Evolutionary and functional analysis ofCyclina sinensis c-Jun AP-1 gene in response to LPS stimulation

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The transcription factor activator protein-1 (AP-1) plays an essential and critical role in the regulation of numerous downstream genes involved in various physiological and chemical responses. In this study, we identified a full-length cDNA of the c-Jun AP-1 gene (termed Csc-Jun) from the transcriptome library in Cyclina sinensis. The cDNA contains an 825-bp open reading frame that encodes a 274-amino acid protein sequence, including a characteristic Jun transcription factor domain and a highly conserved basic leucine zipper (bZIP) signature that shares 90% identity to that of Ruditapes philippinarum. Furthermore, a phylogenetic analysis using MrBayes and PhyML software (with Bayesian and maximum likelihood approaches, respectively) revealed that the c-Jun AP-1 family genes might be involved in adapting to various environments in different invertebrates. We implemented the PAML software with the maximum likelihood method to further select and verify the positive selection sites (PSSs) in the Mollusca c-Jun AP-1 genes, and we detected four PSSs located in the Jun transcription factor domain. In addition, a spatial expression analysis showed that the Csc-Jun cDNA transcript was ubiquitously expressed in all of the tested tissues and was strongly expressed in the hepatopancreas and weakly expressed in the tissues of the hemocytes, gill filaments, mantle and adductor muscle. Quantitative real-time PCR showed that the expression profiles of Csc-Jun were significantly upregulated at different times in all of the tested tissues when challenged with lipopolysaccharide (LPS). Furthermore, knockdown of Csc-Jun by RNA interference resulted in a higher mortality of C. sinensis following LPS exposure. Finally, we explored the function of the TLR13-MyD88 signaling pathway in the innate immunity of C. sinensis by RNA interference and immune challenges. The results revealed that the mRNA expression levels of Csc-Jun were all decreased (P<0.01) in normal and stimulated C. sinensis hemocytes. These data collectively indicated that the c-Jun AP-1 gene might play vital roles in innate immunity and provide new evidence for the evolutionary patterns of innate immune genes in Mollusca.

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