摘要
采用基因定位突变的方法，在体外把来自ｐＢＲ３２２ 的四环素抗性（Ｔｃｒ） 基因片段插入由ｐＳＴ１１４２ 质粒所携带的阴沟肠杆菌ｎｉｆＬ基因３’端的Ｓｍａ Ⅰ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Ｔｃｒ 基因插入ｎｉｆＬ的转录方向相反。经分析显示，Ｅ１３（ ｎｉｆＬ－ ） 由于Ｔｃｒ 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
E. cloacae E26 nifL mutation in chromosome was constructed by in vitr o insertion of Tc r gene into the Sma Ⅰ site located in the nifL 3' terminus of plasmid pST1142 and by in vivo homologous DNA recombination (Fig.1). Recombinants (E12 and E13) were obtained by selection, both had Tc r gene inserted in the chromosome nifL in the opposite transcription orientation. A promoter like nucleotide sequence ( TTT CATA ) was produced in the upstream region of the nifA in E13. We investigated the effect of the nifL mutation on the nif gene transcription in E. cloacae by monitoring β galactosidase synthesis and assaying acetylene reduction. Recombinant E12 has lost the nitrogen fixation activity. The gene nifA was expressed constitutively in E13, and nitrogenase activity was derepressed to a low level in the presence of NH + 4 . In both E13 and E12, carrying a multicopy plasmid pBF101 with the constitutive nifA , a nitrogenase activity as high as 66% in an excess of NH + 4 was constitutively expressed (Tables 2,3). The recombinant strains ( nifL -A c) thus obtained will be useful in enhancing associative nitrogen fixation in the rhizosphere of rice plants.
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