腾冲嗜热厌氧菌热休克蛋白60及其基因表达的热激动态变化分析  

Dynamic analysis of the heat shock protein 60 and its gene expression in Thermoanaerobacter tengcongensis induced by temperature increase

作　　者：孟博[1] 王敬强[1] 李娜[1] 马延和[2] 刘斯奇[1] 

机构地区：[1]中国科学院北京基因组研究所,北京101318 [2]中国科学院微生物研究所,北京100080

出　　处：《微生物学报》2006年第2期269-274,共6页Acta Microbiologica Sinica

基　　金：国家"973项目"(2004CB719605)~~

摘　　要：热休克蛋白60(HSP60)是细菌体内一种非常重要的分子伴侣,其可以协助蛋白质或肽链的正确折叠和构型,防止变性和降解。基于本实验室的早期观察,腾冲嗜热厌氧菌的HSP60是一个典型的温度相关蛋白质,在80℃的表达水平最高。为了进一步了解嗜热菌应急的分子机制,继续进行了在热激后HSP60基因表达的动态研究。将最适温度(75℃)下培养的腾冲嗜热厌氧菌迅速地转移至80℃继续培养,然后在不同的时间点上分别取样,并通过双向电泳、Western blot和Real_time PCR等方法,分析了HSP60在mRNA和蛋白质水平上的表达量的改变。试验结果表明,在80℃热处理4h内的短期应急过程中,HSP60蛋白水平一直呈上升趋势,而它的mRNA水平则表现为先升高后下降的一个非对称性的峰形变化。HSP60的mRNA和蛋白质的对温度的应答快慢程度是不同的。HSP60的mRNA水平的显著变化在1h内便可观察到,而蛋白质水平的显著改变要延迟3h左右。此外,HSP60的mRNA和蛋白质对温度的应答量变大小也是不同的。Heat shock protein 60 （HSP60） is one of the important chaperonins, which can assist proper protein folding and configuration,and can prevent denaturation and degradation of proteins as well. It was found that HSP60 in Thermoanaerobacter tengcongensis （T. tengcongensis） was typically temperature-dependent with the highest protein expression at 80℃. To understand the molecular mechanisms of thermophiles in their responding to heat stress, further investigation was conducted to examine the dynamic expression of HSP60 gene induced by higher temperature. The T. tengcongensis cells cultured at optimal temperature （75℃）were transferred to 80℃ followed by taking the aliquots at different time intervals. To monitor the expression levels of HSP60 mRNA and protein, these bacterial gene products were analyzed by two-dimensional electrophoresis, MALDI-TOF, HPLC-MS/MS, Western blot and quantitative PCR. The experimental data were calculated statistically and quantitatively, which indicate that the HSP60 protein increases steadily and significantly within 4h period after temperature raised, but its mRNA level is shown as a peak shape change within 1h. Obviously, the expression response of HSP60 protein to heat stress is significantly slower than that of its mRNA expression. Additionally, the expression extent of HSP60 mRNA and protein induced by raised-temperature is very different. In E.coli strains, the expression of HSP60 is usually regulated by the σ^32 factor, which disassociates with DnaK immediately when the environmental temperature elevated. Since the genome of T. tengcongensis contains two σ^32 genes, thus, it is likely that both bacteria share the similar mechanisms which regulate HSP60 transcription responding to the changes of temperature. Interestingly, the mRNA of HSP60 drops down about 70% within 30m responding to heat shock, but goes to the increased expression after then. Although this phenomenon is hard to explain based upon the current theory, it is plausible that the transcription in T.

关 键 词：热休克蛋白60 热激动态变化 腾冲嗜热厌氧菌 嗜热菌 

分 类 号：Q936[生物学—微生物学]