【作者】 纪芳；

【导师】 邓子新； 周秀芬；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2001， 硕士

【摘要】 野生型变铅青链霉菌具有一种不同于甲基化修饰的新型DNA硫修饰系统，使DNA在电泳时易遭到氧化双链切割而导致降解(Dnd, DNA degradation)表型(Zhou et al., 1988, 1994, 1999)。己证实该菌株染色体上一段8.3kb的区域与Dnd表型有关，序列分析显示该区域包含了3个ORFs—dndA, dndB和dndC(Zhou et al., 1999；李爱英，2000)。为了验证这三个基因与Dnd表型的关系，构建了一系列衍生于整合型载体pSET152的质粒：pHZ2150，pHZ2151，pHZ2152和pHZ2153。它们均携带包含3个ORFs的区域(除了pHZ2153在dndB和dndC内缺失了2kb)，但已分别在各ORF上发生单基因中断(pHZ2150，pHZ2151和pHZ2152上外源片段的中断分别发生在dndA，月和C上)。将它们导入Dnd表型缺失突变株ZX1中进行基因互补实验，结果证明变铅青链霉菌and基因簇上的3个ORFs均为Dnd所必需。 对野生型变铅青链霉菌dnd基因簇的生物学功能进行了初步探索。以野生型变铅青链霉菌和各dnd基因突变株为研究对象，对它们在极端培养条件(极端pH，高温，高盐和低氧)下的生长状况和对11株噬菌体的感染性进行了比较，结果在测试条件下，没有发现野生型菌株和不同dnd基因突变株之间存在明显差异。提取各菌株低氧条件下培养物的DNA进行电泳检测，结果揭示该生长条件不能改变这两种菌株原有的Dnd表型。 在PFGE中发现耻垢分枝杆菌mc~2155也具有Dnd表型。将变铅青链霉菌的dnd基因簇与mc~2155的基因组序列(未测完)进行同源比较后，发现mc~2155中存在dndA的同源基因(暂命名为mddl)，且二者在氨基酸水平上的同一性为38％。根据该基因的序列设计了两对特异引物，以mc~2155 DNA为模板，扩增得到0.9kb和1.1kb的产物。其中的0.9kb产物经测序验证与mddl的序列一致。以pHZ132为载体，构建了mc~2155的基因组文库。以0.9kb的扩增产物为探针从文库中钓出10个阳性克隆子。在此基础上构建了用于在mc~2155中进行mddl基因中断的载体pHZ2157。更多还原

【Abstract】 A novel DNA modification discovered in Streptomyces lividans 1326 is different from DNA methylation. This unusual modification causes wild type S. lividans 1326 DNA sensitive to site-specific oxidative double-strand cleavage (Dnd phenotype). Preliminary study revealed that such DNA modification involves incorporation of sulphur. The entire functional dnd cluster, 8.3kb in size, including 3 ORFs-dndA, dndB and dndC was involved in this DNA modification. Four plasmids derived from an integrative vector pSET152 were constructed which contain the whole dnd cluster but each ORF has been interrupted individually, and introduced into 5. lividans ZX1, a mutant strain with a chromosome deletion including dnd cluster for complementation test. All of three ORFs were demonstrated to be necessary for the Dnd phenotype.For the survey of the biological relevence, different 5". lividans strains with Dnd+ and Dnd" phenotype were used for testing phage infections, growth under different pH, temperature, concentration of NaCl and an extremely low concentration of O2. No obvious differences could be tested from these strains. The detection of the Dnd phenotype of the total DNA isolated from the mycelie growing at an extremely low concentration of O2 showed no difference from that at normal condition.The Dnd phenotype was also found in DNA of Mycobacterium smegmatis mc2155 by PFGE. Query of the amino acid sequence of dnd cluster from S. lividance to the uncompleted genome sequence of me2155, a homologous gene with a 38% identity to dndA was found and was tentatively named mddl. Two pairs of primers were designed based on the sequence of mddl to get a 0.9kb and an 1.1kb PCR products which were confirmed to be expected sequence from mc2155 by DNA sequencing. The me2155 genomic library was constructed using pHZ132 as the cosmid vector, and 10 positive clones were fished out using 0.9kb PCR product as probe. A construct derived frompHZ2157 is now ready for the constrction of mddl mutant by gene disruption experiment.更多还原