| Apoptosis is a universal mechanism of cellular suicide that plays an important role in normal development and homeostasis, which is conserve in evolution of animals. The regulations of apoptosis have been studied extensively in model species of nematodes, fruit flies and mice, while similar studies in other insects, especially in mosquitos and Tribolium castaneum, lag far behind. This study is based on lap antagonist- Iap- Caspase regulatory pathway in insect. The combination methods of bioinformatics and molecular biology were taken in functional analysis of Culex tritaeniorhynchus lap and its antagonist. Meanwhile, we had successfully expressed T. castaneum iap gene (Tciap) in prokaryotic and eukaryotic systems, and analyzed the relationship of spatial structure and functional activity between C. tritaeniorhynchus lap antagonist Michelob_x (Ctmx) and Tciap1 and Tciap2.In this study, Ctmx was found to have cell-killing activity, just like the Mx from Anopheles gambiae and Culex quinquefasciatu. Further apoptosis detection indicated that Ctmx can induce apoptosis of C6/36 cells, which appeared typical apoptosis characteristics. Not only homologous species Ctiap1. but also heterologous species of Drosophila Diapl and Tciaps can inhibit apoptosis induced by Ctmx, suggesting that the spatial structure of combination between Mx and different species laps are very conservative. Mx three-dimensional structure was predicted by the method of bioinformatics. To change the spatial structure of Ctmx, a series of truncations deleted alpha helix were constructed, and then were transfected into C6/36 cells, respectively. The results showed that the stability of spatial structure maintained by three alpha helixs of Ctmx are necessary for its apoptosis-promoting activity, although the N-terminal IBM motif (IAP-binding motif) of Ctmx is essential to its function, result from no cell-killing function of the truncation deleted the IBM motif. However, we also found that the Agmx of An. gambiae kept the cell-killing activity, after the first alpha helix of the C-terminal Agmx was deleted. Hence, the study on three-dimensional structure and function of the mosquito Mx will not only provide a new way for the synthesis of short peptide with high efficient cell-killing activity in vitro, but also provide a new direction to create new biological insecticides.Tciap1 and Tciap2 showed anti-apoptosis function to inhibit apoptosis induced by Ctmx and Cqmx in C6/36 cells. To define the key domain within Tciapl/2 that is responsible for the anti-apoptotic, a series of truncations were constructed for transfection. The BIR2 of Tciap1 was found to play a key role in the function of anti-apoptotic, while the BIR1 and BIR3 of Tciap2 were both key domains to maintain the function of anti-apoptotic, consistent with phylogenetic analysis of BIR domains.In this paper, we also investigated the capacity that laps inhibited apoptosis induced by virus. It was found that Tciapl can suppress the apoptosis induced by AfMNPV in SL-ZSU-1 cells.In prokaryotic systems we successfully expressed and purified Tciapl and Ctmx proteins, making foundation for the research of the interaction between lap and Mx for the future.In a word, we preliminarily investigated the relationship between spatial structure and function of laps and its antagonists, which will help us to combine lap regulatory pathways and biological control, and provide a potential new target to create biological insecticides or new mosquitocide. Therefore, this research has important theoretical significance and potential application value. |
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