不吸水链霉菌基因文库的构建及初步筛选  

Construction and Initial Screening of Gene Library of the Streptomyces ahygroscopicus

作　　者：桓明辉[1] 陈飞[1] 吴红艳[1] 关艳丽[1] 王志[1] 程永涛[2] 

机构地区：[1]辽宁省微生物科学研究院,朝阳122000 [2]辽宁省朝阳市卫生学校,朝阳122000

出　　处：《生物技术通报》2009年第1期134-137,共4页Biotechnology Bulletin

摘　　要：采用碱裂解法从不吸水链霉菌提取染色体DNA,用Sau3AI对染色体DNA进行部分水解,利用透析袋法回收3.5～9kb之间的DNA片段。以pUC18为载体,用BamHI对其进行酶切,再用热敏磷酸酶去磷酸化,酶切产物与外源DNA按一定比例混合后,加入T4DNA连接酶进行连接。连接产物用受体菌E.coliDH5α进行转化,根据α-互补性质产生的颜色反应,挑选白色菌落,构建了相应的不吸水链霉菌基因文库。分别利用特异性探针2-1-1及1-2-1对不吸水链霉菌基因文库进行筛选,利用DIG-Ⅱ-dUTP对特异性探针进行标记,与基因文库中的阳性转化子进行Southern杂交,通过显色反应对杂交结果进行检测,由于时间关系,暂未筛选出阳性克隆,但这些工作对以后的相关实验研究具有很好的借鉴作用。The plasmid DNA of Streptomyces ahygroscopicus was extracted by soda spht.The plasmid DNA was partial hydrolyzed by Sau3AI,and 3.5-9 kh DNA fragment was wllected by dialyze bag. The camer-PUC18 was digested by BamHI,and the phosphate groups were removed off by Antarctic Phosphatase. Linking the DNA firagment and PUC18 by T4 DNA ligase. According to the color reaction of α-complementation,the gene hbrary was constructed by transforming the linking product into DH5α. The gene library of Streptomyces ahygroscopicus was screened by the specific probes 2-1-1and 1-2-1. Specific probes by DIG- Ⅱ -dUTP, and Southern hybridizated with masculine transformant were also involved,and results of which were examined by the colour reaction. Although the results of hybridization were not as expected,but still being worth for reference for associated research in the future.

关 键 词：不吸水链霉菌 基因文库 地高辛探针标记 SOUTHERN杂交 菌落原位杂交 

分 类 号：TQ455.5[化学工程—农药化工]