肌肉生長抑制素 (Myostatin) 屬於轉化生長因子-beta家族，為調節細胞之增殖以及分化的因子，具有抑制肌肉生長發育的功能。在蛋白功能分析上，Myostatin主要包括三個區域：（1）作為釋放訊息的N端部分，（2）高度保留性的蛋白切割位RXRR，（3）以及半胱氨酸豐富的C端活性區域，其中C端活性區域被認為是較重要之區域。利用一套免疫剔除 (immune-depletion) 技術降低魚體內內生性肌肉生長抑制素，藉此了解肌肉抑制基因於魚體內的功能；在啟動子分析方面，石斑魚肌肉生長抑制基因啟動子全長為1,936 bp，其上具有10個由CANNTG短核苷酸組成的序列，可能為肌肉生長相關調節區段，將其全長及系列區段剔除的片段接入帶有螢火蟲螢光蛋白報導基因的質體(pGL3-Basic)，再轉染進入石斑魚鰭細胞株 (GF-1 cell line) 裡觀察其功能表現，找出啟動子最重要的調控區域及探討可能調控啟動子的相關調節因子。

Myostatin (GDF-8), which belongs to TGF-ß superfamily, is a secreted growth and differentiation factor. It functions as a negative regulator for the growth of skeletal muscle. In the functional analysis of Myostatin, the open reading frame is very conservative, including three domains: (1) N-terminus domain, it functions as secretory signal peptide, (2) conserved proteolytic processing site RXRR, and (3) C-terminus cysteine-rich region as mature bioactive region. Among them, C-terminus cysteine-rich region was determined the most important region. We developed an immunodepletion technique, which is sufficient to induce anti-Myostatin immunoresponse, to demonstrate the function of Myostatin in the grouper. In addition, orange-spotted grouper (Epinephelus coioides) myostatin promoter is defined as 1936-bp and contains ten putative E-Box (CANNTG) motifs. Various constructs of the grouper myostatin promoter were transfected into the grouper cells, regulatory regions on myostatin promoter and the relative regulatory factors were analysed.

[1]Acosta, J., Carpio, Y., Borroto, I., González, O., and Estrada M.P. Myostatin gene
silenced by RNAi show a zebrafish giant phenotype. J. Biotechnol. 119,
324-331 (2005)

[2]Alvarez-Pellitero, P. Fish immunity and parasite infections: from innate immunity to
immunoprophylactic prospects. Vet. Immunol. Immunopathol. 126, 171-198
(2008)

[3]Bergstrom, D.A., Penn, B.H., Strand, A., Perry, L.S., Rudnicki, M.A. and Tapscott, J.
Promoter-specific regulation of MyoD binding and signal transduction cooperate to pattern gene expression. Mol. Cell. 9, 587-600 (2002)

[4]Chalfie, M., Tu, Y., Euskirchen, G.,Ward, W.W., and Prasher, D.C. Green fluorescent
protein as a marker for gene expression. Science. 263, 802-8055 (1994)

[5]Chaudhary, J., Cupp, A.S., and Skinner, M.K. Role of basic-helix-loop-helix transcription factors in Sertoli cell differentiation: identification of an E-box response element in the transferrin promoter. Endocrinology. 138, 667-675 (1997)

[6]Chaudhary, J. and Skinner M.K. Basic helix-loop-helix proteins can act at the E-box
within the serum response element of the c-fos promoter to influence hormone-induced promoter activation in Sertoli cells. Mol. Endocrinol. 13, 774-786 (1999)

[7]Chen, Y.M, Wei, C.Y., Chien, C.H., Chang H.W, Huang S.I, Yang H.L, and Chen T.Y. Myostatin gene organization and nodavirus-influenced expression in orange-spotted grouper (Epinephelus coioides). Comp. Biochem. Physiol. D, Genomics. Proteomics. 2, 215-227 (2007)

[8]Chi, S.C., Lin, S.C., Su, H.M., and Hu, W.W. Temperature effect on nervous necrosis virus infection in grouper cell line and in grouper larvae. Virus. Res. 63, 107-114 (1999)

[9]Cleary, M.A., Stern, S., Tanaka, M., and Herr, W. Differential positive control by Oct-1 and Oct-2: activation of a transcriptionally silent motif through Oct-1 and VP16 corecruitment. Genes. Dev. 7, 72-83 (1993)

[10]Cody, C.W., Prasher, D.C., Westler, W.M., Prendergast, F.G., and Ward, W.W. Chemical structure of the hexapeptide chromophore of the Aequorea green-fluorescent protein. Biochemistry. 32, 1212-1218 (1993)

[11]Daugherty, A.L., McKee, M.L., FitzGerald, D.J., and Mrsny, R.J. Epithelial application of Pseudomonas aeruginosa exotoxin A results in a selective targeting to cells in the liver, spleen and lymph node. J. Control. Release. 65, 297-302 (2000)

