The Effects Of IIS Signaling Pathway On Cell Apoptosis And Proliferation In The Metamorphosis Of Haliotis Diversicolor

Haliotis diversicolor is an economic shellfish with settlement and metamorphosis in its life cycle.On the base of H.diversicolor transcriptome database,we have cloned the full-length cDNA sequences of insulin induced protein 2 and insulin-degrading enzyme,which were named HdINSIG2 and HdIDE,by SMART-RACE.qRT-PCR,prokaryotic expression,ELISA,RNAi and in situ hybridization were applied to reveal the role of HdINSIG2,HdIDE,HdIGFBP7 and HdMIRP on cell apoptosis and proliferation in larva of H.diversicolor.1)The full-length cDNA of HdINSIG2 is 1400 bp,with 123 bp 5’UTR,677 bp 3’UTR and600 bp of ORF.The deduced protein is 199 amino acids,with 5 transmembrane domains.The qRT-PCR results showed that the expression level of HdINSIG2 was high relatively in cleavage stage of larvae,also higher than post embryonic stage（P<0.05）.2)The full-length cDNA of HdIDE is 4244 bp,with 118 bp 5’UTR,1054 bp 3’UTR and3072 bp ORF.The deduced protein is 1023 amino acids with a typical peptidase M16 domain and two peptidase M16C domains.In addition,it has a highly conservative motif of HxxEH,a Zn²⁺catalytic site and a combine site with substrate.The qRT-PCR showed HdIDE was expressed in all embryo developmental stage,with an expression pattern of high-low-high-low-high.3)The prokaryotic expression vector of pGEX-4T-2-HdIGFBP7 and pGEX-4T-2-HdMIRP were successfully constructed and transformed into E.coli BL21.After inducing with IPTG,we detected the expression of recombinant HdIGFBP7 and HdMIRP proteins with SDS-PAGE,then we obtained target protein of about 51 kDa and 39 kDa,respectively.Target proteins were purified by Ni affinity chromatography column.The polyclonal antibody of Hd IGFBP7 and HdMIRP were prepared and the antibody titers were 51200 and 6400 respectively.4)The experiment of RNAi showed that the settlement rate and mortality rate of the larvae were not observably changing compared with the control group while adding dsRNA of HdINSIG2 and HdIDE（P>0.05）.When added GABA,the settlement rate of the larvae was significantly increased than that of negative control group（P<0.01）,adding the recombinant protein of HdIGFBP7 increased the settlement rate（P<0.05）.and there was not significantly increased when added the recombinant protein of HdMIRP（P>0.05）.Besides the mortality rate of the larvae in group of adding GABA,HdIGFBP7 and HdMIRP were significantly decreased than that of negative control（P<0.05）.5)Apoptosis and cell proliferation of the larvae were detected by method of in situ hybridization.The color position and depth of HdCaspase3,HdH3 and HdPCNA had no significant differences between adding HdINSIG2-dsRNA and its control.The expression of HdCaspase3 was detected in the ventral of visceral mass and near the dorsal of visceral mass in adding HdIDE-dsRNA,but the positive signal only detected from the ventral of visceral mass in negative control and blank control group;HdH3 and HdPCNA were detected at the same positions but lighter positive signal than control group.The expression of HdCaspase3 was detected in the ventral of visceral mass and near the dorsal of visceral mass in adding HdIGFBP7and HdMIRP fusion protein,however positive signal only detected from abdominal visceral mass in negative control and blank control group;HdH3 and HdPCNA were detected at the same positions but deeper positive signal than control group.The chromogenic results were similar to group GABA.