头状链轮丝菌染色体复制起始区的克隆和对变铅青链霉菌的转化  

Cloning of the Replication Origin (oriC) of Streptoverticillum caespitosus and Transformation of Streptomyces lividans ZX7

作　　者：马伟[1] 茅翔[1] 卢捷[1] 姜卫红[1] 焦瑞身[1] 覃重军[1] 赵国屏[1] 

机构地区：[1]中国科学院上海生命科学研究院,植物生理生态研究所,上海200032

出　　处：《生物工程学报》2002年第6期662-666,共5页Chinese Journal of Biotechnology

基　　金：国家自然科学基金重点项目资助 (批准号 :10 0 396 30 0 10 )~~

摘　　要：头状链轮丝菌 (Streptoverticillumcaespitosus)ATCC2 742 2是抗肿瘤药物———丝裂霉素A的产生菌 ,根据高GC含量革兰氏阳性菌染色体复制起始区两端基因序列的保守性 ,采用PCR方法从该菌中克隆了一段包含染色体复制起始区 (oriC)的 1 3kb片段。序列分析发现 ,克隆片段与天蓝色链霉菌的oriC及邻近区域的同源性达 80 %以上 ;头状链轮丝菌oriC中含有 2 2个DnaA box ,保守序列为TTGTCCACA。以克隆片段构建的质粒可以跨属转化变铅青链霉菌 (S .lividans)ZX7,原生质体转化效率为 3 2× 10 2 个 μgDNA ;质粒在S .lividansZX7中能以低拷贝形式稳定存在 ;转化子的菌落和菌丝形态均正常。头状链轮丝菌oriC序列与几种链霉菌的oriC有较高的同源性 ,以及在变铅青链霉菌中仍具有复制起始活性等说明链轮丝菌属与链霉菌属在系统发生上关系较近 ;但采用最大似然法分析oriC序列构建的头状链轮丝菌与几种链霉菌的系统进化树表明 ,头状链轮丝菌与几种链霉菌之间的分化距离远大于链霉菌之间的分化距离。Streptoverticillum caespitosus ATCC27422 is a producing strain of mitomycin A for cancer therapy. Taking the advantage of the conserved sequences of genes flanking the oriC of high G+C Gram-positive bacteria, a 1.3 kb DNA fragment containing oriC and its flanking region was cloned by PCR. Nuleotide sequence comparisons revealed that the cloned fragment is more than 80% identical to the same region of S. coelicolor. There are 22 DnaA-boxes in the oriC region, and the conserved sequence of DnaA-box is TTGTCCACA. The plasmid containing the oriC of S. caespitosus was constructed (pMJ9), and it was able to transform the protoplast of Streptomyces lividans ZX7 at the frequency of 3.2×10 2 transformants/μg plasmid DNA. The colony and mycelias morphology of the transformants are normal. The constructed plasmid can exist stable in the host as a low copy extra-chromosome replicon. The high rate of the homology and the cross genus replication initiation activity suggests close relationship between Streptomyces and Streptoverticillum in the evolution. While the maximum likelihood phylogenetic tree based upon the oriC of S. caespitosus and several Streptomyces spp. revealed that S. caespitosus differed extensively from the Streptomyces spp. This result supports the separation of Streptoverticillum from Streptomyces.

关 键 词：头状链轮丝菌 染色体 复制起始区 克隆 变铅青链霉菌 转化 

分 类 号：Q785[生物学—分子生物学]