链霉菌M1033 D-木糖异构酶的分子克隆  

Cloning of D-xylose (D-glucose) Isomerase Gene in Streptomyces

作　　者：朱学良[1] 王玉珍[1] 黄震[1] 刘兢[1] 崔涛[1] 董婉治 牛立文[1] 徐洵[1] 

机构地区：[1]中国科学技术大学生物系

出　　处：《生物工程学报》1992年第3期232-236,共5页Chinese Journal of Biotechnology

基　　金：“863”高技术基金

摘　　要：链霉菌M1033染色体DNA经BamH Ⅰ酶解后电泳,Southern转移。根据自测的链霉菌ML033 D-木糖异构酶氨基酸序列设计合成寡聚核苷酸探针X-2、X-3,以X-2、X-3及Ampullariella sp3876 D-木糖异构酶基因(1.17kb)为探针进行杂交,确定与上述探针杂交最强处在15kb左右。从胶上分离出9—20kb大小的片段,克隆到EMBL 3载体中,经杂交筛选后,得到0.6%的阳性噬斑,其插入大小为13kb。将插人DNA的Sal Ⅰ酶解片段(2.5kb)进一步亚克隆于pUC18,得到重组质粒pUB 1,经酶解图谱、部分序列分析和互补实验确定pUB1含有完整的M1033Genomic DNA from Streptomyces M1033 was digested with BamH I, then electrophoresed, Southern transfered and hybridized. The probes were Ampullariella sp. strain 3876 D-xylose isomerase (D-XI) gene and oligonucleotides corresponding to the amino acid sequences of Streptom- yces M1033 D-XI. A strong hybridizing band of about 15 kb was found. The 9—20 kb DNA fragments were, recovered from the gel, cloned into EMBL 3 vector and screened with the same probes. The 13 kb insert DNA from a positive plaque was digested with SalI. A 2.5 kb DNA segment of the digestion was then subcloned into pUC18 (Assigned pUB1). Restriction mapping, partial sequence analysis of pUB1 displayed high homology with the S. violaceoniger. On the other hand, pBU1 was found to exhibit complementation with xyl-E, coli strain HB 101 on M_9 medium in which D-xylose was the sole carbon source. Therefore, it is concluded that there is Streptomyces M1033 D-XI gene in the pUB1 clone.

关 键 词：克隆 链霉菌 木糖异构酶 

分 类 号：Q785[生物学—分子生物学]