Cloning of TRIM25 Gene in Rainbow Trout (Oncorhynchus mykiss) and Its Expression Analysis
LEI Ming-Quan1, HUANG Jin-Qiang1, LI Yong-Juan1,2,*, WU Shen-Ji1, ZHAO Lu1, PAN Yu-Cai1, SUN Tong-Zhen1
1 College of Animal Science and Technology, Gansu Agricultural University, Lanzhou 730070, China; 2 College of Science, Gansu Agricultural University, Lanzhou 730070, China
Abstract：Tripartite motif containing 25 (TRIM25), a member of the TRIM family, plays an important role in the host antiviral immune response. To understand the molecular characteristics of TRIM25 in rainbow trout (Oncorhynchus mikiss) and its expression changes during infection with Infectious hematopoietic necrosis virus (IHNV), in this study, the full-length cDNA sequence of TRIM25 (GenBank No. OL692831) was obtained by rapid amplification of cDNA ends (RACE) technique and bioinformatically analyzed. At the same time, the qPCR technique was used to detect changes in TRIM25 expression in different tissues of healthy rainbow trout and at different time points (0, 6, 12, 24, 48, 72, 96, 120 and 144 h) after infection with IHNV in the intestine, liver, spleen, skin, head kidney and gill. The results showed that the full-length sequence of the rainbow trout TRIM25 cDNA, 4 745 bp, had an ORF of 2 028 bp and encoded 675 amino acids. Bioinformatics analysis showed that the TRIM25 protein had a molecular weight of 76.04 kD, a theoretical pI of 8.79, an instability coefficient of 49.2 and an average hydrophilic coefficient of -0.591, indicating TRIM25 was an unstable hydrophilic protein. Analysis of the structural domains of TRIM25 protein revealed that the protein included RING, B-boxes, B-Box C-terminal domain and PRY-SPRY structural domains. The homology and phylogenetic tree analysis showed that rainbow trout had the highest homology (94.96%) with salmon (Oncorhynchus keta) and was closely related, but had low homology with mammals and amphibians. qPCR showed that TRIM25 was widely expressed in all tissues of healthy rainbow trout, with its highest expression in liver, followed by that in head, kidney and brain, and the lowest expression in skin. After infection with IHNV, the expression of TRIM25 gene in liver, head kidney, spleen, intestine and skin peaked at 48 h, and gill reached the peak at 96 h. Expression of each tissue at the peak was extremely significant compared to the control (P<0.01), with the most significant changes in expression in head and kidney and spleen, which were 5.72 and 4.33 times of the control group, respectively. The above results suggested that TRIM25 played an indispensable role in the immune response of rainbow trout against IHNV infection. This study provides basic data for an in-depth study of the role of TRIM25 in the regulation of antiviral immunity in fish.
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