Cloning And Sequence Analysis Of Slpi Apoptosis Protease Activating Factor--Apaf-1

Apoptosis, or programmed cell death, is the spontaneous death process which will happen after physiological and pathological stimulation. Apoptosis is an active, highly ordered, gene controlled and a series of enzymes involved process. Now, apoptosis is a hot spot of research. The studies with nematodes is the pioneer of cell apoptosis. The mechanism of apoptosis in insects and mammals is similar, but there are also some differences. Three major pathways of cell apoptosis is generally accepted by people now. They respectively are mitochondria pathway, endoplasmic reticulum pathway, and death receptor pathway.The three pathways are not separated.They regulate cell apoptosis together. In this study, we have cloned Spodoptera litura Apaf-1gene full-length cDNA,in hopes to further explore the role of the Apaf-1gene in cell apoptosis, and further study the interactions of a variety of protein in apoptosome.In this study, according to the highly conserved genomic sequence of Bombyx mori Apaf-1cDNA,partial CDS sequence of Manduca sexta Apaf-1and Danaus plexippus putative Apaf-1sequence, we designed and synthesized some pairs of specific primers. The cDNA encoding Apaf-1of Spodoptera litura was amplified from the total RNA extracted from SL-1cells or SL-1worm by RT-PCR technique. By the way, SL-1cell was treated with actinomycin D for12h to induce cell apoptosis.Firstly, we get a778bp fragment of Apaf-1gene. And then,based on this sequence, we designed a number of specific primers respectively in order to extend3’and5’end fragment of Apaf-1gene.We get a582bp fragment in5’end of Apaf-1gene and a3057bp fragment in3’ end of Apaf-1gene. Finally, based on this fragment, pairs of3’-and5’-RACE specific primers were designed. We cloned5’end and3’end of Spodoptera litura Apaf-1by SMART RACE. By sequence spicing, the full length cDNA sequence of Apaf-1was got. The full length cDNA was5246bp which contains189bp untranslated region of5’end and413bp untranslated region (not including poly A sequence) of3’end.According to the analysis,we know the sequence contains an ORF (open reading frame) consisted of4626bp and encoded1541amino acids. We forecast that the molecular weight of the protein is about175.8KD. The amino acid sequence of Spodoptera litura Apaf-1was submitted to NCBI to conduct proteins and protein (BLASTP) homologous comparison. The result of corresponding sequence comparisons showed that the sequence had the highest homology with Bombyx mori Apaf-1,the homo logy rate was62%, and the sequence was also homologous with some other species.Molecular phylogenetic tree of12Apaf-1amino acid sequence from12species were constructed by MEGA3.1.Result showed that Spodoptera litura and Bombyx mori Apaf-1was exactly the same.It had high homology rate with mammal,too.Protein structure prediction and function analysis of Spodoptera litura Apaf-1by SMART illustrates that there is a CARD sequence at azyl end of Apaf-1protein. NB-ARC domain follows CARD. The protein contains five WD40domains.However, no signal peptide sequence is found. The successful clone of Spodoptera litura Apaf-1gene play an important role in the further study of Spodoptera litura Apaf-1gene structure, gene expression and regulation and its effect on apoptosis.