Gene Cloning and Primary Functional Analysis of TRIF in Nile Tilapia (Oreochromis niloticus)
LIU Jie1,2, GAO Feng-Ying2, CHEN Gang1,*, LU Mai-Xin2,*, CAO Jian-Meng2, LIU Zhi-Gang2, WANG Miao2, HAN Xue-Qing2,3
1 College of Fisheries, Guangdong Ocean University, Zhanjiang 524025, China; 2 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; 3 College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China
Abstract：As an adaptor in Toll-like receptor (TLR) signaling pathway, TIR domain-containing adaptor inducing interferon-β receptor (TRIF) mediates downstream signaling cascades and plays an important role in host innate immune responses. In order to investigate the potential role of TRIF in anti-disease immune response of Nile tilapia (Oreochromis niloticus), the cDNA sequence (GenBank No. MT199561) of TRIF gene was obtained by reverse transcription PCR (RT-PCR) and fragment amplification, and bioinformatic analysis was also performed. Real time quantitative PCR (qRT-PCR) was used to detect TRIF expression in healthy individuals and those with intraperitoneal injection of Streptococcus agalactiae, lipopolysaccharides (LPS) and polyinosinic polycytidylic acid (Poly I:C). The recombinant eukaryotic expression vector was constructed to analyze the subcellular localization of TRIF in 293T human (Homo sapiens) embryonic kidney cells and the activation of nuclear factor κ B (NF-κB). The results showed that TRIF cDNA was 3 135 bp in length which contained an ORF of 1 653 bp encoding a polypeptide with 550 amino acid residues. The deduced amino acid sequence contained Toll/IL-1 receptor (TIR) domain. In the phylogenetic tree, Nile tilapia was clustered with other fish and then clustered with zebrafish (Danio rerio) and mammals. The TRIF expression were widespread in all the tested tissues and organs (liver, skin, heart, kindey, stomach, gill, brain, spleen, intestine, blood and muscle), with the highest expression in the muscle and blood. After challenged with pathogenic bacteria S. agalactiae, the TRIF gene expression increased in the spleen and kidney, and then reached the peak at 24 h post infecton (hpi). After infection with LPS, TRIF was up-regulated in the liver and spleen, while down-regulated in the intestine and kidney. TRIF was up-regulated in the intestine and liver, and down-regulated in the kidney after infection with Poly I:C. The result of subcellular localization showed that TRIF protein distributed in the cytoplasm and could significantly increase NF-кB activity. Taken together, the above findings suggested that TRIF gene might play an important role in immune responses of Nile tilapia. This study could provide a reference for further studying on the function of TRIF gene in immunity evolution of teleost.
刘洁, 高风英, 陈刚, 卢迈新, 曹建萌, 刘志刚, 王淼, 韩雪晴. 尼罗罗非鱼TRIF基因克隆及功能初步分析[J]. 农业生物技术学报, 2020, 28(6): 1039-1051.
LIU Jie, GAO Feng-Ying, CHEN Gang, LU Mai-Xin, CAO Jian-Meng, LIU Zhi-Gang, WANG Miao, HAN Xue-Qing. Gene Cloning and Primary Functional Analysis of TRIF in Nile Tilapia (Oreochromis niloticus). 农业生物技术学报, 2020, 28(6): 1039-1051.
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