Gene Cloning And Expression Analysis Of Insulin-like Peptides And Their Receptors In The Green Rice Leafhopper,Nephotettix Cincticeps(Uhler)(Hemiptera:Cicadellidae)

The green leafhoppers,Nephotettix cincticeps(Uhler)(Homoptera:Cicadellidae),is one of the important insect vectors to spread rice dwarf virus(RDV).The complex interaction relationship among rice,RDV and N.cincticeps is formed in long-term evolution.The previous studies showed biological changes like the extension of nymph development,the shortening of female adult longevity,and the decreases of fecundity and offspring survival rate in N.cincticeps infected with RDV.In order to explore the underlying mechanisms,we primarily studied the genes and their functions of insulin-like peptides and their receptors in the interaction.The main results are shown as follows:1.Gene cloning and sequence analysis of insulin-like peptides and their receptors in N.cincticepsThe genes of 10 insulin-like peptides(NcILP1-10)and 2 insulin receptor(NcInR1-2)were obtained via screening from the transcriptome database of N.cincticeps in the laboratory.These genes contain a signal peptide fragement with 18?26 amino acid residues,which means they are all secretory proteins.The full-length cDNA sequences of these genes were cloned by RT-PCR.There is an evolutionary diversity in the sequences of insulin-like peptides and their receptors.2.Expression analysis of insulin-like peptides and their receptors in N.cincticepsThe temporal and spatial transcription of insulin-like peptides and their receptors in N.cincticeps were characterized by real-time quantitative PCR.Except for NcILP5 with high expression levels in eggs,nymphs and adults,the other insulin-like peptides and their receptors are mainly expressed in nymph and adult stages.As for the tissue distribution of gene expression,NcILP1 and NcILP3 are found mainly in the midgut;NcILP2 and NcILP5 in the ovary;NcILP4,NcILP6 and NcILP8 in the brain;NcILP9 in brain and midgut;and NcILP10 in brain,ovary and midgut.NcInRl is expressed mainly in the brain and ovary.NcInR2 is expressed in the brain,midgut and ovary,in which the expression is significantly higher in the ovary.RDV can significantly inhibit the gene expression of NcILP4 in N.cincticeps.The diversity of temporal and spatial expression distributions of insulin-like peptides may reveal the similarity and difference in the function of these genes.3.Preliminary exploration of NcILP4 function in N.cincticepsThe transcription level of NcILP4 gene decreased significantly in N.cincticeps after RNAi.The content of trehalose increased slightly with NcILP4 gene silence,while the contents of glycogen and triglyceride decreased significantly.It indicates that NcILP4 may be involved in trehalose metabolism,and glycogen and fat synthesis process.Based on these results,it suggests that RDV may impact the biology of N.cincticeps through interaction with NcILP4.The results not only provide a basis for further research on the interaction mechanism among rice,RDV,and N.cincticeps,but also for further understanding of insect insulin genes and their biological function,as well as for development of new pest control technology.