植物瞬时表达体系真空侵染法的优化

Optimization of Vacuum Infiltration of Transient Expression System in Plants

病毒能够激活宿主的防御途径,诱导宿主大量基因表达。辅助成分-蛋白酶(helper-component protease, HC-Pro)是RNA病毒编码的基因沉默抑制子,关于HC-Pro诱导植物内源基因表达的分子机制并不十分清楚。本研究利用农杆菌介导的真空侵染法在拟南芥(Arabidopsis thaliana)小苗中瞬时表达了外源基因HC-Pro,并对影响基因表达效率的真空侵染条件进行了优化。结果表明农杆菌重悬液浓度OD600=1.5,真空压力为0.08 MPa,HC-Pro基因表达效率最高(95%)。半定量RT-PCR检测结果表明在真空侵染14 d后,HC-Pro在拟南芥(Arabidopsis thaliana)中得到了瞬时表达。通过优化了农杆菌介导的真空侵染法,外源基因快速、高效在拟南芥中得到了表达。本研究结果为研究病毒抑制子HC-Pro的功能提供了帮助。

Virus can activate host defence pathway and induced a number of genes expression in host. HC-Pro is a gene silencing suppressor encoded by RNA virus, the molecular mechanism that HC-Pro induced endogenous genes expression in plants is not very clear. Here, we transient expressed foreign gene HC-Pro in Arabidopsis thaliana seedings by Agrobacterium-mediated vacuum infiltration, and the vacuum infiltration conditions that influence gene expression efficiency were optimized. The results showed that the expression efficiency of HC-Pro gene is highest(95%) when the concentration of Agrobacterium resuspension OD600=1.5 and the vacuum pressure is 0.08 Mpa.The results of semi-quantitative RT-PCR had shown that HC-Pro was transiently expressed in Arabidopsis plants on 14 day past infiltration. Our study optimized Agrobacterium-mediated vacuum infiltration and obtained rapid and high-efficiency of foreign gene expression in Arabidopsis plants. The results of this study provides a basis for the functions study of viral suppressor HC-Pro.