The Application Of RNAi Technique In The Process Of Gynoparaous Morph In Cotton Aphid

Cotton aphids(Aphids gossypii Glover),the destructive insect pests in the agriculture,horticulture and forestry,are capable of reproducing asexually and sexually upon environmental change;Cotton aphids also have the wing dimorphism.RNA interference(RNAi)technology has been widely concerned since its discovery and has now become an important tool for reverse research on gene function.This method has been studied in a variety of model organisms,such as honeybee,cricket,drosophila,and pea aphid.But the interference efficiency varies widely among different species of organisms.RNAi technology is rarely studied in cotton aphid.Insulin signal transduction plays a major role in many important physiological processes,including growth,development,metabolism,reproduction and aging.Therefore,it is urgent to clarify the molecular mechanism of insulin signaling pathway in regulating metamorphosis,so that provide important theoretical basis for insect pest control.In this study,cotton aphids as the material,choose the gene of insulin signaling pathway RNA interference and detection interference efficiency,in order to explain the effects of insulin receptor on the reproductive development of cotton aphid.The gene include Insulin receptor1 and Insulin receptor 2(InR1and InR2),Foxo,Akt and PI3 K.RNAi uses a method of microinjection.The dsRNA used for injection is also chitosan-embedded,transmembrane protein and transfection reagent mixed.The concentration and location of the injection were based on the results of the previous study: the optimal dose is 10 ng for 3rd instar of aphid;the optimal site is the conjunctive between the mesothorax and metathorax.Injection the ds In R1 and dsInR2,detection the interference efficiency of G2 generation 5d,10 d and adult with qRT-PCR.The results show that injection with dsIn R1 has significant interference efficiency and the interference efficiency in the adult period can reach 73%,in the tenth day can reach 60%;In addition,we found that there also have an impact on the reproduction and wing type of cotton aphid.There were 16% wingless cotton aphids in the G2 generation adult.Anatomical cotton aphid and observation the ovary,we found the number of ovarioles was significantly reduced.However,there was almost no interference efficiency of G2 after injection with dsInR1 embedded by chitosan or mixed with transmembrane protein.Injection the dsIn R1,detection the interference efficiency of G1 generation 11 d and 12 d with qRT-PCR.The results show that there only a little interference efficiency,but not significant.In addition,interference with Foxo,Akt and PI3 K by the same method,the results cannot be achieved RNAi in G1.However,further studies have shown that there was a70% interference efficiency in G1 generation 12 days after birth,after injected the dsInR1 mixed with transfection Reagents-Biofection.This result provides an effective method for studying the molecular mechanism of gynoparpus formation.