异形胞分化蓝细菌dnaA温度敏感型突变体的构建  

Construction of dnaA Temperature-Sensitive Mutants of Heterocyst Differentiated Cyanobacterium

作　　者：魏新元[1] 丑敏霞[2] 闻盼盼[1] 

机构地区：[1]西北农林科技大学食品科学与工程学院,陕西杨陵712100 [2]西北农林科技大学生命科学学院,陕西杨陵712100

出　　处：《西北植物学报》2010年第3期459-465,共7页Acta Botanica Boreali-Occidentalia Sinica

基　　金：陕西省自然科学基金(SJ08B12);西北农林科技大学人才基金(01140408)

摘　　要：以能分化异形胞的蓝细菌(Anabaenasp.PCC7120)为材料,采用重组PCR在体外对控制DNA复制起始的dnaA基因进行定点突变后克隆到整合质粒中,再通过三亲本杂交将整合质粒转移到Anabaena PCC7120中,以分离和筛选温度敏感型突变体。结果成功获得Anabaena PCC 7120 dnaA高温敏感性突变体。研究表明,利用重组PCR技术可在体外实现对Anabaena PCC 7120的dnaA的定点突变,并可通过同源重组双交换成功实行整合质粒中突变基因对野生型基因的置换,使突变基因插入到细胞染色体中,进而成功构建温度敏感型突变菌株。To construct temperature-sensitive mutant of heterocyst developmental cyanobacterium Anabaena sp.PCC 7120,mutations were introduced into dnaA,the initiation gene of DNA duplication,by means of recombinant polymerase chain reaction （PCR） in vitro.The mutational dnaA was inserted into integration plasmid and the result plasmid was subsequently transferred into Anabaena PCC 7120 through three-parent conjugation to isolate and screen the temperature-sensitive mutants.The result showed that the high temperature-sensitive mutants of Anabaena PCC 7120 were successful constructed.The natural dnaA in Anabaena PCC 7120 chromosome could be replaced by mutative dnaA through homologous recombination double-crossover following overlap PCR mediated site-directed mutagenesis of dnaA in vitro.The study suggested that temperature-sensitive mutant could be constructed by dnaA mutating.

关 键 词：温度敏感型 DNAA 定点突变 异形胞 

分 类 号：Q789[生物学—分子生物学]