耐辐射球菌recQ双突变株的构建及逆境分析  

Construction of the recQ double mutants and analysis of adversity in Deinococcus radiodurans

作　　者：黄丽芬[1] 张韶文[2] 华孝挺[1] 高冠军[1] 华跃进[1] 

机构地区：[1]浙江大学原子核农业科学研究所农业部核农学重点开放实验室 [2]浙江大学校医院,杭州310027

出　　处：《微生物学报》2006年第2期205-209,共5页Acta Microbiologica Sinica

基　　金：国家"973项目"(2004CB19604);国家自然科学基金重点资助项目(30330020);国家杰出青年基金项目(30425038)~~

摘　　要：RecQ解螺旋酶是生物有机体在进化中高度保守的SF1超级家族解螺旋酶的一个亚族,它对维持基因组的稳定性有重要的作用。耐辐射球菌野生型菌株R1有两个具有特殊结构的解螺旋酶DR1289和DR2444,运用PCR突变法克隆具有自身groEL启动子、KAT启动子与卡那霉素抗性基因、氯霉素抗性基因融合的DNA片段反向重组到基因组中,首次构建并鉴定了卡那霉素抗性完全突变株ΔDR1289,氯霉素抗性完全突变株ΔDR2444,双突变株ΔrecQ。辐射条件下和H2O2氧化压力下突变株生存率结果表明:ΔDR2444与R1存活率趋势线基本一致,而ΔDR1289和ΔrecQ双突变株较为敏感。根据上述结果推测,DR1289是一个对R1保持极端抗性的必须基因,而DR2444则是极端抗性的非必须基因。As a subfamily member of SFI superfamily, the RecQ helicases are highly conserved in evolution and are required for maintaining genome stability in all organisms. Loss of RecQ helicase function leads to a breakdown in the maintenance of genome integrity, in particular hyper-recombination. Named after the recQ gene of Escherichia coil, lower eukaryotic species generally only contain a single RecQ family representative; for example, Sgsl in the budding yeast, Saccharomyces cerevisiae, and Rqhl in the fission yeast, Schizosaccharomyces pombe. There are, however, multiple members in most higher organisms, with five being present in humans. Defects in three of these human RecQ helicases give rise to defined clinical disorders associated with cancer predisposition and variable aspects of premature aging. Deinococcus radiodurans encodes two recQ genes with unusual domain= DR1289 and DR2444, whose functions, however, remain obscure currently. DR1289 contains three tandem copies of the C-terminal helicase-RNase D （HRDC） domain, instead of the single copy present in all other bacteria except Neisseria that similarly possesses three copies. DR2444 contains a HRDC domain and a domain homologous to cystathionine gamma-lyase; this is the first example of an HRDC domain that is not associated with either a helicase or a nuclease. In this study, a fusion DNA fragment carrying kanamycin resistance gene with the D. radiodurans groEL promoter, chloramphenicol resistance gene with KAT promoter was cloned by PCR amplification and reversely inserted into the recQ locus in the genome of the wild-type strain RI. Three resulting recQ-deficient strains, designated △DR1289, △DR2444 and △recQ （double mutation）, were constructed, Results show that △DR1289 and △ recQ were very sensitive to ionizing radiation and H2O2, while △ DR2444 and wild strain R1 were not. The phenotype of △ DR1289 was similar to many RecQ helicase mutants, Therefore, it was presumed that DR1289 was the necessary gene in maintaining the extreme resi

关 键 词：耐辐射球菌 RECQ 突变 

分 类 号：Q936[生物学—微生物学] Q78