| Ostrinia furnacalis(Asian corn borer)is an important pest of corn,and the application of biological control bacteria is one of the crucial methods to control this insect pest in integrated pest management(IPM).Transcription factors Relish,a kind of immune related gene,was cloned by the method of Rapid Application of cDNA End(RACE)in O.furnacalis larvae in this study.Three kinds of bio-control bacteria,such as Pseudomonas aeruginosa,Micrococcus luteus and Bacillus thuringiensis Kurtaki(Bt)were used to treat O.furnacalis larvae by using the method of injection or feeding the larvae.The gene repressions of Relish and four kinds of antimicrobial peptides(AMPs),such as Cecropin A,Gloverin,Moricin and Attacin were analyzed after the treatment of three kinds of insect pathogens by real time PCR.The regulation mechanism of Relish gene was studied using RNA interference.This study.lays a foundation for exploring the function of Relish,and provides a theoretical basis for the further development of insect immune regulating pesticides in future.The main results are as follows:The cDNA sequence of Of-Relish gene in O.furnacalis larvae was successfully obtained using 3'-RACE and 5'-RACE method.The full length of this gene is 3165 bp,and the 2882 bp open reading frame encodes a 956-residue protein with a theoretical molecular mass of 98 kDa,including an 81 bp 3' non-coding region(UTR)and a 153 bp 5'-UTR.The start codon is ATG and the stop codon is TAG.Bioinformatics analysis on deduced amino acids showed that Of-Relish has no transmembrane domain,and has two glycosylation sites,and 101 phosphorylation sites.Of-Relish was hydrophilic protein.The three-dimensional structure analysis showed that there are 4 alpha helices and 16 beta sheets in Of-Relish.The strains of P.aeruginosa and M.luteus were injected into 4th instars larvae,respectively.The gene expression levels of Of-Relish and four kinds of antimicrobial peptides,such as Cecropin A,Gloverin,Moricin and Attacin were detected using quantitative real time PCR(qPCR).The results show that Of-Relish,Cecropin A,Gloverin,Moricin and Attacin gene expression levels increased significantly(P<0.05),after infection of P aeruginosa or M luteus in the body of 5th instar larvae compared with the control group.The injection of P.aeruginosa or M.luteus can induce the Of-Relish gene to regulate the gene expression of four kinds of AMPs,resulting in immune defense responses in O.furnacalis larvae.Using RNA interference,Of-Relish gene expression level in 3id instar O.furnacalis larvae injected with dsRNA-Relish were down-regulated to 42.08%,42.61%,47.69%and 64.85%of those in the control group post injection of 84 h,96 h,108 h and 120 h,respectively.The results showed that down-regulation of the expression of Of-Relish gene was successful with RNAi.After down-regulating the expression of Of-Relish gene with RNAi,the larvae were further injected with P aeruginosa or M luteus respectively.The results show that the expressions of Of-Relish,Cecropin A,Gloverin,Moricin and Attacin genes were also significantly inhibited,compared with the separate injection of bacteria,which illustrated that the Of-Relish can regulate the expressions of four kinds of antibacterial peptide genes.Bt was mixed with artificial diet,and then was used to feed 4th instar O.furnacalis larvae.The results of qPCR showed that Of-Relish,Cecropin A,Gloverin,Moricin and Attacin expression levels increased obviously,which showed that feeding with Bt can also induce Of-Relish gene to regulate gene expression of four kinds of AMPs,and induce the immune defense responses in O.furnacalis larvae. |
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