类胡萝卜素在耐辐射奇球菌辐射抗性中的作用  

Effects of Carotenoids on The Radioresistance of The Extremely Radioresistant Bacterium Deinococcus radiodurans

作　　者：杨桥[1] 张晓玲[3] 张磊[2] 代俊[2] 张俊祥[2] 焦炳华[1] 

机构地区：[1]第二军医大学生物化学与分子生物学教研室,上海200433 [2]病毒学国家重点实验室,武汉大学生命科学学院,武汉430072 [3]中国水产科学研究院东海水产研究所,上海200090

出　　处：《生物化学与生物物理进展》2009年第6期715-721,共7页Progress In Biochemistry and Biophysics

基　　金：国家高技术研究发展计划(863)资助项目(2007AA091501,2006AA09Z416,2006AA09Z425);中国博士后科学基金资助项目(20080431365)~~

摘　　要：为研究耐辐射奇球菌(Deinococcus radiodurans)中类胡萝卜素的生化合成基因及其在该细菌抗辐射机制中的生物学作用,通过有机溶剂提取及LC-MS技术分析了D.radiodurans所产类胡萝卜素物质的主要组分,运用PCR及基因同源重组技术,对该菌中类胡萝卜素生化合成途径的八氢番茄红素合成酶(phytoene synthase,crtB)及八氢番茄红素脱氢酶(phytoene desaturase,crtI)基因进行了缺失突变,通过表型观察及HPLC定量分析突变株所产类胡萝卜素的组分变化确证突变株构建成功.野生株及crtB与crtI基因缺失突变株对电离辐射和H2O2的敏感性差异比较分析显示,和野生株相比,两种突变株对不同剂量电离辐射和不同浓度H2O2的敏感性更强.crtB及crtI基因功能研究表明,这两个关键性合成基因的缺失,导致突变株不能催化合成类胡萝卜素生化合成途径中的重要中间体——番茄红素及一系列下游产物.通过λ原噬菌体紫外线诱导系统、电子自旋共振(ESR)及DMPO自旋捕集技术,分别在体内和体外评价了其类胡萝卜素的抗氧化能力.结果表明,两种类胡萝卜素对超氧阴离子(O·2)及羟自由基(·OH)均表现出较强的清除作用.上述研究结果为探究D.radiodurans的类胡萝卜素合成基因和生物学功能,及类胡萝卜素在D.radiodurans抗辐射机制中的作用提供了新的直接实验证据.To investigate the biosynthetic genes and the biological functions of carotenoids on the radioresistant mechanisms of the extremely radioresistant bacterium, Deinococcus radiodurans, the carotenoids of this bacterium were extracted with cold methanol and acetone, then were isolated and analyzed by LC-MS technique. Two colorless mutants knocking out of the phytoene synthase （crtB） and phytoene desaturase （crtI） genes were constructed by PCR and in vivo homologous recombination. The successful construction was confirmed by phenotype observation and carotenoids analysis by high performance liquid chromatography （HPLC）. Comparative analysis of the sensitivities among the two colorless mutants and wild R1 showed that the two colorless mutants were more sensitive to ionizing radiation （IR） and ultraviolet （UV）, indicating that the deletion of crtB and crtI genes made the mutants unable to biosynthesize the crucial intermediate lycopene and series of downstreaming carotenoids. The scavenging abilities of the two carotenoids were measured using the ultraviolet induction of bacteriophage k （UIB） system and electron spin resonance （ESR） spin trapping technique in vivo and in vitro, respectively. The results showed that the main two carotenoids exhibited significant antioxidant capacities to superoxide anion （O2） and hydroxyl radicals （-OH） which were two main types of flee radicals produced during IR. This study has provided for the first time a new and direct evidence to explore the biosynthesis genes of carotenoids and their biological functions for the radioresistant mechanisms ofD. radiodurans.

关 键 词：耐辐射奇球菌 类胡萝卜素 抗辐射 自由基 

分 类 号：Q93[生物学—微生物学]