Identification And Expression Analysis Of Immune Response Gene From The Chinese Oak Silkworm, Antheraea Pernyi

As invertebrates, insects lack the adaptive immune system, and rely on develoed innate immune to defend against the invasion of alien pathogens. The innate immune system is composed of cellular and humoral immunity. Both of them together kill and eliminate the pathogens by phagocytosis, nodulation, encapsulation, coagulation, and melanization. Cellular immunity refers to the phagocytosis and encapsulation of foreign invaders conducted by hemocytes. The humoral immunity is mainly composed of two anti-microbial peptides expressing pathways, Toll and Imd, and the melanization cascade catalyzed by phenoloxidases. Antheraea pernyi (Lepidoptera:Saturniidae) is one of the most well-known species among wild silkworms and commercially cultivated for silk production in China, India, and Korea. The pupae of A. pernyi are considered to be a source of high-quality protein foods containing all the essential amino acids. A subtractive cDNA library of A. pernyi pupae was constructed by suppression subtractive hybridization (SSH). A total of four hundred clones were randomly sequenced and analysed using BLASTX software and classified for the result. Some randomly selected immune response-related genes were confirmed by semi-quantitative reverse transcription-PCR (RT-PCR) and real-time quantitative reverse transcription-PCR (qRT-PCR). These identified immunity-related genes provide important information to understand the innate immunity mechanism in A. pernyi. The studies and the main results are as followings:1. Construction of subtractive cDNA libraryA subtractive cDNA library was constructed to screen for immunity-related genes in the fat bodies of pupae challenged with E. coli. A total of four hundred clones from the SSH cDNA library were randomly selected and sequenced,328 ESTs were identified with high sequence quality. These sequences were assembled and clustered using CAP3 program and 202 unigenes were obtained. Using Blastn in the NCBI, these unigenes were annotated and assorted into nine groups, including 50 immune response-related genes, three cytoskeleton genes, eight cell cycle and apoptosis genes, five respiration and energy metabolism genes, five transport genes,40 metabolism genes, ten stress response genes, four transcriptional and translational regulation genes and 77 unknown genes. In addition, the full cDNA length sequences of cecropin-like 4 and lysozyme of A. pernyi were obtained according to sequences assembled. Multiple sequence alignments and phylogenetic analysis show that they have highly homologue and close to other saturniidae insects.2. Expression profile analysis of some randomly selected immune response-related genesIn order to detect the expression of the immune response genes in the fat body of pupa challenged by E. coli at different time, semi-quantitative RT-PCR and qPCR were carried out. Among PRRs, both PGRP and βGRP were highly expressed at 0.5 h, and The expression levels of PGRP-A and hemolin like protein were strongly induced almost 80 fold and three fold at 3 h, respectively. Immunlectin A was down-regulated and hemolin was significantly expressed before injection. AMPs are the crucial component of insect innate immunity. The expression levels of eight AMP genes were rapidly induced by E. coli and kept at the high levels. The expression of attacin-1 reached the peak at 3 h, the highest expression levels of the cecropin like 4 and lysozyme were detected at 12 h, and the lebocin 2 had the highest expression at 24 h. In addition, microvitellogenin, DOPA decarboxylase and glutathione S-transferase theta also were expressed more than 2 fold at 0.5 h. Protease inhibitors 5,6 and 8 were significantly expressed at 3 h, protease inhibitors 3, serine protease like protein 1 and serpin like 4 were highly up-regulated at 24 h.3. Cloning and expression analysis of Apolipophorin-Ⅲ gene from Anthraea pernyiIn this study, an ApoLp-Ⅲ gene from the fatbody of A. pernyi pupae was isolated and characterized and named as Ap-apoLp-Ⅲ. The full-length cDNA of Ap-apoLp-Ⅲ is 687 bp, including a 5’-untranslated region (UTR) of 40 bp,3’-UTR of 86 bp and an open reading frame (ORF) of 561 bp encoding a polypeptide of 186 amino acids which contains an Apolipophorin-Ⅲ precursor domain (PF07464). The deduced Ap-apoLp-Ⅲ protein sequence reveals 68,59, and 23% identity with its homologue of Manduca sexta, Bombyx mori, and Aedes aegypti, respectively. Phylogenetic analysis showed that the Ap-apoLp-Ⅲ is close to Bombycoidea. qRT-PCR analyses revealed that Ap-apoLp-Ⅲ gene was expressed during the four developmental stages and in some tested tissues. After microorganism infections, expressions of the Ap-apoLp-Ⅲ gene were up-regulated significantly at different time points. The Ap-apoLp-Ⅲ was amplified by RT-PCR, ligated to pET-28a(+) expression vector and transformed into Escherichia coli BL21(DE3), induced by IPTG under different concentrations. Through SDS-PAGE electrophoresis and Western blot analysis, the induced fusion protein was successfully expressed and the purified recombinant protein was also performed. These results will provide the foundation for further study function of Ap-apoLp-Ⅲ gene.