Molecular Cloning, Immune Correlation Analysis Of Lipase And Heat Shock Protein In The Chinese Oak Silkworm, Antheraea Pernyi

The Chinese oak silkworm (Antheraea pernyi) is a kind of valuable economic insect. Because Antheraea pernyi is reared in wild, its growth is influenced by a varity of factors,including external environment, the quality of leaf, climate conditions and so on. The immune system in insects is different from that in vertebrates, they rely on their natural immune system, which is formed in the long-term survival evolution, to resist various exogenous pathogens in wild environment.In this study, we took eggs, larva, pupae and moths as the experimental materials, based on the migut transcriptome data of Antheraea pernyi infected with ApNPV contructed in our laboratory, genes encoding lipase, and Hsp70 from Antheraea pernyi are cloned. Using bioinformatics methods to analyze the structure and function of Aplipase sequence.The semi-quantitative PCR is used to analyze the profile in different development stages and tissues; the Real-time PCR is used to analyze the relative expressions induced by exogenous stimulus over periods of time.The results are as follows:1. PCR cloing technology was used to get the lipase gene cDNA sequence.Sequence analysis revealed that the open reading frame of Aplipase is 1527 bp with an open-rading frame encoding 509 amino acids. Using BLAST analysis, the amino acid sequence of Aplipse shared 79% identity with Bombyx mori, and the homology of Danaus plexippus and Papilio xuthus ranged from 50% to 60%. The deduced amino acid sequence of Aplipase exhibited a high similarity with those of Lepidoptera.Semi-quantitative PCR results indicated that Aplipase is mainly expressed in all stages and tissues of midgut and fatbody in 5th instar. Real-time PCR results indicated that relative expressional level of Aplipase in fatbody was increased at 48 h after pathogen infection, and then decreased.2. Two heat shock protein 70 genes ApHSP70-1 and ApHSP70-2 were cloned from the fat body of A.pernyi pupae by RT-PCR technology. The open reading frames of ApHSP70-1 and ApHSP70-2 are all 1905 bp,encoding 634 amino acids.The encoded proteins containing cytoplasmic characteristic motif are predicted to be inducible heat shock protein.The sequence similarity between ApHSP70-1 and ApHSP70-2 is 86.92%, and the sequence similarity of them with ApHSC70 is 71.82% and 70.14% respectively. Semi-quantitative RT-PCR indicated that the expressional level of ApHSP70-1 and ApHSP70-2 genes were increased obviously after high temperature (43 ℃) treatment,while the expressional level of ApHSC70 gene had little change. Real-time PCR detection showed that the expression level of ApHSP70-2 in fat body of A.pernyi pupae was up-regulated by 6.32 times after high temperature (43 ℃) treatment, and that of ApHSP70-1 was up-regualted by 4.18 times.