摘要
选用 pGEX 6P 1表达载体 ,构建GST与线粒体小分子热激蛋白的融合蛋白表达载体 ,使用谷胱苷肽Sepharose 4B亲合柱 ,纯化大肠杆菌中表达的融合蛋白 ,利用ProScission蛋白酶 ,将线粒体小分子热激蛋白从融合蛋白中切出 ,获得纯化的线粒体小分子热激蛋白。分析线粒体小分子热激蛋白对柠檬酸合成酶体外热稳定性的影响 ,发现线粒体小分子热激蛋白可以减缓柠檬酸合成酶的热变性 ,并促进热变性的柠檬酸合成酶复性 ,说明高温下线粒体小分子热激蛋白对柠檬酸合成酶有保护功效。利用转基因方法 ,将线粒体小分子热激蛋白基因导入烟草 ,在CaMV35S启动子的驱动下 ,导入烟草的线粒体小分子热激蛋白基因可在烟草细胞中组成性地表达 ,比较正常烟草线粒体和转基因烟草线粒体的氧化磷酸化效率 ,发现高温下转基因烟草线粒体的氧化磷酸化效率高于对照 ,说明线粒体小分子热激蛋白对线粒体具有保护作用。此外 ,转基因烟草花粉粒的高温萌发率也高于对照 ,说明线粒体小分子热激蛋白不但可以增加线粒体的耐热性 。
A fusion protein expression vector including GST gene and the gene of mitochondrial small heat shock protein was constructed with the pGEX 6P 1 vector. The fusion protein was induced in E.coli and purified with a glutathione Sepharose 4B affinity column. Small heat shock protein cleaved out of the fusion protein by ProScission proteinase was purified(Fig.1). The effect of mitochondrial small heat shock protein on the heat stability of citrate synthase in vitro was analyzed. The results showed that mitochondrial small heat shock protein decelerated the heat denaturation of citrate synthase as well as enhanced the renaturation of the heat denatured citrate synthase, suggesting that mitochondrial small heat shock protein prevented the citrate synthase from heat denaturation(Fig.2). The tomato mitochondrial small heat shock protein gene with CaMV35S promoter was introduced into tobacco and expressed constitutionally in tobacco(Fig.3). The efficiency of the mitochondrial oxidative phosphorylation in vitro of the transgenic tobacco was higher than that of normal mitochondria(Fig.4). The results indicated that mitochondrial small heat shock protein protected mitochondria from heat damage. Furthermore, the germination ratio of transgenic tobacco pollen at high temperature was higher than that of normal tobacco pollen(Fig.5), suggesting that the expression of mitochondrial small heat shock protein increased the heat tolerance of not only mitochondria but also plant cell as whole.
基金
国家自然科学基金资助课题 (3 9770 0 76)
关键词
线粒体
小分子热激蛋白
柠檬酸合成酶
氧化磷酸化
花粉粒萌发
耐热
高温
mitochondria, small heat shock protein, citrate synthase, oxidative phosphorylation, pollen germination, heat tolerance
