基于热驯化的草鱼肌球蛋白的性质变化及其分子机理

为了探明草鱼肌球蛋白的结构和性质伴随栖息环境温度变化的时间过程及分子机理、实现对草鱼的热驯化改性,本文对冬季草鱼进行了30℃的热驯化试验。通过对不同热驯化阶段草鱼肌球蛋白Ca^2＋-ATPase变性速率常数（KD）的测定和对肌球蛋白的DSC分析,考察了肌球蛋白热稳定性的变化趋势;进一步运用RT-PCR考察了已知的3种草鱼肌球蛋白重链同工型基因在不同热驯化阶段草鱼肌肉中的表达模式。结果显示,热驯化至5周左右,草鱼肌球蛋白Ca^2＋-ATPase的KD值由热驯化前的41.8×10^-4/s下降至3.25×10^-4/s;DSC分析结果显示,肌球蛋白的热变性温度（Tm）由驯化前的37℃上升至43.6℃;RT-PCR结果表明,热驯化前和热驯化2周的草鱼肌肉中只表达低温型（gc-10）,热驯化3至5周时,3种同工型基因都有表达,而热驯化5周以后表达的是中间型（gc-I）和高温型（gc-30）,以后者居多。这些结果较清楚地证明了草鱼肌球蛋白热稳定性发生急剧变化所需的热驯化时间约为5周左右;对冬季草鱼热驯化3至5周左右;其肌肉中肌球蛋白重链同工型基因的表达模式已开始发生变化,即肌球蛋白发生重建,从而导致其热稳定性的改变。

Character Changes of Myosin from Thermally Acclimatized Grass Carp and Molecular Mechanisms

To clarify the time-effect rules and molecular mechanisms of changes in the structures and properties of grass carp （Ctenopharyngodon idellus） myosin with the environment temperature, and improve properties of grass carp protein, the thermal acclimation experiment （30 ℃） for winter grass carp was carried out. The changes in thermal stability of myosin from grass carp at various stages of thermal acclimation were examined by determining the inactivation rate constants （KD） of myosin Ca^2＋-ATPase and using DSC analysis. Furthermore,RT-PCR was performed to examine the expression patterns of the three types of myosin heavy chain isoform gene in muscles from grass carp at various stages of thermal acclimation. The results indicated that the KD value declined from 41. 8 × 10^-4/s （before thermal acclimation ） to 3. 25 × 10^-4/s （thermally acclimatized for five weeks）, and accordingly, DSC analysis showed the denaturation temperature （Tm） of myosin increased from 37 ℃ to 43. 6 ℃. The results of RT- PCR indicated that the gc-10 type was only expressed in muscles from grass carp thermally acclimatized for 0 and two weeks, the three types of isoform genes were all expressed in muscles from grass carp thermally acclimatized for three to five weeks, whereas the gc-I and gc-30 types were expressed in muscles from grass carp thermally acclimatized for over five weeks. These results demonstrated that it took about five weeks to have dramatic changes in thermal stability of myosin for thermally acclimatized grass carp, and when the winter grass carp was thermally acclimatized for three to five weeks,the changes of expression patterns of myosin heavy chain isoform gene occurred, which resulted in reconstructing of myosin and subsequent changes in its thermal stability.