题目: Molecular cloning and expression analysis of an apoptosis-associated gene Daxx from zebrafish,Danio rerio

作者: Lin Qi ,Zhiming Xiang*

刊物: Fish & Shellfish immunology

刊号: 2015,45:59-66

摘要: The death domain-associated protein Daxx exerts many functions including the induction and inhibition of apoptosis, regulation of chromatin remodeling and gene transcription. In this report, we have cloned and characterized a Daxx ortholog from the zebrafish, Danio rerio. The bioinformatics analysis results indicated that the open reading frame (ORF) of zebrafish Daxx is 2,151bp long and encodes a putative protein of 716 amino acids containing Daxx domain. Though quantitative PCR analyses, Daxx mRNA was detected in embryonic development from 6 h to 120 h and in all 11 selected zebrafish tissues, and the expression of Daxx was increased first and then decreased during megalocytivirus infectious spleen and kidney necrosis virus (ISKNV) infection. Fluorescence microscopy indicated that the full-length protein was located in the nuclei of the tested Hela cells uniformly but punctiform distribution in HEK293T. In the luciferase report assays, the GAL4-Daxx fusion protein inhibited the transcriptional activity of L8G5- Luc reporter gene showed that Daxx might act as a transcriptional repressor, following the overexpression in HEK293T, the activation of NF-kB-Luc and p53/p21-Luc reporter genes were repressed by the protein. These results suggested that Daxx might play definite role in apoptosis and innate immunity in zebrafish.

题目：ChAkt1 involvement in orchestrating the immune and heat shock responses in Crassostrea hongkongensis:Molecular cloning and functional characterization

作者: Wang F, Xiao S, Zhang Y, Zhang Y, Liu Y, Yan Y, Xiang Z*, Yu Z*.

刊物: Fish & Shellfish immunology

刊号: 2015,47:1015-1023

摘要: G-protein-coupled receptors (GPCRs) are the largest class of cell-surface receptors and play crucial roles in virtually every organ system. As one of the major downstream effectors of GPCRs, Akt can acquire information from the receptors and coordinate intracellular responses for many signaling pathways, through which the serine/threonine kinase masters numerous aspects of biological processes, such as cell survival, growth, proliferation, migration, angiogenesis, and metabolism. In the present study, we have characterized the first Akt1 ortholog in mollusks using the Hong Kong oyster, Crassostrea hongkongensis (designed ChAkt1). The full-length cDNA is 2223 bp and encodes a putative protein of 493 amino acids that contains an amino-terminal pleckstin homology (PH) domain, a central catalytic domain, and a carboxy-terminal regulatory domain. Quantitative real-time PCR analysis showed that ChAkt1 mRNA is broadly expressed in various tissues and during different stages of the oyster's embryonic and larval development. Upon exposure to two stressors (microbial infection and heat shock), the expression level of ChAkt1 mRNA increases significantly. Furthermore, ChAkt1 is located in the cytoplasm in HEK293T cells, where the over-expression of ChAkt1 regulates the transcriptional activities of NF-kB and p53 reporter genes. Taken together, our results indicate that ChAkt1 most likely plays a central role in response to various stimuli in oysters and has a particular response to microbial pathogens and high temperature.

题目：CgIκB3,the third novel inhibitor of NF-kappa B (IκB) protein,is involved in the immune defense of the Pacific oyster,Crassostrea gigas.

作者: Xu,F.,J. Li,Y. Zhang,X. Li,Y. Zhang*,Z. Xiang,Z. Yu*

刊物: Fish & Shellfish immunology

刊号: 2015,46:648-655

摘要: Inhibitor of NF-kB (IkB), the important regulator of NF-kB/Rel signaling pathway, plays the crucial role in immune response of both vertebrates and invertebrates. Here, a novel homologue of IkB was cloned from Crassostrea gigas, and designated as CgIkB3. The complete CgIkB3 cDNA was 1282 bp in length, including a 942 bp open reading frame (ORF), a 51 bp 50 UTR and a 289 bp 30 UTR. The ORF encodes a putative protein of 313 amino acids with a predicted molecular weight of approximately 34.7 kDa. Sequence analysis reveals that CgIkB3 contains a conserved degradation motif but with only five ankyrin repeats. Neither a PEST domain nor a C-terminal casein kinase II phosphorylation site was identified through either alignment or bioinformatic prediction. Phylogenetic analysis suggested that CgIkB3 shares common ancestor with CgIkB1 rather CgIkB2, and theoretically it may originate from one duplication event prior to divergence of CgIkB1 and CgIkB2. Tissue expression analyses demonstrated that CgIkB3 mRNA is the most abundant in gills and heart. The expression following PAMP infection showed that CgIkB3 was significantly up-regulated in a similar pattern when challenged with LPS, HKLM or HKVA, respectively. Moreover, similar to CgIkB1 and CgIkB2, CgIkB3 can also inhibit Rel dependent NF-kB activation in HEK293 cells in a dose-dependent manner. In summary, these findings suggest that CgIkB3 can be as the functional inhibitor of NF-kB/Rel and involved in the host defense of C. gigas. The discovery of the third IkB emphasizes the complexity and importance of the regulation on NF-kB activation.