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

注射金属蛋白酶的特异性抑制剂1，10-二氮杂菲显著抑制了罗非鱼的嫩化，提示基质金属蛋白酶参与了罗非鱼的宰后嫩化过程。然后，从罗非鱼中分离出基质金属蛋白酶(MMPs)及其组织抑制因子(TIMP)编码基因。实际上，重组的基质金属蛋白酶降解了肌肉结缔组织的主要成分--胶原，并且这种活性被TIMP的加入所抑制。这些事实表明，MMPTIMP系统在鲜鱼肌肉的嫩化过程中起着非常重要的作用，转基因表达TIMP可能会抑制鲜肉的嫩化。我们获得了一株高表达TIMP的转基因青蛙。组织学分析表明，转基因表达的TIMP可以有效地抑制胶原蛋白的降解。为了将这项技术应用于养殖鱼类，我们选择了重要的鱼类养殖物种红鳍。为了构建表达载体，还分离了三个不同的α-肌动蛋白基因(两个骨骼肌型和一个心肌型)。这些肌动蛋白基因由8个外显子组成，上游有E盒和Carg盒，但在鱼类中的表达模式明显不同。以绿色荧光蛋白为报告基因，构建了含有这些基因启动子区域的表达载体。利用这些载体，我们将TIMP基因导入到真核表达载体中，将TIMP基因导入真核表达载体。我们现在饲养了200条可能带有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.