hpRNA的制备新方法及其在RNAi载体中的应用

A Prepared Method of hpRNA and Its Application for RNAi Vector

[目的]验证制备新方法所获得的稳定hpRNA是否可用于RNAi载体的构建并达到基因表达干扰的效果。[方法]利用制备新方法(2μL DNA与8μL H_2O混匀,95℃5 min,37℃15 min)制备hpRNA,后采用REGS方法构建Zm MDAR RNAi载体,通过Grx1-ro GFP2和荧光实时定量PCR检测基因表达干扰的效果。[结果]利用制备新方法制备50 nt hpRNA。Zm MDAR基因的表达量变化和Grxl-ro GFP2探针的荧光比值变化表明hpRNA介导的干扰载体引起了原生质体氧化还原状态的变化。[结论]通过制备新方法所制备的hpRNA为RNAi载体所介导的干扰作用奠定基础。

[ Objective ] The aim was to test a method if it is applied for the RNAi vector and lead to interference effect. [ Method] We prepared hpRNA by the new method （2 μL DNA and 8 μL H20 mixed, 95℃, 5 min, 37℃ ,15 min）, constructed ZmMDAR RNAi vector with REGS method, and tested the interference effect of gene expression by Grxl-roGFP2 and mall-time PCR. [ Result] Using new method for preparing 50nt hpRNA, change of Zm MDAR expression and fluorescence ratio of Grxl-roGFP2 indicated that hpRNA mediated interference vector caused the change of redox state of protoplast. [ Conclusion ] The prepared hpRNA by new method will lay a foundation for interference effect mediated by RNAi vector.