CAJ | 학술논문

以能分化异形胞的蓝细菌(Anabaena sp.PCC 7120)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 PCC 7120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.
이능분화이형포적람세균(Anabaena sp.PCC 7120)위재료,채용중조PCR재체외대공제DNA복제기시적dnaA기인진행정점돌변후극륭도정합질립중,재통과삼친본잡교장정합질립전이도Anabaena PCC 7120중,이분리화사선온도민감형돌변체.결과성공획득Anabaena PCC 7120 dnaA고온민감성돌변체.연구표명,이용중조PCR기술가재체외실현대Anabaena PCC 7120적dnaA적정점돌변,병가통과동원중조쌍교환성공실행정합질립중돌변기인대야생형기인적치환,사돌변기인삽입도세포염색체중,진이성공구건온도민감형돌변균주.
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.