Study On Cloning And Expression Of Calcium Activated Potassium Channel Gene From Turbot (Scophthalmus Maximus L)

Ion channel is a membrane protein located on the cell membrane,providing gate for current of ion across cell membrane. The main function of ion channel is to initiate the cell signal system, stimulating various metabolic activities inside the cell. It is always the target of many new drug and is the hot research point in biomedical field. In the immune response of animal, the ion channels regulate the activation and prolixferati on of the immune cell and start immune response of the cell, playing a very important role in keeping normal function of the animal immune system.Calcium activated potassium channel (KCa) is an important channel located on the membrane regulating functions of immune cell. Through comparing the homology of different KCa gene sequences in GenBank, a special set of primers was designed and applied in RT-PCR, an expected segment was obtained using this specific primers and RNA template of the turbot blood. The sequence of cloned segment was identified as a new member of the calcium activated-potassium channel family and named as TSKCa after sequencing and bioinformatics analyzing. The 5'end and 3'end of the TSKCa gene segment were extended using 5' Race and 3'Race approaches. The full-length sequence of this gene was assembled,structural characters and phylogenetic analysis was performed using bioinformatics approaches. The full-length of TSKCa gene is 1698bp.The open reading frame is 1632bp, coding a protein of 544 amino acids. Phylogenetic analysis was conducted between turbot TSKCa protein with those of other species. Transmembrane domain of turbot TSKCa protein was predicted using SeqFacts and DAS Server and structural characters were compared with that of other species, TSKCa protein has a structure of ion channel containing a pore region and six transmembrane domains of S1 to S6. The expression patterns of TSKCa gene in different organs and tissues of turbot were studied.We found TSKCa gene exists in majority of organisms of turbot.After Vibrio anguillarum infection The amount of TSKCa gene increases obviously in brain,headkidney,spleen and blood, but increases slightly in intestines and liver,indicating the important role of TSKCa in immune defense response of the fish.