MOLECULAR CLONING OF TRPV1 IN SPINIBARBUS SINENSIS AND THE RESPONSES TO STIMULATION OF HOT TEMPERATURE AND LPS

TRPV1 is a multifunctional ion channel in mammals, and one of its most critical functions is to sense high temperature. According to the available data, the function of TRPV1 in fish temperature perception is controversial: TRPV1 of zebrafish (Danio rerio) and rainbow trout (Oncorhynchus mykiss) can sense heat, while TRPV1 of other fish like ice-fish (Chionodraco hamatus) and Antarctic fish (Trematomus bernacchii) are reported to be insensitive to temperature changes. In order to test whether the expression of TRPV1 in freshwater fish in China is sensitive to temperature, Spinibarbus sinensis was selected as the experimental animal in the present study. TRPV1 gene was cloned by RT-PCR, and then bioinformatic analyses were carried out to predict the secondary and tertiary structure or TRPV1 protein. Tissue expression profile of this gene was also studied by RT-qPCR. Since mammalian TRPV1 can sense both the environmental temperature and the inner body inflammation, we detected the expression of TRPV1 mRNA in the diencephalon and the medulla oblongata of S. sinensis, which were treated by PBS or LPS injection and then put in water of different temperatures (25℃ and 35℃) for 1h, 6h and 24h.　　In the present study, the cloned fragment of TRPV1 gene is 2023 bp in length, encoding a peptide consisting of 673 amino acid residues. The predicted TRPV1 protein of S. sinensis shares a highest similarity of 88.80% with that of common carp (Cyprinus carpio), and it contains a predicted Ankyrin repeat domain (ANK), 6 transmembrane domains, and an ion transmembrane domain (Ion_trans). Based on the online SWISS-MODEL, the functional protein of TRPV1 in S. sinensis might be a homotetramer. The TRPV1 gene of S. sinensis is widely expressed in various tissues, including all parts of brain (telencephalon, mesencephalon, diencephalon, cerebellum, medulla oblongata), and internal organs such as kidney, liver, spleen and gut. However the expression levels in the skin and muscle are very low. High temperature and LPS injection can induce the significant changes of this gene in both the diencephalon and the medulla oblongata, however there are opposite alteration patterns in these two tissues, in a time specific manner. Taking all the above-mentioned information together, it is clear that the expression of TRPV1 in S. sinensis is temperature and inflammation sensitive, with a tissue and time specific pattern.