| Apoptosis is a course which plays a central role in development and homeostasis of all multicellular organisms, DNA damage reaction and many types of human pathologies, including cancer, ischemia, autoimmune diseases, as well as neurodegenerative disorders. The IAP antagonist-IAP-cysteine protease pathway was reported to be one of major regulation in mosquito apoptosis. Mosquitoes have extensive distribution in the world, and they are the media of many epidemics such as malaria, yellow fever, dengue fever and various cephalitis. The research on pathway of mosquito apoptosis focused more on apoptosis caused by virus infection, infectious diseases and so on, while there is few research on relationship between spatial structure and function of IAP antagonist. The molecular biological studies of IAP and its antagonist of Culex tritaeriorhynchus has not been reported. So the research of apoptosis regulatory factors in mosquito will inspire a novel idea in the area of biological control of mosquitoes, and it has a promising prospect in the building of a resource-conserving and environment-friendly society today.The combination of bioinformatics and molecular biology was taken in this work. Inhibitor of apoptosis proteins (iap) genes and mx genes (the antagonist of iap) from C tritaeriorhynchus and Culex quinquefasciatu were cloned and sequced respectively. The Ctiap gene from C. tritaeriorhynchus is1230bp in length, and two iap gene called Cqiapl and Cqiap2from C. quinquefasciatu are1230bp and1518bp in length, respectively. Both putative proteins of Ctiap and Cqiap1contain2BIR domains and1RING finger domain, as well as code the same409amino acids, while Cqiap2containes3BIR domains,1RING finger domain, and encodes505amino acids. Ctmx gene and Cqmx genes are312bp in the same length, encoding103amino acids. The putative proteins of Ctmx and Cqmx are highly similar to the Mx protein of the published C. quinquefasciatu (Gene ID:6034303). There are only4amino acids differences between CtMx and Mx, and their similarity is96%; while CqMx and Mx have3amino acids differences, the similarity is97%. Both Mx proteins from these two mosquitoes contain an invariable N-terminal IAP-binding motif (IBM) that is almost identical to that of the Drosophila Grim protein, but alignmental Mx sequences outside the IBM region showed little similarity. To study the function and interaction of cloned IAPs and its antagonists, we transfected the iap and mx gene into Aedes albopictus C6/36cells, and the survival percentage of these cells were detected by MTT method. The result showed that cloned Ctmx and Cqmx could result in significant cell death, identical with the function of reported mx gene from Aedes aegypti. For functional analysisi of cloned lap genes, mx and iap plasmids of C. tritaeriorhynchus were mixed with the ratio of1:1,1:3,1:5,1:7, separately, then cotransfected into C6/36cells. The result showed that survival percentages of cells raised from the ratio of1:1to1:3, and survival percentages of cells with the ratio of1:3,1:5,1:7have not significant diffirence with that of vector control, suggesting that Ctiap could completely resume the cell killing of Ctmx. To further study the function mechanism of Mx, the3D structure of Ctmx was predicted by relative software. The result showed that except for IBM, Ctmx contains3alpha helixes. We have cloned the fragements which contain one alpha helix and two alpha helix, and then transfected into C6/36cells, respectively. The results indicate that there is no significant difference of cell mortality among them and complete Ctmx, while when the fragement without the IBM was transfected into C6/36cells, cell mortality greatly reduced, indicating that its proapoptotic activity is depended on IBM. The clone only containing IBM was undergoing.iap and mx genes from C. tritaeriorhynchus and C. quinquefasciatu were cloned and fuctional analysis, furthermore relationship between spatial structure and function of IAP antagonist were performed in this text. Our work will not only enrich the study on mosquito apoptosis, but also provide a potential new target for the research and development of biological insecticides and new mosquitocide. |
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