过刊浏览

阴沟肠杆菌(nifL~(-)A~(c))工程菌株的构建

邱德义, 沈炳福

中科院上海植物生理研究所.上海 200032

摘要

采用基因定位突变的方法,在体外把来自pBR322的四环素抗生(Tc~(r))基因片段插入由pST1142的质粒所携带的阴沟肠杆菌nifL基因3^-端的SmaⅠ位点,再经细胞体内同源基因片段的重组交换,选择获得了在染色体上nifL突变的阴沟肠杆菌E12和E13,两者Tc~(r)基因插入nifL的转录方向相反。经分析显示,E13(nifL~(-))由于Tc~(r)基因插入后,在nifA上游产生具有启动子功能的核苷酸序列(-TTTCATA-),激活nifA的表达。当E13和E12被引入多拷贝组成型nifA质粒pBF101后,在有氨条件下nif基因去阻遏表达,呈高的固氮酶活力,与野生型E26(pBF101)比较,其比活力提高近1倍。

关键词： 基因表达; nifL; nifA; 阴沟肠杆菌

Construction of genetically engineered strains of Enterobacter cloacae (nifL~(-)A~(c)) by gene targeting mutation

Qiu Deyi, Shen Bingfu

Shanghai Institute of Plant Physiology,Chinese Academy of Scioences.Shanghai 200032

Abstract

E. cloacae E26 nifL mutation in chromosome was constructed by in vitro insertion of Tc~(r) gene into the Sma I site located in the nifL 3^-terminus f plasmid pST1142 and by in vivo homologous DNA recombination. Recombinants (E12 and E13) were obtained by selection, both the Tc~(r) gene inserted in the chromosome nifL in the opposite transcription orientation. A promoter-like nucleotide sequence (-TTTCATA-) was produced in the upstream region of the nifA in E13.

Key words: Gene expression;nifL;nifA;Enterobacter cloacae

收稿日期：　　录用日期：

通讯作者：　　E-mail:

引用本文：

邱德义, 沈炳福. 阴沟肠杆菌(nifL~(-)A~(c))工程菌株的构建[J]. 生理学报 1999; 51 (3): .

Qiu Deyi, Shen Bingfu. Construction of genetically engineered strains of Enterobacter cloacae (nifL~(-)A~(c)) by gene targeting mutation. Acta Physiol Sin 1999; 51 (3): (in Chinese with English abstract).