[12]Donnelly, J.J., Ulmer, J.B., Hawe, L.A., Friedman, A. SHI, X.P., Leander, K.R., Shiver, J.W., Oliff, A.I., Martinez, D. Montgomery, D. and Liu, M.A. Targeted delivery of peptide epitopes to class I major histocompatibility molecules by a modified Pseudomonas exotoxin. Proc. Natl. Acad. Sci. U. S. A. 90, 3530-3534 (1993)

[13]Du, R., Chen, Y.F., An, X.R., Yang, X.Y., Ma, Y., Zhang, L., Yuan, X.L., Chen, L.M., and Qin, J. Cloning and sequence analysis of myostatin promoter in sheep. DNA. Seq. 16, 412-417 (2005)

[14]Du, S.J., Gao, J., Anyangwe, V. Muscle-specific expression of myogenin in zebrafish embryos is controlled by multiple regulatory elements in the promoter. Comp. Biochem. Physiol. B, Biochem. Mol. Biol. 134, 123-134 (2003)

[15]Escobar, J., Van Alstine, W.G., Baker, D.H., and Johnson, R.W. et al. Decreased protein accretion in pigs with viral and bacterial pneumonia is associated with increased myostatin expression in muscle. J. Nutr. 134, 3047-3053 (2004)

[16]Gao, J., Li, Z., and Paulin, D. A novel site, Mt, in the human desmin enhancer is necessary for maximal expression in skeletal muscle. J. Biol. Chem. 273, 6402-6409 (1998)

[17]Gonzalez-Cadavid, N.F., Taylor, W.E., Yarasheski, K., Sinha-Hikim, I., Ma, K., Ezzat, S., Shen, R., Lalani, R., Asa, S., Mamita, M., Nair, G., Arver, S., and Bhasin, S. Organization of the human myostatin gene and expression in healthy men and HIV-infected men with muscle wasting. Proc. Natl. Acad. Sci. U. S. A. 95, 14938-14943 (1998)

[18]Goodbourn, S., Didcock, L., and Randall, R.E. Interferons: cell signalling, immune modulation, antiviral response and virus countermeasures. J. Gen. Virol. 81, 2341-2364 (2000)

[19]Guttridge, D.C., Mayo, M.W., Madrid, L.V., Wang, C.Y. and Baldwin, A.S. NF- B-Induced Loss of MyoD Messenger RNA: Possible Role in Muscle Decay and Cachexia. Science. 289, 2363-2366 (2000)

[20]Hseuh, K.H., and Shang, H.F. Engineering of Pseudomonas exotoxin A into useful proteins for disease treatment. J. Chinese. Biochem. Soc. 23, 135-151 (1994)

[21]Jafar-Nejad, H. and Bellen, H.J. Gfi/Pag-3/senseless zinc finger proteins: a unifying theme? Mol. Cell. Biol. 24, 8803-8812 (2004)

[22]Kambadur, R., Sharma, M., Smith, T.P.L., and Bass, J.J. Mutations in myostatin (GDF8) in double-muscled Belgian Blue and Piedmontese cattle. Genome Res. 7, 910-916 (1997)

[23]Karim, L., Coppieters, W., Grobet, L., Valentini, A., and Georges, M. Convenient genotyping of six myostatin mutations causing double-muscling in cattle using a multiplex oligonucleotide ligation assay. Anim. Genet. 31, 396-399 (2000)

[24]Kerr, T., Roalson, E.H., and Rodgers, B.D. Phylogenetic analysis of the myostatin gene sub-family and the differential expression of a novel member in zebrafish. Evol. Dev. 7, 390-400 (2005)

[25]Lassar, A.B., Buskin, J.N., Lockshon, D., Davis, R.L., Apone, S., Hauschka, S.D., and Weintraub, H. MyoD is a sequence-specific DNA binding protein requiring a region of myc homology to bind to the muscle creatine kinase enhancer. Cell. 58, 823-831 (1989)

[26]Lu, L., Zhou, S.Y., Chen, C., Weng, S.P., Chan, S.M., and He, J.G. Complete genome sequence analysis of an iridovirus isolated from the orange-spotted grouper, Epinephelus coioides. Virology. 339, 81-100 (2005)

[27]Maccatrozzo, L., Bargelloni, L., Radaelli, G., Mascarello, F., and Patarnello, T. Characterization of the myostatin gene in the gilthead seabream (Sparus aurata): sequence, genomic structure, and expression pattern. Mar. Biotechnol. 3, 223-230 (2001)

[28]Malmgaard, L. Induction and regulation of IFNs during viral infections. J. Interferon. Cytokine. Res. 24, 439-454 (2004)

[29]McPherron, A.C. and Lee, S.L. Double muscling in cattle due to mutations in the myostatin gene. Proc. Natl. Acad. Sci. U. S. A. 94, 12457-12461 (1997)

[30]Melaine, N., Lienard, M.O., Elisabeth, G., Annick, R., Nathalie, D.R., and Bernard, J. Production of the antiviral proteins 2'5'oligoadenylate synthetase, PKR and Mx in interstitial cells and spermatogonia. J. Reprod. Immunol. 59, 53-60 (2003)

