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

【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 8.7-24.39 μg and 7.48-22.4 μg dsgfp.Using internal standard mixed pure dsRNA method, the fermentation of pET-2P and L4440 expression vector induced bacteria solution was calculated to produce 8.7-24.39 μg 7.48-22.47 μg 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.