Study On Lysozyme Gene Expression And Analysis From Hemolymph Induced By Different Microbes In Antheraea Pernyi

The Chinese oak silkworm, Antheraea pernyi, which is an important economic insect is fed in the wild. Because of its feeding ways, a variety of pathogens in the external environment may lead to the occurrence of disease and cause huge economic losses to breeders. In recent years, central issue is focused on immune research of A. pernyi and some progress has been made. Compared with higher animals, humoral immunity of insect is absence of specific antigen-antibody reaction. It formates a complete open defense system which play the role by activity factors, such as lectin and lysozyme, fitting blood cells. Recently, large insects such as Bombyx mori and A.pernyi are used as test material. The experimental direction focuses on immune activity factors, for example its inducers, separation and purification, dynamics, antibacterial mechanisms, structural analysis, cloning. In this paper, we take A. Pernyi pupae as the experiment object. Firstly, antibacterial substances are detected from A. pernyi haemolymph induced by microbes. Secondly, by SDS-PAGE electrophoresis and mass spectrometry, protein differential expression bands are obtained and analysed to certain interest protein in the next study, namely lysozyme. Then the method of molecular biology is used to research deeply in two aspects which include gene cloning and real-time quantitative PCR. The results are as follows:1. All the haemolymph of four induced A. pernyi pupa are taken as the experiment object. We resprctively use inhibition zone for the determination of lysozyme activity. It showed that all the haemolymph of four induced treatment groups can generate the antimicrobial activities to M. lysodeikticus, and the antimicrobial effect varied remarkably among different inducers. There were significant antimicrobial zones after72hours, the antimicrobial zone diameter of treatment groups’ ascending order is sterile water, E. coli, C. militaris and N. pernyi. Meanwhile, the antimicrobial activity of the haemolymph from the female pupae of A. pernyi was stronger than that of the male pupae.2. Protein electrophoresis results show that the molecular mass of approximatele28KD and44KD protein bands are significangly deepened.The consequence indicates that differentially expressed protein bands appear after the hemolymph immuned. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis identified a total of117non-repetitive protein in two which are marked as the AP28and AP44. Based on coverage, the identification number of peptides and identification of the number of spectra,26high crediting proteins are screened from the identified proteins.GO analysis shows that26total proteins are annotated to35GO entries in three GO main bodies. Respectively, there are five proteins to participate in immune system of Antheraea pernyi and stimulate response process on AP28and AP44.3. TA cloning technology is used to get the lysozyme gene cDNA sequence. Sequence analysis revealed that total length of A. Pernyi lysozyme is670bp, which includes44bp of the5’end non-coding region,422bp of the coding region and204bp of the3end non-coding region. Meanwhile, it has a422bp of opening up reading frame and encoding140amino acids. Its amino acid sequence homology analysis shows that the homology with lepidopteran insects is75%-82%, compared with the other species of the class Insecta, the homology of the range is40%-50%, compared with the higher animals, the homology of40%or less.4. The pupae of A. pernyi induced by three microbes including Escherichia coli, Cordyceps militaris, Nosema pernyi and sterile water were used as material. The gene expression change of lysozyme were analyzed on the haemolymph from the pupae of A. pernyi induced by different exogenous substances by using real-time fluorescence quantitative PCR. The results showed that in four induced treatment groups, lysozyme expression of haemolymph were up-regulated rapidly from4to8hours. And the highest expression levels can be obtained at24hours in female pupae and from8to12hours in male pupae. The A. pernyi lysozyme expression level was up-regulated rapidly and short duration induced by E. coli. However, the A. pernyi lysozyme expression levels were increased by using C. militaris and N. pernyi as the inducers. It was shown that there were some differences in immuned response time and relative gene expression levels in A. pernyi induced by the different inducers, and there were differences between male and female individuals in A. Pernyi.