STUDY OF REGULATORY REGION OF FISH TIMP GENE

鱼类TIMP基因调控区的研究

• 批准号: 12660185
• 负责人: KINOSHITA Masato
• 金额: $ 2.62万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12660185/
• 关键词: Japanese flounder; proteinase; inhibitor; tenderization; meat

The toughness is one of the most important elements, which define the commercial value of raw meat of fish. The degradation of extracellulr matrix is thought to be a cause of post-mortem tenderization of fish meat. Our previous study suggested that this tenderization is caused mainly by the metalloproteinases. Especially, matrix metalloproteinases (MMPs) are thought to be the most possible candidates for post-mortem tenderization of fish meat.To elongate the toughness of fish meat during chilled storage, we proposed new technique mentioned below. There are endogenous proteinase inhibitors specific for MMPs. They are designated as tissue inhibitore of metalloproteinases (TIMPs). We attempt to elevate the quantity of TIMPs in fish meat during alive. At present, gene manipulated food has not get public acceptance yet So, we try to find suitable treatments which promote gene expression of TIMPs with the DNA sequence information of regulatory region of TIMP2b gene.Using the BAC library of cloned Japanese flounder genomic DNA, we isolated two BAC clone containing TIMP2b gene. As a result of investigation of 5' flunking region of TIMP2b gene, we found a cis-element called CRE-BP1, which is known to response to stress such as UV, heat-shock, and osmotic pressure. To investigate the function of CRE-BP1, the luciferase expression vector which contains a series of deleted 5' flunking region of TIMP2b.The 5' flunking region of TIMP2b responded to hyper-osmotic stress, but do not to hypo-osmotic, heat-shock, and UV stress. These results suggested that the possiblity to elongate the toughness of fish meat durng chilled storage.

鱼肉的韧性是决定其商业价值的重要因素之一。细胞外基质的降解被认为是鱼肉嫩化的原因之一。我们以前的研究表明，这种嫩化主要是由金属蛋白酶引起的。特别是基质金属蛋白酶（matrix metalloproteinases，MMPs）被认为是鱼肉宰后嫩化最有可能的候选酶。存在对MMPs特异的内源性蛋白酶抑制剂。它们被称为金属蛋白酶组织转运蛋白（TIMPs）。我们试图提高鱼肉中TIMP的含量。目前，基因操纵食品还没有得到广泛的认可，因此，本研究试图利用TIMP 2b基因调控区的DNA序列信息，寻找合适的促进TIMP 2b基因表达的处理方法。通过对TIMP 2b基因5'端侧翼区的研究，我们发现了一个顺式元件CRE-BP 1，该元件对紫外线、热休克和渗透压等胁迫有应答作用。目的：研究TIMP 2b 5'端缺失片段的荧光素酶表达载体CRE-BP 1的功能。TIMP 2b 5'端缺失片段对高渗胁迫有反应，而对低渗、热休克和紫外线胁迫无反应。这些结果表明，冷藏可以延长鱼肉的韧性。

项目成果

M.Kinoshita, T.Yabe, M.Kubota, K.Takeuchi, S.Kubota, H.TYoyohara, M.Sakaguchi: "cDNA cloning and characterization of two gelatinases from Japanese flounder"Fisheries Science. (In press). (2002)

M.Kinoshita、T.Yabe、M.Kubota、K.Takeuchi、S.Kubota、H.TYoyohara、M.Sakaguchi：“来自日本比目鱼的两种明胶酶的 cDNA 克隆和表征”渔业科学。

M. Kinoshita, T. Yabe, M. Kubota, K. Takeuchi, S. Kubota, H. Toyohara, M. Sakaguchi: "cDNA cloning and characterization of two gelatinases from Japanese flounder"FISHERIES SCIENCE. (in press). (2002)

M. Kinoshita、T. Yabe、M. Kubota、K. Takeuchi、S. Kubota、H. Toyohara、M. Sakaguchi：“来自日本比目鱼的两种明胶酶的 cDNA 克隆和表征”渔业科学。

M.Kinoshita, T.Yabe, M.Kubota, K.Takeuchi, S.Kubota, H.TYoyohara, M.Sakaguchi: "c DNA cloning and characterization of two gelatinases from Japanese flounder"Fisheries Science. (in press).

M.Kinoshita、T.Yabe、M.Kubota、K.Takeuchi、S.Kubota、H.TYoyohara、M.Sakaguchi：“来自日本比目鱼的两种明胶酶的 c DNA 克隆和表征”渔业科学。