The Function Of Cathepsins In Immune Response And Metamorphosis Of Antheraea Pernyi

Cathepsins are a group of proteases located in lysosome and play many vital roles in many physiological processes including individual development of insects.As an important lepidoptera wild silkworm,Antheraea pernyi was lived outdoors and more likely to be influenced by the environmental conditions as well as other insects and pathogens.Therefore,the research on development and immune process was necessary for further understanding of the related metabolic processes of Antheraea pernyi.In the present study,cathepsin O(Ap-Cathepsin O)and cathepsin L-like(Ap-Cathepsin L-like)were identified from Antheraea pernyi,also their functions in immune process and metamorphosis were investigated.The results of the experiments were listed as follows:1.Cloning,prokaryotic expression and antiserum preparation of Ap-Cathepsin O and Ap-Cathepsin L-like.The open reading frame(ORF)of Ap-Cathepsin O is 1170 bp,encodes a protein with 390 amino acid residues,including a conserved 129 inhibitor domain and a peptidase CIA(clan CA of cysteine proteases,papain family C1 subfamily)domain.Multiple sequence alignment and phylogenetic analysis showed that Ap-Cathepsin O was more similarly and closely related to B.mori cathepsin O.The ORF of Ap-Cathepsin L-like is 1155 bp,encodes a protein with 385 amino acid residues.The sequence analysis indicated that Ap-Cathepsin L-like had no signal peptide,and the tertiary structure showed that there were two different active sites(Asp 179,Ser185)existed compared with B.mori cathepsin L.The expression vector Pet-28a(+)-Ap-Cathepsin O and Pet-28a(+)-Ap-Cathepsin L-like were constructed and the recombinant proteins were expressed by IPTG induction.The recombinant proteins were purified using nickel column and then used to prepare the rabbit polyclonal antibodies for further research.2.Expression distribution of Ap-Cathepsin O and Ap-Cathepsin L-likeExpression levels of Ap-Cathepsin O and Ap-Cathepsin L-like were determined in different tissues of the third day of the fifth instar larvae.The results showed that Ap-Cathepsin O and Ap-Cathepsin L-like were expressed in all the examined tissues with high expression levels in fat body.The expression patterns of target genes in different development stages showed that Ap-Cathepsin O was expressed higher in pupae and moth stages,and Ap-Cathepsin L-like was highly in molting and puape stages.These results indicated that Ap-Cathepsin O and Ap-Cathepsin L-like may play roles in metabolic process and metamorphosis.3.Expression patterns of Ap-Cathepsin O under pathogens and 20E challenges.The immune challenges using M.luteus,E.coli,AP-NPV,B.bassiana were performed to analyze the expression patterns of Ap-Cathepsin O in the third day of the fifth instar larvae.The results showed that Ap-Cathepsin O was signaficantly upregulated up to 10 times compared with control in fat body and hemocytes with different expression patterns,indicating Ap-Cathepsin O was involved in immune process of Antheraea pernyi.20E was injected to analyze the expression patterns of Ap-Cathepsin O under hormone challenge in pupal stage.The results showed that the expression of Ap-Cathepsin O was increased 4 times and then down-regulated,indicating Ap-Cathepsin O was related with the hormone regulation.4.Ap-Cathepsin L-like is involved in the 20E regulated apoptosis process in metamorphosis.he expression level of Ap-Cathepsin L-like was signaficantly upregulated under 20E injection from 24 h to 72 h.Expression level of apoptosis related genes in pupal stages were determined after dsRNA injection and the results showed that the caspase-3(a apoptosis effector gene)was decreased and the Bcl-2(a apoptosis inhibitor gene)was not changed significantly.These results indicated that Ap-Cathepsin L-like participated in the 20E-regulated apoptosis process.5.Ap-Cathepsin L-like is involved in the fat body dissociation process in pupal stage.Ap-Cathepsin L-like was knocked down by RNAi in fat body cultured in vitro and then the dissociated fat body cells were counted.The results showed that the dissociated fat body cells were significantly decreased after Ap-Cathepsin L-like RNAi compared with the GFP RNAi group,indicating Ap-Cathepsin L-like may involve in the fat body dissociation process in pupal stage.