Functional Study Of Two Acetylcholinesterase Genes And Their Relationship In Bombyx Mori Using RNA Interference

Acetylcholinesterase (AChE) plays a key role in neurotransmission, in which itmaintains the transmission of nerve impulses by catalyzing acetylcholine in the neuronalsynapse. Acetylcholinesterase in Lepidoptera is encoded by two ace genes, ace1and ace2.To study their functions and relationship, we used RNA interference. The results are asfollows.In this study, we used RNA interference (RNAi) to individually knock down theexpression of ace1or ace2in a Bombyx mori cell line (BmN) to explore their functions andrelationship. Knockdown efficiency was confirmed by quantitative real-time PCR. We alsomonitored transcription levels of ace1and ace2at different time points after B. morinucleopolyhedrovirus (BmNPV) infection. We found that when the expression of ace1wasknocked down to37.5%, the expression of ace2increased by66%; when ace2expressionwas reduced to33.4%, the ace1expression was similarly reduced to26.7%.Acetylcholinesterase activity was decreased to86.2%when ace2expression was inhibited.However, no significant difference was observed in AChE activity when ace1expressionwas knocked down. After BmNPV infection, ace1expression was gradually increasing,while ace2expression was gradually decreasing. Our study indicates that ace2plays animportant compensatory role for the loss of ace1in maintaining AChE activity byincreasing its expression level. In addition, ace2may also regulate transcription of ace1.On the other hand, we used RNA interference (RNAi) to knock down their expressionto explore their functional relationship. The transcription levels of these two genes in brain,fatbody, midgut and silk gland were determined by quantitative real-time PCR. We alsoexamined the activity of AChE and the resistance of B. mori to insecticides after RNAi.When the expression of ace1in brain was knocked down to14.8%of its normal level,ace2’s expression was increased by3.12-fold. However, when the expression of ace2wasreduced to22.1%of its normal level, ace1’s expression was also reduced to5.8%. In thefatbody, midgut and silk gland, the expression of ace2was inhibited with ace1RNAi. Simultaneous injection of siRNA-ace1and siRNA-ace2resulted in reduced expressionlevels of both genes, although the reduction was less dramatic. The activity of AChE in thebrain was higher than that in other tissues. The activity was decreased to73.5%when ace2expression in brain was inhibited, while knockdown of ace1failed to cause significantchanges in AChE activity. In the midgut and silk gland, inhibition of either ace1’s or ace2’sexpression resulted in significantly decreased AChE activity. When both ace1and ace2’sexpression was knocked-down, significantly reduced A four tissues tested. Furthermore,phomix-resistanChE activity was observed in allce assay revealed that interference of ace2alone or both AChE genes dramatically increased the mortality to75%and80%,respectively. Overall, our findings help elucidate the relationship between the two acegenes in Lepidoptera.