关键词: 二化螟, 碱性神经酰胺酶, 中性鞘磷脂酶, RNAi, 显微注射法, 荧光纳米粒子介导喂食法, 浸泡法

Abstract: 【Aim】 To establish and optimize the RNAi system for alkaline ceramide (saCER) and neutral sphingomyelin (snSMase) genes of Chilo suppressalis by comparing the RNA interference efficiencies based on different methods and different target genes.【Methods】 Double-stranded specific RNA (dsRNA) of Drosophila daCER gene was introduced into Drosophila S2 cells by soaking in dsRNA-containing medium. Specific dsRNA of saCER and snSMase genes of C. suppressalis were synthesized in vitro and introduced into the 3rd instar larvae (micro-injection) and the 2nd instar larvae (fluorescent nanoparticle-mediated feeding) of C. suppressalis, respectively. The expression levels of these target genes were measured by qPCR for investigating the RNAi efficiency among different methods as well as different genes. 【Results】 The expression level of daCER gene decreased by about 84% by soaking Drosophila S2 cells in dsRNA-containing medium at a concentration of 15 ng/μL for 72 h. The RNAi efficiency of saCER gene was the highest (41%) at 60 h post injection into the 3rd instar larvae at the dsRNA concentration of 5 000 ng/μL, and that of snSMase gene was the highest (47%) at 48 h post injection at the dsRNA concentration of 2 500 ng/μL. The highest RNAi efficiencies of saCER and snSMase genes were obtained on the 7th day (32%) and the 8th day (52%)， respectively, after feeding the 2nd instar larvae with dsRNA (48 μg/d). For aCER gene in Drosophila S2 cells, the interference efficiency with soaking method was significantly higher than those with injection and feeding method of C. suppressalis. There was no significant difference in the RNAi efficiency of saCER and snSMase genes using the same introduction method, and between methods of micro-injection and fluorescent nanoparticle-mediated feeding for the same gene (saCER or snSMase) of C. suppressalis. 【Conclusion】 The aCER and nSMase genes are sensitive to RNAi by introducing dsRNA both in vivo and in vitro. The protocols of RNAi via micro-injection and fluorescent nanoparticle-mediated feeding developed and optimized in this study are feasible for the basic research of enzyme genes of the sphingolipid metabolism. The RNAi efficiencies of aCER gene in C. suppressalis larvae using injection and feeding methods were significantly lower than that in Drosophila S2 cells using soaking method, further confirming that the interference efficiency of dsRNA into the C. suppressalis cells is greatly reduced due to the rapid degradation of dsRNA by RNase in hemolymph and the barrier of midgut pericardial membrane.

Key words: Chilo suppressalis; alkaline ceramidase, neutral sphingomyelinase, RNAi, micro-injection method, fluorescent nanoparticle-mediated feeding method, soaking method