| Summary: | 碩士 === 國立臺灣海洋大學 === 生物科技研究所 === 94 === Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family that includes NGF, NT3, NT4/5 and NT6. BDNF play a critical role in the regulation of neuron survival and growth. BDNF gene has a complex structure with multiple regulatory elements and multiple promoters that are differentially expressed in central or peripheral nerve tissues. In this study, we have designed primers for the amplification of BDNF exon 2 and used this DNA fragment as a probe to screen the zebrafish genomic library. We obtained three BDNF clones from zebrafish genomic library. After genomic structure analysis, we found that one of the BDNF clones included 20 kb insert, which contained the BDNF multiple promoter regions. To analyze the promoter activity of BDNF-1C, we designed primers to amplify a 4 kb promoter region and ligated this DNA fragment into pEGFP-1 vector. The resulting plasmid was named pBDNF-1C EGFP. After transfection of pBDNF-1C EGFP into grouper brain (GB3) and grouper heart (GH) cells, the expression of EGFP was observed, which revealed BDNF-1C promoter functions in the two cells. By microinjection of pBDNF-1C EGFP into zebrafish fertilized eggs, the expression of EGFP was also observed in muscle, gut, epidermis cell and somite of 72-hr embryos. Whole mount in situ hybridization was performed to further confirm the expression of BDNF during zebrafish development. The expression of BDNF was observed in brain, tail, muscle and common cardinal vein.
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