RNA Interference Of Two Acetylcholinesterases In Plutella Xylostella L.

Acetylcholinesterase (AChE, EC3.1.1.7) is a key enzyme in terminating neurotransmission by rapidly hydrolyzing the neurotransmitter acetylcholine (ACh) in the synaptic cleft. AChE is the target of organophosphate and carbamate insecticides and its mutations confer insecticides resistance. AChE has atypical functions including inducing axon growth,synapsesformation and differentiation, cell migration and interaction, cell adhesion and apoptosis, as well as enhancing hematopoietic cell formation and aggregate amyloid.Two acetylcholinesterase genes have been found in many insects. AChEl is considered as the key target of insecticide in most of pest. However, Cyclorrapha suborder and Leptinatarsa decemlineatahaveonlyace2. It was suggested that ace1is lost in evolution.RNA interference (RNAi) is a mechanism which can result in function-lost via turning off target gene expressionby exogenous or endogenous small double-strand RNA. RNAi exists in a wide range of organisms which is an old defense mechanism against the virus and transposon. As an efficient tool of gene function analysis and gene therapy, RNAi become more and more popular nowadays.In this work, we used RNAi technology to silence two acetylcholinesterase genes in Plutella xylostella. The gene functions of two ace genes in P. xylostella were analyzed.1. FAM labeled siRNAs were injected into the second instars at the back abdomenof diamondback moth. FAM labeled siRNAs were observed in the head at24hours after siRNA injecting, suggesting that siRNA can passed through the blood-brand barrier and reached the central neural system.The mortalities of siRNA-treated insects increased with the time after injecting si-pxace, indicating that RNAi in this pest was effective.2. Real time-PCRshow that the mRNA abundance of ace1in siPxace-1treated insects was reduced by90%at72hoursafter injecting, and the mRNA abundance of ace2in si Pxace-2treated insects was reduced by50%. The AChE activity of si Pxace-1, si Pxace-2and si Pxace-1&2treated groups are decreased by64%,21%,47%at96hours afer injecting, separately.3. Feeding response experiment indicated that interfering px-ace1but not px-ace2led to low creeping speed and long feeding time. Interfering AChE genes also resulted in autotomy in intraspecies. The body-lengths and body-weights in siPxace-1treated group were significantly lower than those in si Pxace-2and the control. There was no significant difference in si Pxace-2group.The growth inhibition rate was about50%in siPxace-1treated insects. Moreover, abnormal phenotypes such as gastropod undeveloped at one or both sides, head and gastropod growing inhibition, were also observed insi Pxace-1treated group, suggesting that AChE in diamondback mothmight have atypical functions.4. After silencing AChE gene by RNAi, all tested insects were treated with chlorpyrifos andspinosad followed by bioassay. The mortalities were increased with the time after siRNA injection. A significant difference was observed at48hours post-inection, suggesting that AChE-interfered diamondback moths were more sensitive to organophosphorus insecticide andspinosad.