Molecular Cloning And Functional Analysis Of The Apoptosis Inhibitor BmAPI5 In The Silkworm,Bombyx Mori L.

Apoptosis plays an important role in life activities of organisms,which process is regulated by diverse genes.The apoptosis inhibitor-5?API5?,as a recently discovered apoptosis inhibitor,can effectively block cell apoptosis,but has different structure with the known inhibitors of apoptosis?IAPs?.At present,the studies on API5 have mainly focused on human carcinogenesis because its high level of expression can promote the development of cancer cells.A homologous gene,BmAPI5?GenBank number: XM₀04927989?,was found in the silkworm,Bombyx mori through the blast analysis of gene sequence.However,the role of BmAPI5 in the silkworm has not been reported yet.In this thesis,the full length cDNA of BmAPI5 was cloned,and its bioinformatics,temporal and spatial distribution in both transcriptional and translational levels,and effects on apoptosis in B.mori were analyzed.The main results are shown as follows.1.Molecular cloning and expression of BmAPI5 geneThe full-length cDNA sequence of BmAPI5 gene was cloned,which has an open reading frame?ORF?of 1,671 bp encoding 557 amino acid residues with a molecular weight of 63.13 kD and an isoelectric point of 6.09.The BmAPI5 protein is mainly composed of ?-helix and random coil,which has Lxx LL motif and leucine zipper domain.The similarity of amino acid sequence of BmAPI5 to the homologue Aac11 in Drosophila melanogaster and human was 46% and 47%,respectively.The recombinant BmAPI5 gene was inserted into the expression vector,pET-32 a and expressed in Escherichia coli.The open reading frame of BmAPI5 was cloned into the prokaryotic expression vector pET-32 a,and the recombinant protein was obtained for the preparation of rabbit polyclonal antibody.2.Temporal and spatial expression of BmAPI5 geneThe transcription and expression levels of BmAPI5 gene at different developmental stages,in various tissues and on exposure to microbial infection were determined using both real time quantitative PCR and Western hybridization,respectively.The different levels in both transcription and translation of BmAPI5 were detected in the eggs,larvae in different instar,and pupae,among which the transcriptional level in the 5th instar larvae was significantly higher.The transcription and expression levels of BmAPI5 were significantly higher in the fat body.The infection of Bombyx mori nucleopolyhedrovirus?BmNPV?and E.coli could significantly induce the transcription and expression of BmAPI5 gene,in which the former induction was more obvious.It suggests that BmAPI5 may be involved in BmNPV-host interaction.3.Effects of BmAPI5 gene on apoptosisThe enhanced expression and inhibited expression systems of BmAPI5 in BmN cells were constructed,respectively.The effects of expression variation of BmAPI5 gene on apoptosis of BmN cells were further analyzed using flow cytometry and Caspase assay.The results of flow cytometric determination showed that the enhanced expression of BmAPI5 gene can significantly reduce the level of apoptosis in the BmN cells induced by starvation,while conversely,the inhibited expression results in a significantly higher level of apoptosis in the starvation treated cells.The results of Caspase assay were consistent with the results of flow cytometric assay.This suggests that BmAPI5 may play an important role in inhibiting apoptosis.The results of this study can provide not only a basis for further research of the regulation mechanisms of BmAPI5 on apoptosis,but also new ideas for exploration of BmAPI5 function in BmNPV-host interaction and antiviral prevention in sericulture.