Functional Analysis Of Insulin Receptor And Its Signaling Pathway Genes In Hyphantria Cunea(Drury)by RNAi Technology

In recent years,Hyphantria cunea is a quarantine pest that seriously harms forests and crops.Currently,the control of Hyphantria cunea is still dominated by chemical control,but the long-term usage of pesticides has caused environmental damage.With the development of molecular biology,it has become a new research field to seek new molecular targets for developing molecular inference or creating new insecticides for green control.Insulin signaling pathway is closely related to the nutrition metabolism,growth,reproduction and lifespan of individuals.Nowadys,the physiological function of insulin signaling pathway in Hyphantria cunea lacks extensive and in-depth researches.In this study,we used H.cunea to study the effects of key factors in insulin signaling pathway on the growth and development by RNAi technology.These key factors include Insulin receptor(Ins),Protein Kinase B(AKT/PKB),Forkhead box-containing protein(FOXO),Phosphotidylinositol-3-kinase(PI3K)and mammalian target of rapamycin(mTOR).Detailed research results are as follows:1.The HcInsR,HcAKT,HcFOXO,HcPI3K and HcmTOR gene sequences were cloned from the genome of H.cunea,and the sequences were verified by RT-PCR.Bioinformatics analysis showed that the open reading frame length of the five genes was 4617,1626,1506,3102 and 7296bp,respectively,encoding 1538,541,501,1033 and 2431 amino acids.Conserved domain analysis indicated that HcInsR,HcAKT and HcPI3K are members of the protein kinase C family while HcmTOR and HcFOXO is a member of the PIKK family and the FH superfamily,respectively.2.The expression levels of HcInsR,HcAKT,HcFOXO,HcPI3K and HcmTOR genes were different during developmental stages of H.cunea.The expression levels of HcInsR,HcAKT,HcPI3K and HcmTOR genes in egg stage were all higher than those in other developmental stages,and the expression levels in larval stages were much lower.Tissue specific expression results showed that five insulin signaling pathway genes were highly expressed in the digestive system of larvae,especially in the midgut and the hindgut,which were up to 85.26-fold of that in the control.It is speculated that the key genes of insulin signaling pathway play an important role in the secretion of digestive enzymes and nutrient absorption of H.cunea.The dsRNA injection successfully silenced the HcInsR gene of H.cunea,and the silencing efficiency reached 64.77%,64.21%and 57.82%at 24h,48h and 72h.Silencing of HcIn.sR gene interfered with the normal growth and development of larvae.The weight of the control group at 24h,48h and 72h was 1.19-,1.17-and 1.16-fold of those in the treatment group,respectively.Their mortality rate was 6.67%in the control group and 53.33%in the injection group.The starting time of 4th instar larvae to 5th instar larvae and 5th instar larvae to 6th instar larvae were delayed by 1d.3.The genes of HcInsR,HcAKT,HcFOXO,HcPI3K and HcmTOR were successfully silenced by siRNA injection,with the silencing range of five different sites ranging from 10.60%to 93.15%,among which the silencing efficiency of HcFOXO gene was the highest with 93.15%.The larvae injected with siRNA showed growth retardation.The weight of the larvae in the injection group was lower than that of the control group,and the weight of AKT1474 was 24.27mg lower than that of control group at 72h.The mortality rate of the injection group was up to 42.5%,which was significantly different from the control group(2.5%).The injection of siRNA into the 5th instar larvae resulted in a 2?4.5 d prolongation of the time from the 5th instar to the end of pupation.These results indicated that the key genes of the insulin signaling pathway are involved in the regulation of the growth and development of the H.cunea larvae.The insulin signaling pathway can affect the absorption of sugars,the synthesis of fats and the storage of proteins in cells.When these necessary substances for life activities are disturbed,the weight,duration and longevity of the insects are also affected.The results enrich the function of the key genes in the insulin signaling pathway and contribute to better understand the growth and development mechanisms of H.cunea.