Expression Analysis of Antheraea pernyi Lysozyme Gene Induced by Different Microbes
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Lysozyme is an important antimicrobial protein of insect humoral immunity and plays a significant role in resisting the invasion of exogenous substances.The pupae of Antheraea pernyi treated by Escherichia coli,Cordyceps militaris,Nosema pernyi and sterile water were used as material to determine the antimicrobial activity of A.pernyi pupal haemolymph against Micrococcus lysodeikticus upon induction by various exogenous substances.Moreover,the sequence of A.pernyi lysozyme gene(GenBank accession No.DQ353869) was used to design primers to analyze expressional variation of lysozyme gene in A.pernyi pupae induced by different exogenous substances using real-time fluorescence quantitative PCR.The results showed that haemolymph of all four treatment groups could generate antimicrobial activity against M.lysodeikticus,and the antimicrobial effect was significantly different.A.pernyi pupal haemolymph displayed remarkable antimicrobial effect at 72 h after treatment.The diameter of antimicrobial zone was in the ascending order of sterile water,E.coli,C.militaris and N.pernyi.Meanwhile,antimicrobial activity of the haemolymph from female pupae of A.pernyi was higher than that of the male pupae.In all four treatment groups,expression of lysozyme gene in haemolymph of A.pernyi pupae was up-regulated rapidly at 4~8 h after treatment.The highest expression level was observed at 24 h in female pupae and 8~12 h in male pupae.E.coli could induce rapid up-regulation of lyzozyme gene expression but its effect could only sustain in short duration.High expression levels of A.pernyi lysozyme gene could be induced by C.militaris and N.pernyi.It was shown that the immune response time and expression level of immune-related gene in A.pernyi varies with different inducing agents,and the response is also different between male and female individuals of A.pernyi.
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