4种原核表达双链RNA的dsRNA提取方法效果评价

Comparative Study on the Effect of 4 kind of dsRNA Extraction Methods form Prokaryotic Expression Double-stranded RNA

【目的】构建表达不同片段长度和GC含量的dsegfp片段,比较4种dsRNA提取方法,获得最佳的提取方法以及dsRNA相对最优的表达载体,为dsRNA的降解研究提供基础。【方法】以pET-2p和L4440为载体,分别构建5组不同长度、不同CG含量的gfp-201-30CG、gfp-201-50-CG、gfp-423-30CG、gfp-423-50CG和gfp-423-70CG为目的片段的dsgfp表达载体,运用Trizol法、酚氯仿法、RNA-easy提取液以及75%酒精沉淀法分别提取1和50 mL经异丙基硫代-β-D-半乳糖苷(IPTG)诱导菌液表达的dsgfp,核酸检测A260/A280、A260/A230比较纯度,以体外合成dsgfp为对照,评价4种方法提取效率及2种载体dsgfp表达能力。【结果】4种提取方法经多重方差分析,其中提取纯度最高的是Trizol法和酚氯仿法A260/A280、A260/A230均值分别可达1.83、1.79;1.78、2.1,损失率分别为23.83%、21.44%。其次是75%酒精沉淀法A260/A280、A260/A230均值可达1.8、1.78,损失率为32.3%。提取纯度最低的为RNA-easy提取液,其A260/A280、A260/A230均值可达1.56、1.56,损失率为37.62%。利用内标混合纯dsRNA的方法,计算获得每毫升pET-2P和L4440表达载体诱导菌液可发酵产生8.7~24.39μg、7.48~22.47μg dsgfp。【结论】提取pET-2p和L4440表达的dsgfp,其中酚氯仿法可用于快速提取高质量、高纯度原核表达的dsgfp,pET-2p为dsgfp最优表达载体。

【Objective】Prokaryotic expression of dsRNA has a wide range of potential applications in the field of pest control.【Methods】Using pET-2p and L4440 as vectorsconstruct five groups of gfp-201-30CG,gfp-201-50-CG,gfp-423-30CG,gfp-423-50CG and gfp-423-70CG of different length and different CG content as the target fragments of dsgfp expression,Trizol method,phenol chloroform method,RNA-easy extraction solution and 75%alcohol precipitation method to extract 1ml and 50 mL of dsgfp induced by isopropylthio-β-D-galactoside(IPTG)in bacterial solution,respectively Nucleic acid detection A260/A280,A260/A230 compare the purity,with in vitro synthesis of dsgfp as a control,to evaluate the extraction efficiency of the four methods and the expression ability of the two vectors dsgfp.【Results】The four extraction methods were analyzed by multiple variance analysis.Among them,the Trizol method and the phenol-chloroform method A260/A280 and A260/A230 with the highest extraction purity reached 1.83 and 1.79 respectively;1.78 and 2.1,respectively,he loss rate was 23.83%,21.44%.Followed by the 75%alcohol precipitation method,the average values of A260/A280 and A260/A230 can reach:1.8,1.78,and the loss rate s 32.3%.The lowest extraction purity is s the RNA-easy extract,whose average values of A260/A280 and A260/A230 can reach 1.56 and 1.56,and the loss rate is s 37.62%.Using the method of mixing pure dsRNA with internal standard,it was calculated that per milliliter of pET-2P and L4440 expression vector induced bacterial liquid can ferment to produce 8.7-24.39μg and 7.48-22.4μg dsgfp.Using internal standard mixed pure dsRNA method,the fermentation of pET-2P and L4440 expression vector induced bacteria solution was calculated to produce 8.7-24.39μg 7.48-22.47μg dsgfp.per ml.【Conclusion】Four methods re used to extract dsgfp expressed by pET-2p and L4440 The he phenol chloroform method can be used to quickly extract high-quality and high-purity prokaryotic dsgfpquickly pET-2p is the optimal expression of dsgfp he carrier provides technical support for the subsequent study of the degradation degree of dsgfp in different environments and the analysis of degradation products.