The Functional Analysis Of ERK Gene In Defense Against Cry1Ca Protein In Chilo Suppressalis

The striped rice borer,Chilo suppressalis(Walker),is one of the major serious rice pests all around the world.The insecticidal crystal protein produced by Bacillus thuringiensis(Bt)has insecticidal activity and can be applied to the control of pests through the use of biological pesticides and transgenic Bt crops.Thus,it is important to know the defense mechanism of target insect pests against Bt protein.Mitogen-activated protein kinase(MAPK)signaling pathway is widely found in organisms and plays an important role in cell growth,development,differentiation,and apoptosis.ERK genes play an important role in the MAPK signaling pathway.In the present study,RNAi technology was used to study the function of CsERK genes in the defense of Cry1Ca protein in C.suppressalis.The main results are as follows:1.The full length of ERK genes were cloned by RT-PCR combined with RACE technology.The CsERK1 gene sequence is 1,892 bp in length,the open reading frame(ORF)sequence is 1,209 bp,and encodes 403 amino acids,and the predicted protein molecular mass is 44.51 kDa.The full length of CsERK2 gene is 2,015 bp in length,and the ORF sequence is 1,092 bp.It encodes 364 amino acids and the predicted protein has a molecular weight of 41.96 kDa.Sequence analysis found that the internal homology of the two sequences was only 21.5%.2.The real-time fluorescence quantitative PCR technique was used to analyze the temporal and spatial expression patterns of ERK gene in different periods and tissues of C.suppressalis larvae.The results of time-expression model showed that the CsERK1 gene had the highest expression level in the pupa and adult;the CsERK2 gene had higher expression in the adult than other periods.Spatial expression patterns showed that both CsERK1 and CsERK2 genes had the highest expression in the midgut.3.Different concentrations of Cry1Ca protein were mixed into diet to feed third instar larvae of C.suppressalis,and the expression of ERK genes was detected by realtime fluorescence quantitative PCR.The results showed that fed on 20 ?g/g of Cry1Ca artificial diet for 30 mins,the expression of CsERK1 gene was rapidly up-regulated by 3 fold,while the expression of CsERK2 gene was not changed significantly.After fed on 60 ?g/g of Cry1Ca artificial diet for 30 mins,the expression of CsERK1 gene did not show any significant changes,however,the expression of CsERK2 gene was significantly down-regulated and then slightly up-regulated after 1 h treatment.4.The ERK genes of the new hatched larvae of C.suppressalis was silenced by feeding dsRNA.After 48 h treatment,the silencing efficiency of CsERK1 gene and CsERK2 gene was 46.2% and 38.5%,respectively.After 48 h of interference,the larvae were transferred to an artificial diet containing 30 ?g/g of Cry1Ca protein or transgenic Bt rice plants which expressing Cry1Ca protein for 7 days.The bioassay results showed that there was no significant difference in larval mortality between the two treatment groups and the control group.Although Cry1Ca protein can change the expression of CsERK genes in C.suppressalis,no significant difference was found on the larval mortality after silencing the CsERK genes.These results indicated that CsERK genes does not participate in the defense mechanism of Cry1Ca protein in C.suppressalis.