The optimized method to isolate heterologous DSRNA expressed in Escherichia coli HT115(DE3)

The effectiveness of RNA interference (RNAi) in suppressing the transcriptional activity of target genes of insect pests, phytopathogenic viruses and fungi determines the interest in the biosynthesis of double-stranded RNA (dsRNA) in bacteria Escherichia coli as a less expensive technology compared to in vitro synthesis. Simplifying the procedure for isolating dsRNA from bacteria could help reduce the cost of dsRNA production and expand the use of RNAi in plant protection. In the present work, a simple and effective method for the isolation and purification of dsRNA from E. coli cultures has been developed to evaluate the prospects for the use of these effector molecules in agricultural practice. Our goal was to create an original vector for the efficient expression of dsRNA molecules in E. coli HT115(DE3), as well as to optimize the conditions for extracting nucleic acids from bacterial cells, treating them with RNase A and subsequent isolation of dsRNA using acid phenol. To construct a new vector producing dsRNA in E. coli HT115(DE3), we used the pRSETа plasmid (Thermo Fisher Scientific, USA) without the region encoding the N-terminal recombinant tag. To integrate the additional T7 promoter into the vector, the plasmid was re-amplified using the forward primer 5´-CCCTATAGTGAGTCGTATTAGAAAGGAAGCTGAGTTGGCTGCTG-3´ (T7 promoter is underlined) and the reverse primer 5´-CTTTGTTAGCAGCGGATCAAG-3´, as well as Phusion Flash High-Fidelity PCR Master Mix (Thermo Fisher Scientific, USA). Phosphorylation of the ends of the PCR products and their ligation using T4 polynucleotide kinase and T4 DNA ligase (Thermo Fisher Scientific, USA) made it possible to obtain an original vector for the expression of dsRNA in bacteria. For the biosynthesis of dsRNA in E. coli , we used fragments of genes encoding the b'-COP subunit of the coatomer complex of the Colorado potato beetle Leptinotarsa decemlineata , one of the endonucleases of the green peach aphid Myzus persicae , as well as fragments of the three L. decemlineata genes (subunits A of the vacuolar ATPase, subunit 7 (Mov34) of the 26S proteasome and actin), which were inserted into the vector multicloning site. The constructed plasmids were electroporated into E. coli HT115(DE3) cells using an electroporator 2510 (Eppendorf, Germany) at 1700 V. Ligation of L. decemlineata and M. persicae gene fragments into a new vector, subsequent transformation of E. coli HT115(DE3) cells with these constructs, expression and isolation of dsRNA demonstrated the high efficiency of their biosynthesis, comparable to that of traditional L4440 vector. Optimization of conditions for the extraction of nucleic acids from bacterial cells before their treatment with RNase A showed the effectiveness of ultrasonication as well as heating in water at 95 °C. To remove single-stranded RNA, the optimal content in the reaction mixture was 0.4 M NaCl and 1.25 μg/ml of RNase A (Thermo Fisher Scientific, USA). Treatment of the reaction mixture with acid phenol (pH 4.0) and then with chloroform is necessary not only for selective extraction of dsRNA, but also for inactivation of stable RNase A. The reaction mixture treatment with chloroform alone did not lead to complete inactivation of the enzyme. The amount of dsRNA isolated from bacteria sonicated before treatment with RNase A under selected conditions with used equipment was 9.1±1.2 μg/ml of culture. It was comparable with the use of a more complex extraction method based on heating with SDS (sodium dodecylsulfate) and subsequent phenol-chloroform extraction (8.8±0.6 μg/ml of culture). When the bacteria were heated for 20 minutes in water at 95 °C before treatment with RNase A, the dsRNA yield was approximately 55 % of the values observed using the other two methods (5.0±1.5 μg/ml culture).