The Cloning Of MAPK From Ostrinia Furnacalis Guenee And The Response Of MAPK Expression To Sublethal Concentration Of Biopesticide Cry1Ab Treatment

The functions of mitogen-activated protein kinase(MAPK)was studied in Ostrinia furnacalis(Guenee)larvae,and the regulation mechanism of CrylAb,a kind of toxic protein of Bacillus thuringiensis,on the expression of MAPK gene and other related immunity and stress response genes in O.furnacalis(Guenee)larvae was explored in this research.The full-length cDNA sequence of MAPK was obtained with RACE method,and the sequence of MAPK was analyzed bioinformaticly.The recombinant MAPK protein could be prokaryotic expressed successfully in Escherichia coli BL21 strain.The target protein was verified by Western blotting analysis.Furthermore,the polyclonal antibody of MAPK fusion protein was prepared,then the cellular and tissue distribution of MAPK was analyzed.The O.furnacalis 4th instar larvae were raised on the artificial diet treated with sublethal concentration of LC30 of activated Cry1Ab,and quantitative real time PCR(qRT-PCR)was applied to analyzed the effect of Cry1Ab-LC30 treatment on the expression of MAPK and related genes on transcription level.The RNA interference(RNAi)was applied to inhibit the expression of MAPK gene,and then qRT-PCR was used to analyze the expression levels of MAPK and other related genes in O.furnacalis larvae.After the Cry1Ab-LC30 treatment,the MAPK-dsRNA was injected to 4th instar larvae,and the expression of MAPK and other related genes was analyzed by qRT-PCR.The results are as follows:(1)The full-length cDNA sequence of MAPK gene in O.furnacalis was cloned by RACE.The full length of the MAPK cDNA sequence is 1592 bp,encoding 364 amino acids,including a 65 bp 5'non-coding region and a 432 bp 3' non-coding area(GenBank accession number:MF797866).The open reading frame(ORF)is about 1095 bp.ATG is the start codon and TAA is the stop codon.Of-MAPK has a calculated molecular weight of approximately 41.9 kDa.It contains a S TKC conserved domain at positions 22-355 bp.The prediction of protein structure of MAPK showed that it has 13 alpha helices and 8 beta sheets.The amino acid sequences of Of-MAPK and Chilo suppressalis ERK2,Amyelois transitella MAPK,Hyposmocoma kahamanoa MAPK,Galleria mellonella MAPK,Papilio xuthus MAPK,Helicoverpa armigera MAPK are highly homologous.(2)Prokaryotic recombinant expression of the MAPK protein was carried out using pET-28a expression system.The results showed that the recombinant protein of Of-MAPK was expressed in the supernatant after the induction at different concentrations of IPTG,and the molecular weight of MAPK was about 41 kDa.The optional conditions for large amount of protein expression in the supernatant were 28? and 0.2 mM IPTG induction.The MAPK recombinant protein was confirmed by Western blotting.After the preparation of the polyantibody of MAPK protein,the distribution of MAPK protein was observed on the surface of hemocytes by FITC labeling.The MAPK protein was also observed in integument of O.furnacalis larvae by immunoelectron microscopy(3)The expression of MAPK and other related genes,such as AKHR,PPO,GSH-Px,Cat and Hsp90,in the 4th instar O?furnacalis larvae were detected by qRT-PCR after the treatment of sublethal concentration LC30 of Cry1Ab.The expression of MAPK and above related genes was also detected after the MAPK-dsRNA injection.Additionally,the expression level of MAPK and other related genes were also analyzed after Cry1Ab-LC30 treatment and successful MAPK RNA interference.The results showed that the expression levels of MAPK and Hsp90 were significantly up-regulated after the CrylAb-LC30 treatment;the expression levels of AHKR and GSH-Px were significantly affected in the early stage,otherwise the expression of PPO and Cat were inhibited.When MAPK-dsRNA was injected into O.furnacalis larvae,the MAPK gene expression was down-regulated from 12 to 72 h.When the expression of MAPK was silenced with RNAi,which could effectively down-regulate the transcription level of MAPK,the expression levels of AKHR was up regulated.Meanwhile,the expression of PPO was down regulated within 0-48 h,and then up regulated,and GSH-Px and Cat expressions were inhibited at 12-24 h,then followed by a significant increase.However,the expression of Hsp90 gene showed continuous interference effects.When the O.furnacalis larvae were fed with the activated Cry1Ab-LC30 and then injected with MAPK-dsRNA,the results showed that there was some correlations between the expression of MAPK gene and the related genes,and the expression of PPO?Hsp90?GSH-Px and Cat genes were positive correlations with the expression of MAPK,while AHKR showed negative correlations to MAPK expression.