Mitochondrial small heat-shock protein protects citrate synthase, mitochondria and pollen at high temperature
Mitochondrial small heat-shock protein protects citrate synthase, mitochondria and pollen at high temperature
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发表时间：
2001
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影响因子：
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通讯作者：
Jian Liu;Shono Mariko
中科院分区：
文献类型：
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作者：
Jian Liu;Shono Mariko
以pGEX 6P 1为载体，构建了GST基因与线粒体小分子热休克蛋白基因的融合表达载体。在大肠杆菌中诱导融合蛋白，并用谷胱甘肽Sepharose 4B亲和柱纯化。用ProScission蛋白酶从融合蛋白中切割出小的热休克蛋白进行纯化（图1）。分析了线粒体小分子热休克蛋白对柠檬酸合酶体外热稳定性的影响。结果表明，线粒体小分子热休克蛋白既能抑制柠檬酸合酶的热变性，又能促进柠檬酸合酶的复性，提示线粒体小分子热休克蛋白能阻止柠檬酸合酶的热变性。将带有CaMV35S启动子的番茄线粒体小分子热激蛋白基因导入烟草，并在烟草中进行了组成型表达。转基因烟草的线粒体氧化磷酸化效率高于正常烟草。结果表明，线粒体小分子热休克蛋白保护线粒体免受热损伤。此外，转基因烟草花粉在高温下的萌发率高于正常烟草花粉（图5），表明线粒体小分子热激蛋白的表达不仅提高了线粒体的耐热性，而且提高了植物细胞整体的耐热性。
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.