Dissertation > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)

Study on Cloning and Expression of Calcium Activated Potassium Channel Gene from Turbot (Scophthalmus Maximus L)

Author HanGuang
Tutor HuangXiaoHang
School First Institute of Oceanography of State Oceanic Administration
Course Marine organisms
Keywords Calcium-activated Potassium channel Turbot Clone Gene Expression
Type Master's thesis
Year 2007
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Ion channels are located in the cell membrane surface of a membrane protein, and to provide a path for the transmembrane transport of various inorganic ions. The main function of the ion channels in cells, start by opening and closing the channel control ion and out of the intracellular signal transduction system, activation of intracellular physiological activities. Ion channels regulate the activation and proliferation of immune cells in the animal immune response process, start the cellular immune response. Therefore, to maintain the normal function of the immune system of the animal plays a very important role of ion channels. Long-term study found that the ion channel is the target of a variety of new drugs, and therefore the study of ion channels is always the forefront of the field of the biomedical community. Calcium-activated potassium channels (KCa) is located in the cell surface of immune cells in regulating cell function of an important class of channels. By comparison in GenBank different KCa genes homologous similarity, design-specific primers, China's marine aquaculture turbot (Scophthalmus maximus L) blood cDNA as a template to clone a gene fragment by RT-PCR. Using bioinformatics approaches were compared and analyzed the gene fragment sequences confirmed the the fragment case of small-conductance calcium-activated potassium channel gene (SKCa) family. Design nested PCR primers by 5 `race clone the 5 'end of the gene sequence, through the 3` race cloning the 3 `end of the gene sequence, three sequences synthesized by biology software stitching turbot TSKCa The full-length sequence of the gene. The the TSKCa gene sequence of full-length 1698bp, the length of the open reading frame of 1632bp, encoded TSKCa protein of 544 amino acids. Bioinformatics software will the protein already SKCa TSKCa proteins in GenBank for homology analysis, phylogenetic trees, to use SeqFact of transmembrane TSKCa protein structure prediction and DAS server, and the basis of the findings speculated TSKCa the structure of proteins, to infer the TSKCa protein by S1-S6 six transmembrane domains and a pore region (P region). In view of the fish immune response SKCa gene studies have reported few cloned TSKCa gene in large turbot expression of different organs and tissues, and expression anguillarum infection cases studied found TSKCa gene in are distributed in various tissues and organs in the majority of subjects turbot, infection anguillarum TSKCa gene expression in various tissues and organs showed a clear upward trend, which in the brain, head kidney, spleen, and blood in the expression amount increased significantly. The relative expression level changes in the liver and intestine smaller that TSKCa gene in turbot immune defense, may play an important role in resistance to pathogen infection process.

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