Effect Evaluation And Stability Analysis Of Extraction Methods For Prokaryotic Expression DsRNA

Prokaryotic induced expression of ds RNA has potential application value in the field of pest control.The cost of producing ds RNA by prokaryotic induction fermentation is low,but the concentration and yield of expressed ds RNA cannot be measured,making this method insufficient in the study of gene function of coleopteran insects;ds RNA is susceptible to nuclease degradation,making its stability and degradation detection technology research awaiting rich.Therefore,in this study,720 bp green fluorescent protein(GFP)was used as the template to construct p ET-2p-dsgfp and L4440-dsgfp expression vectors of different lengths and different GC contents;secondly,the in vitro synthesis of dsgfp was used as a control to compare and analyze 4 methods to extract dsgfp Purity,use the internal standard mixed pure dsgfp method to calculate the loss rate of the extraction method,analyze the expression of p ET-2p-dsgfp and L4440-dsgfp to preliminarily determine the optimal extraction method of dsgfp and the relative optimal expression vector;finally,the EP tube at room temperature After standing,the potato leaves were smeared with dsgfp degradation products for electrophoresis gray-scale analysis and Chip-seq sequencing technology to obtain relatively stable dsgfp.The main research contents and results are as follows:1.Artificially synthesized 5 groups of target fragments: 201 bp length 30% GC content gfp(referred to as 201-30 GC,the same below),201-50 GC,423-30 GC,423-50 GC,423-70 GC.They were cloned into p ET-2p and L4440 vectors to obtain p ET-2p-dsgfp and L4440-dsgfp expression vectors.Grayscale analysis showed that the expression level of p ET-2p-dsgfp was higher than that of L4440-dsgfp.2.Using in vitro synthetic dagfp as a blank control for multiple analysis of variance,the Trizol method and the phenol chloroform method with the highest extraction purity are obtained.The average values of A260/A280 and A260/A230 can reach 1.83,1.79;1.78,2.1,respectively;use internal standard mixing The loss rates of Trizol method,phenol-chloroform method,RNA-easy extraction solution and 75%alcohol precipitation method detected by pure ds RNA method were 23.88%,21.44%,32.3% and 37.62%,respectively;1ml induced bacterial solution was extracted by phenol-chloroform method.The two vectors express the same dsgfp concentration with significant difference(P<0.05).p ET-2p-dsgfp and L4440-dsgfp can induce fermentation to produce 8.7-24.39?g/ml and 7.48-22.47?g/ml dsgfp,respectively.It further shows that the expression ability of p ET-2p-dsgfp is higher than that of L4440-dsgfp.3.The one-way variance statistics show that 423-70 GC is relatively stable under room temperature environment.Using Chip-seq technology to sequence,it was found that the percentage of 423-70 GC successfully aligned sequences was 1794109(3.37%),and the average alignment quality score was 40.23.Both were better than other dsgfp,further indicating that 423-70 GC had the best stability.