Functions Of Sl-Apaf-1 From Spodoptera Litura In Apoptosis

Apoptosis is a protection mechanism of multicellular organisms to maintain in vivo environment,studying on apoptosis has important significance in biology.And Apaf-1(Apoptotic protease activating factor-1)plays the key role in apoptosis pathway according to the studies on mammals.Apaf-1 mainly effect the mitochondrial apoptosis pathway.In humans,cytochrome C is released from mitochondiral membrane,after binding to Apaf-l,recruit procaspase-9,then form a complex named apoptosome.Hence,trigger cell death.However,if's not clear what the function of Apaf-1 is in lepidopteran insect.A new Apaf-l,named Sl-Apaf-1 from Spodoptera litura has already indentified by our lab.And similar to human Apaf-1,Sl-Apaf-1 also contains three domains,CARD domain,NOD and WD motifs.This study demonstrate preliminarily that Sl-Apaf-1 plays an important role in lepidopteran insect apoptosis.According to known gene sequences of Sl-Apaf-1,we discussed the role of Sl-Apaf-1.First of all,we designed three siRNA,and chose the best one for RNAi assay.We also designed qRT-PCR in order to detect the efficiency of RNAi.Based on RNAi experiment,we induced the cells by ActD and calculated the rate of apoptosis by FACS.Secondly,the overexpression of Apaf-1 also took ActD and FACS for observing the changes of the rate of apoptosis.The whole experiment states that Apaf-1 has a great influence on the rate of insect cell apoptosis when the ActD exist,but can not induce apoptosis by itself.Besides,things are different in Drosophila,which is more evolutionary similar to lepidopteran insect than humans.Cytochrome C seems like not playing in apoptosis pathway here,and Dark,the homologue of Apaf-1,don't need any help from cytochrome C to make apoptosome and to transfer the signals of cell death.However,we have the clue that cytochrome C do be released in the process of lepidopteran insect apoptosis.To make clear the interaction of Sl-Apaf-1 and Cyto.C in insect cells,we designed the Co-located assay and BiFC.And just like the Apaf-1 did in humans,Sl-Apaf-1 can bind cytochrome C in cytoplasm of insect cells.It suggests that insect apoptosis is unlike flies,cytochrome.C is also essntial for this type of cell death.Hence,we conclude that Sl-Apaf-1 is very important to lepidopteran insect apoptosis,maybe even the key protein like human,Apaf-1,and it has a interation with cytochrome.C,to transfer the signals of apoptosis just like human-Apaf-1.This study build a foundation for the further study on lepidopteran insect apoptosis pathway.