Studies on the post-mortem fish muscle tenderization -Analysis using the transgenic red seabream

注射金属蛋白酶特异性抑制剂1,10 -菲罗啉可显著抑制红鲷肉的嫩化，提示基质金属蛋白酶参与了红鲷死后肉的嫩化过程。然后，从红鲷中分离到基质金属蛋白酶（MMP）和金属蛋白酶组织抑制剂（TIMP）的编码基因。实际上，重组MMP降解了肌肉结缔组织的主要成分胶原蛋白，并通过添加TIMP抑制其活性。说明MMP-TIMP系统在红鲷死后肌肉嫩化过程中起着重要作用。这些结果提示TIMP转基因表达可能抑制死后肉的嫩化。我们制作了过表达TIMP的转基因medaka。组织学分析证实了TIMP转基因表达对胶原分解的抑制作用。为了将该技术应用于养殖鱼类，我们采用了重要的鱼类养殖品种红鲷。为了构建表达载体，还分离了3种不同的α-actin基因（2种骨骼肌型和1种心肌型）。这些肌动蛋白基因由8个外显子组成，在上游区域具有E Box和CArG Box，但在鱼类中的表达模式明显不同。我们用GFP作为报告基因制作了包含这些基因启动子区域的表达载体。结果表明，所有载体均可用于红鲷的转基因。利用这些载体，将TIMP基因导入红鲷体内。我们现在饲养了200条可能携带TIMP基因的鱼。

By the injection of 1, 10-phenanthroline, a specific inhibitor for metalloproteinase, meat tenderization of red seabream was significantly suppressed, suggesting that matrix metalloproteinase is involved in the post-mortem meat tenderization of red seabream. Then, Genes encoding matrix metalloproteinases (MMP) and tissue inhibitor of metalloproteinases (TIMP) were isolated from red sea bream. Actually, recombinant MMP degraded collagen, a main component of the muscle connective tissue, and the activity was inhibited by the addition of TIMP. These facts suggested MMP-TIMP system plays a very important role in the post-mortem tenderization of red seabream muscle.These findings suggested the transgenic expression of TIMP might suppress the post-mortem meat tenderization. We made a transgenic medaka overexpressing TIMP. Histological analysis demonstrated the effectiveness of the transgenic expression of TIMP for the suppression of collagen breamdown.To apply this technology to cultured fish, we adopted red seabream, an important species for fish culture. For the construction of the expression vector, three distinct α-actin genes (two skeletal muscle types and one cardiac muscle type) were also isolated. These actin genes are composed of 8 exons and having E Box and CArG Box in the up-stream region, but the expression pattern in the fish was clearly distinct. We made the expression vectors harboring the promoter regions of these genes with GFP as a reporter gene. As a result, all vectors were revealed to be useful for making transgenic red seabream.By using these vectors, we introduced TIMP gene into red seabream. We are now keeping 200 fish possibly having TIMP gene.