不带抗药性基因的nifA质粒的构建  

Construction of a nifA containing Plasmid Without Antibotics resistant Genes

作　　者：叶志华[1] 梁建庆[1] 陶家法[1] 丘元盛[1] 

机构地区：[1]广东省微生物研究所

出　　处：《农业生物技术学报》1998年第4期393-397,共5页Journal of Agricultural Biotechnology

基　　金：广东省自然科学基金

摘　　要：本研究克隆了萤光假单胞菌ＡＳ１．５５（ＰｓｅｕｄｏｍｏｎａｓｆｌｕｏｒｅｓｃｅｎｓＡＳ１．５５）的亮氨酸基因（ｌｅｕ＋）ＥｃｏＲＩ片段（～６．６ｋｂ），并获得含有该片段的重组质粒ｐＢＲ３２２－ＬＥＵ。从ｐＢＲ３２２－ＬＥＵ质粒中分离出ｌｅｕ＋ＥｃｏＲⅠ片段，将其插入到ｎｉｆＡ质粒ｐＭＣ７１Ａ的ＥｃｏＲⅠ位点使氯霉素抗性基因（Ｃｍｒ）失活，从而构建了不带抗药性基因的ｎｉｆＡ质粒ｐＭＣ７１Ａ－ＬＥＵ。用ｐＭＣ７１Ａ－ＬＥＵ转化３０株ｌｅｕ－水稻根际固氮菌阴沟肠杆菌Ｅ２６（ＥｎｔｅｒｏｂａｃｔｅｒｃｌｏａｃａｅＥ２６），其中一株Ｅ２６－１能够互补，转化频率为１×１０３转化子／μｇ质粒ＤＮＡ。ｐＭＣ７１Ａ－ＬＥＵ在Ｅ２６－１菌株中不够稳定，在ＬＢ培养基中连续培养３０代后，丢失率为６０％～８０％。Ｅ２６－１（ｐＭＣ７１Ａ－ＬＥＵ）与亲株Ｅ２６（ｐＭＣ７１Ａ）相比，耐铵固氮能力没有变化。A 6.6kb Eco RⅠ fragment of the gene encoding for leucine biosynthetase from Pseudomonas fluorescens strain AS1.55 was cloned using plasmid pBR322 as the vector. The leu + Eco RⅠ fragment was isolated from the plasmid pBR322 LEU and was inserted into the Eco RⅠ site of chloramphenicol resistant gene in the nif A plasmid pMC71A. A resultant nif A plasmid pMC71A LEU was obtained and resulted in the inactivation of the resistance to chloramphenicol. Among thirty strains of the leu diazotroph Enterobacter cloacae E26 in the rice rhizosphere transformed with the plasmid pMC71A LEU, only one strain E26 1 can be complemented with a transformation frequency of 1×10 3 transformants/μg plasmid DNA. The plasmid pMC71A LEU was not stable in the strain E26 1, and the plasmid could be lost at a rate of 60%～80% after thirty generations cultivated in LB medium. As compared to its parental strain E26(pMC71A), the E26 1(pMC71A LEU) strain has no much changed in the capability of nitrogen fixation and the resistance to ammonia.

关 键 词：nifA质粒 基因克隆 pMC71A 质粒构建 阴沟肠杆菌 

分 类 号：S188[农业科学—农业基础科学] S154.381