[31]Mosher, D.S., Quignon, P., Bustamante, C.D., Sutter, N.B., Mellersh, C.S., Parker, H.G., and Ostrander, E.A. A mutation in the myostatin gene increases muscle mass and enhances racing performance in heterozygote dogs. PLoS. Genet. 3, 779-786 (2007)

[32]Phillips, K. and Luisi, B. The virtuoso of versatility: POU proteins that flex to fit. J. Mol. Biol. 302, 1023-1039 (2000)

[33]Pizza, M., Giuliani, M.M., Fontana, M.R., Monaci, E., Douce, G., Dougan, G., Mills, K.H., Rappuoli, R., and Del, G.G. Mucosal vaccines: non toxic derivatives of LT and CT as mucosal adjuvants. Vaccine. 19, 2534-2541 (2001)

[34]Potthoff, M.J. and Olson, E.N. MEF2: a central regulator of diverse developmental programs. Development. 134, 4131-4140 (2007)

[35]Salerno, M.S., Thomas, M., Forbes, D., Watson, T., Kambadur, R., and Sharma, M. Molecular analysis of fiber type-specific expression of murine myostatin promoter. Am. J. Physiol. Cell. Physiol. 287, C1031-C1040 (2004)

[36]Samuel, C. E. Host genetic variability and West Nile virus susceptibility. Proc. Natl. Acad. Sci. U. S. A. 99, 11555-11557 (2002)

[37]Samuel, C.E. Knockdown by RNAi-proceed with caution. Nat. Biotechnol. 22, 280-282 (2004)

[38]Schuelke, M., Wagner, K.R., Stolz, L.E., HÜbner, C., Riebel, T., KÖmen, W., Braun,
T., James, F.T., and Lee, S.J. Myostatin mutation associated with gross muscle
hypertrophy in a child. N. Engl. J. Med. 350, 2682-2688 (2004)

[39]Seliger, H.H. and McELROY, W.D. Spectral emission and quantum yield of firefly bioluminescence." Arch. Biochem. Biophys. 88, 136-141 (1960)

[40]Sharma, M., Kambadur, R., Matthews, K.G., Somers, W.G., Devlin, G.P., Conaglen, J.V., Fowke, P.J., Bass, J.J. Myostatin, a transforming growth factor-beta superfamily member, is expressed in heart muscle and is upregulated in cardiomyocytes after infarct. J. Cell. Physiol. 180, 1-9 (1999)

[41]Spiller, M.P., Kambadur, R., Jeanplong, F., Thomas, M., Martyn, J.K., Bass, J.J., and Sharma, M. The myostatin gene is a downstream target gene of basic helix-loop-helix transcription factor MyoD. Mol. Cell. Biol. 22, 7066-7082 (2002)

[42]Stinckens, A., Luyten, T., Bijttebier, J., Van den Maagdenberg, K., Dieltiens, D., Janssens, S., De Smet, S., Georges, M., and Buys, N. Characterization of the complete porcine MSTN gene and expression levels in pig breeds differing in muscularity. Anim. Genet. 39, 586-596 (2008)

[43]Vopalensky, V., Masek, T., Horvath, O., Vicenova, B., Mokrejs, M., and Pospisek, M. Firefly luciferase gene contains a cryptic promoter." RNA. 14, 1720-1729 (2008)

[44]Weintraub, H., Tapscott, S.J., Davis, R.L., Thayer, M.J., Adam, M.A., Lassar, A.B., and Miller, A.D. Activation of muscle-specific genes in pigment, nerve, fat, liver, and fibroblast cell lines by forced expression of MyoD. Proc. Natl. Acad. Sci. U. S. A. 86, 5434-5438 (1989)

[45]Whittemore, L.A., Song, K., Li, X., Aghajanian, J., Davies, M., Girgenrath, S., Hill, J.J., Jalenak, M., Kelley, P., Knight, A., Maylor, R., O' Hara, D., Pearson, A., Quazi, A., Ryerson, S., Tan, X.Y., Tomkinson, K.N., Veldman, G.M., Widom, A., Wright, J.F., Wudyka, S., Zhao, L., and Wolfman, N.M. Inhibition of myostatin in adult mice increases skeletal muscle mass and strength. Biochem. Biophys. Res. Commun. 300, 965-971 (2003)

[46]Williams, B.R. PKR; a sentinel kinase for cellular stress. Oncogene. 18, 6112-6120 (1999)

[47]Ye, H.Q., Chen, S.L., Sha, Z.X., and Liu, Y. Molecular cloning and expression analysis of the myostatin gene in sea perch (Lateolabrax japonicus). Mar. Biotechnol. 9, 262-272 (2007)

[48]粘茂偉, 利用TR-PCR發展石斑魚神經壞死病毒次單位疫苗，國立成功大
學生物科技研究所碩士論文，台南 (2003)。

[49]魏志嶽, 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 (2005)。

[50]簡正修, 點帶石斑魚肌肉倍增基因功能分析與其基因啟動子之調節，國立成功大學生物科技研究所碩士論文，台南 (2007)。