肌肉倍增基因(myostatin)，其轉譯之蛋白為第八號生長分化因子，屬於TGF-ß superfamily的成員。對肌肉發育而言，Myostatin扮演負向調控的角色，可以藉著抑制其功能，使得生物體產生肌肉倍增的表現型。本論文所研究之點帶石斑魚Myostatin功能分析方面，利用免疫剔除技術抑制石斑魚內生性Myostatin，的確可以使魚隻產生肌肉倍增表現型，證明石斑魚的Myostatin也是具有抑制肌肉生長發育功能。而由組織切片及H&E染色結果，得知石斑魚肌肉倍增表現型是因為肌肉纖維變大所造成。
Myostatin啟動子分析方面，證明所選殖到的啟動子具有活性及方向性之後，再以系列剔除的方式證明啟動子最重要的功能區域，是坐落於E6及E1的兩個調控位置。接著於GF-1細胞實驗探討，其它影響Myostatin啟動子活性的因子，包括神經壞死病毒感染的逆境因素及血清濃度。在神經壞死病毒對Myostatin啟動子的研究中發現，病毒會抑制Myostatin啟動子活性，而且可能是由於病毒複製過程時產生之dsRNA效應所導致的結果。而血清濃度對Myostatin啟動子的研究中發現，高血清濃度會誘導Myostatin啟動子活性。所以點帶石斑魚Myostatin基因的表現量，是會受環境的影響而有不同的結果。
Myostatin是種分泌性的生長調節因子，亦研究Myostatin是否具有自我調控功能。利用西方墨點法證明點帶石斑魚之Myostatin具有被分泌到細胞外之功能，且其訊號因子坐落於前二十個胺基酸序列。再將myostatin基因及由Myostatin啟動子所驅動之luciferase報導基因的兩個構築載體，共同轉染至GF-1細胞內，發現Myostatin啟動子之活性會被抑制。相同的結果，在細胞培養液施加重組Myostatin蛋白，亦會抑制啟動子的活性。證實點帶石斑魚之Myostatin對於其本身之啟動子具有自我調控之負向回饋抑制的功能。

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.

張馨文 (2004) 石斑魚肌肉倍增基因之選殖與應用，國立成功大學生物科技研究所碩士論文，台南。

魏志嶽 (2005) 石斑魚肌肉生長抑制素之基因選殖與功能分析，國立成功大學生物科技研究所碩士論文，台南。

歐明昌 (2006) 石斑魚神經壞死病毒B2基因功能分析及檢測方法開發，國立成功大學生物科技研究所碩士論文，台南。

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