| Summary: | 碩士 === 國立成功大學 === 生物科技研究所碩博士班 === 95 === Myostatin (GDF-8) is a secreted growth and differentiation factor which belongs to TGF-ß superfamily. It is a negative regulator for muscle growth. Animals with mutations of myostatin have marked double-muscling phenotype. In the aspect of myostatin functional assay in orange-spotted grouper (Epinephelus coioides), we have created double-muscling groupers by using immuno-depletion technique to block the grouper’s endogenous myostatin. Our results showed that grouper’s myostatin also possesses the function of inhibiting muscle development. Furthermore, by using histological analysis we had proved that the grouper’s double-muscling phenotype is due to hypertrophy of myofibers.
In the aspect of myostatin promoter regulation analysis, after proving the myostatin promoter had activity, we then used series truncation technique to determine the most important regulatory position of myostatin promoter and found that it was located in E6 and E1 elements of myostatin promoter. In addition, In GF-1 cells in vitro assay, we had identified two factors that can regulate myostatin promoter activity. One is environmental stress such as Nervous necrosis virus (NNV) infection, the other is serum concentration. NNV infection can down-regulate myostatin promoter activity, and this mechanism was probably due to the effect of virus dsRNA during virus replication. On the other hand, high serum concentration can up-regulate myostatin promoter activity. According to these results, grouper’s myostatin gene expression is influenced by viral infection and serum concentration.
Myostatin is a secreted protein. We also determine whether myostatin contains auto-regulation function. By using western blot analysis, we found that grouper’s myostatin can be secreted to outside of the cell and we also identified the probable secretion signal was located within 1-20 of the polypeptide chain. Besides, we had co-transfected two constructs, myostatin gene and a reporter gene luciferase driven by myostatin promoter, into GF-1 cells. Our results showed that myostatin promoter activity can be down-regulated. Adding recombinant myostatin to GF-1 cells culture medium also got the same results. These indicated that grouper’s myostatin possesses auto-regulation function, and myostatin promoter can be feedback inhibited.
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