Effects Of Sublethal Tebufenozide And Methoprene On The Expression Of Dopa Decarboxylase From Ostrinia Furnacalis Guenée Larvae

In order to investigate the regulation mechanism of Dopa decarboxylase(DDC)ralated to insects' innate immunity,DDC gene was cloned from the Ostrinia furnacalis(Guenee)larvae with RACE,and then the sequence was bioinformaticly analyzed,The expression profiles of DDC gene after tebufenozide and iethoprene treatments with sublethal concentration of LC30.from 5th instar O.furnacalis larvae were analyzed by real time PCR.To get the optimal conditions of prokaryotic expression,the DDC protein was expression in Escheirichia coli BL21(DE3)strain.The taget expressed protein was identified with Western blot.The main results are as follows:(1)In this study,RT-PCR and RACE(Rapid amplification of cDNA ends)methods were used for the cloning of full length cDNA encoding DDC from the 5th instar larvae of O.furnacalis.The full length cDNA of DDC was 1866 bp(GenBank accession number:KT428965),containing an ORF of 1431 bp,a 5'UTR of 202 bp and a 3'UTR with polyadenylation signal of 55 bp.The open reading frame starts from the 203th nucleotide and ends at the 1633th nucleotide.The deduced amino acid sequence starts with Met with the length of 476 amino acids.The DDC was predicted with molecular weight of 53.8 kDa and p1 value of 5.95.Bioinformatics analysis showed that there was no possible transmembrane protein model and no signal peptide in the N terminal of the peptide,and there is a glycosylation sites which located 32th aimno acid,and 36 phosphorylation sites were uniformly distributed throughout the polypeptide chain.BlastP analysis showed that the amino acid sequence of the cloned DDC cDNA from O.furnacalis was highly similar to those of Spodoptera exigua DDC?Clanis bilineata DDC?Mythimna separata DDC?Antheraea yamamai DDC?Danaus plexippus DDC?Operophtera brumata DDC and Spodoptera litura DDC.(2)The pET-28a vector was used to express DDC protein.After the IPTG inducement at different concentrations,the DDC protein was expressed in E.coli BL21,and the molecular weight of the recombinant DDC protein was about 53kDa which is same as the predicted.After 1.2 mmol/L IPTG induced in 37?,1.2 mmol/L IPTG induced in 28? and 1.0 mmol/L,1.2 mmol/L IPTG induced in 37?.the pET-28a-Of-DDC recombinant protein was expressed in the precipitation and supernatant.And then the expressed protein was identified by Western blot.(3)The real time PCR was used to analyzed the expression profileof DDC gene after the larvae treated with tebufenozide and methoprene at LC30 concentration.The results show that DDC expression levels showed a tendency of increase in the control group with the development,and after 48 hours,the expression of DDC began to decrease in the control group.In the treatment group with tebufenozide LC30,the expression of DDC post 0-24 h-treatment was not differenent significantly with that in the control group.However,post 48-72 h,the expression of DDC was inhibited remarkablely.In the treatment group with methoprene LC30,the expression of DDC increased obviously post 0-24 h treatment,and the expression of DDC gene reached its peak post 72 h treatment.