Molecular Cloning and Expression Analysis of Transforming Growth Factor TGF-β1 and TGF-β3 in Half-smooth Tongue Sole (Cynoglossus semilaevis) Following Stimulation with Vibrio harveyi
Abstract：Transforming growth factor β (TGF-β) superfamily plays critical roles in immune cell functional regulation, cell proliferation and differentiation, tissue wound repair. The half-smooth tongue sole (Cynoglossus semilaevis), with excellent properties such as fast growth, individual large, meat delicious and nutrient-rich, is an important economic marine fish species in the North of China. It has become a new kind of excellent breeding object in recent years. But with the enlargement of the farming scale, a variety of infectious diseases is becoming more and more frequently, especially the bacterial disease such as the Vibrio harveyi infection, fast onset and spread quickly. These diseases bring great economic losses to the aquaculture industry. Therefore, in order to carry out the half-smooth tongue sole immune genetic research and explore its immune response mechanism with Vibrio harveyi infection, the TGF-β1 and TGF-β3 genes were cloned from liver in the half -smooth tongue sole. Sequence analysis showed that TGF-β1 and TGF-β3 amino acid sequence consisted of multiple N-glycosylation sites and a TGF-β family signature. Analysis of phylogenetic tree indicated that TGF-β1 and TGF-β3 of the half-smooth tongue sole had a high homology with that of fish. The qRT-PCR analysis showed that TGF-β1 and TGF-β3 expressed in a diverse array of tissues including heart, kidney, intestine, muscle, skin, brain, gill, liver and spleen of healthy half-smooth tongue sole and there were significant differences. The highest levels of TGF-β1 were detected in skin, the higher levels were discovered in kidney and the lowest levels were found in muscle. TGF-β3 expressed the highest in skin, higher in brain and the lowest in muscle. After the Vibrio harveyi infection, the expression levels of TGF-β1 showed uptrend in liver, peaked at 48 h after infection, with 3.17 times of the control group, then fell to control levels. In the spleen, TGF-β1 and TGF-β3 showed similar expression trend , rising in the first stage and then decreasing. The expression of TGF-β1 and TGF-β3 reached maximum after infection 24 and 12 h, respectively, with 1.70 times and 1.61 times of the control group (P＜0.05). In the kidney, the expression trend of TGF-β1 and TGF-β3 were still similar, both peaked at 12 h after infection, respectively, with 1.23 times and 1.42 times of the control group (P＜0.05), then showed a downward trend. In the gill, TGF-β1 expression quantity did not change significantly comparing with the control group. TGF-β3 expression quantity rose in the first stage and then decreased, peaked at 24 h after infection, with 4.71 times of the control group (P＜0.05). The above results suggested that TGF-β1 and TGF-β3 may play an important role in the immune against bacterial infections in the half-smooth tongue sole. This study provides a strong evidence for the participation of TGF-β1 and TGF-β3 in the immune regulation mechanism and also a theoretical basis for molecular immune research in the half- smooth tongue sole.
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