Cloning, Expression and Functional Analysis of TRAF4 Gene in Nile Tilapia (Oreochromis niloticus)
HAN Xue-Qing1,2, GAO Feng-Ying1,*, LU Mai-Xin1,*, LIU Zhi-Gang1, CAO Jian-Meng1, WANG Miao1, YI Meng-Meng1, ZHANG De-Feng1
1 Pearl River Fisheries Research Institute, Key Laboratory of Tropical ＆Subtropical Fishery Resource Application ＆Cultivation, Ministry of Agriculture, Chinese Academy of Fishery Science, Guangzhou 510380, China; 2 College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China
Abstract：Tumor necrosis factor receptor associated factors 4 (TRAF4) is a special member of the TRAFs family, and its biological functions are numerous and complex. In order to investigate the immune function of TRAF4 gene in Nile tilapia (Oreochromis niloticus), the cDNA sequence (GenBank No. MH986338) and genomic sequence of TRAF4 gene were obtained by reverse transcription PCR and fragment amplification. In addition, the biological information such as gene and protein structure were also analyzed. Then qRT-PCR was used to detect the tissue distribution of the gene in healthy individuals, the expression of the gene at different developmental stages after fertilization, and the expression characteristics of the gene after artificial infection with Streptococcus agalactiae. Finally, the recombinant eukaryotic expression vector was constructed to analyze the subcellular localization of TRAF4 in 293T human embryonic kidney cells and its role in nuclear factor κB (NF-κB) pathway. The results showed that the TRAF4 cDNA was 4 483 bp in length which contained an ORF of 1 413 bp encoding 470 amino acid residues. There were 7 exons and 6 introns among the genomic sequence. The putative protein of TRAF4 contained 3 characteristic domains conserved in the TRAF family, including 1 N-terminal RING-finger, 3 zinc-fingers and 1 C-terminal meprin and TRAF-homology (MATH) domain. To examine the basal expression of TRAF4, qRT-PCR analysis was carried out in brain, gill, liver, spleen, intestine, heart, kidney, stomach, skin, muscle, and blood. To eliminate the individual variation, 6 fishes were sampled and analyzed separately by qRT-PCR, and their mean values were considered. The results showed that the expression of TRAF4 was detected in all the examined tissues. And the highest expression level of TRAF4 was detected in brain, whereas the lowest expression was detected in spleen and blood. The expression at different developmental stages after fertilization was also detected and each assay was performed in triplicate. The results showed that the expression of TRAF4 was detected in all the developmental stages. qRT-PCR was also used for TRAF4 expression when Nile tilapia were challenged with Streptococcus agalactiae. In the infected fish gill and blood, TRAF4 gene expression significantly increased and reached its peak at 72 h post infection (hpi). However, the expression levels of TRAF4 in the intestine and kidney were all down-regulated when compared with their own control group (0 h group). The TRAF4 ORF was amplified and subcloned into the plasmid vector (pcDNA3.1/CT-GFP-TOPO®). Then the result of subcellular localization showed that TRAF4 protein distributed mainly in the cytoplasm, and overexpression of TRAF4 could significantly increase the NF-кB activity in 293T cells. The TRAF4 gene might play an important role in the immune response of Nile tilapia. This study provides basic data for further investigation of the TRAF4 gene and understanding anti-infective immune mechanism of Nile tilapia.
韩雪晴, 高风英, 卢迈新, 刘志刚, 曹建萌, 王淼, 衣萌萌, 张德锋. 尼罗罗非鱼TRAF4基因的克隆、表达及功能分析[J]. 农业生物技术学报, 2019, 27(3): 381-392.
HAN Xue-Qing, GAO Feng-Ying, LU Mai-Xin, LIU Zhi-Gang, CAO Jian-Meng, WANG Miao, YI Meng-Meng, ZHANG De-Feng. Cloning, Expression and Functional Analysis of TRAF4 Gene in Nile Tilapia (Oreochromis niloticus). 农业生物技术学报, 2019, 27(3): 381-392.
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