PVX-MP virus vector had severer symptoms of virus infection than that of PVX virus vector infection only.Inoculation with PVX-MP also caused leaf curling phenomenon in newly growing leaves and necrosis both in infiltrated and systemic leaves while tobacco infected by empty viral vector had only mild symptoms of virus infection and could recover from PVX infection in newly growing leaves.BYDV-MP gene expression was verified by using RT-PCR.[Conclusion] BYDV-MP protein is determinant factor for viral disease which promotes the system infection and enhances its symptom of PVX.In addition,the method of using PVX expression vector to express exogenous MP gene in Nicotiana benthamiana is feasible.