Chen S, Guan Y, Ying J, Li Z, Wang J, Li J
College of Biology, China Agricultural University, Beijing 100094, China.
Wei Sheng Wu Xue Bao. 2001 Oct;41(5):523-9.
The glnB and glnZ genes of A. brasilense have 70% homology at nucleotide sequence. glnB is located in a 3.7 kb Eco RI+ PstI fragment and glnZ is located in a 3.7 kb SalI fragment. Both glnB and glnZ genes were mutagenized by Kmr cassette insertions and glnB- and glnZ- mutants were obtained. glnB- mutant did not have any nitrogenase activity, while glnZ- mutant still has nitrogenase activity. The coding regions of glnB and glnZ were cloned into pVK100 vectors and recombinant plasmids pVK-II and pVK-Z were obtained, respectively. The recombinant plasmids pVK-II and pVK-Z were introduced into glnB- and glnZ- to produce C-glnB and C-glnZ, respectively. C-glnB can restore nitrogenase activity and C-glnZ does not have effect on nitrogenase activity. When pVK-II and pVK-Z were introduced into A. brasilense Yu62 and draT-, respectively, the Yu62-II (containing pVK-II) and draT-II (containing pVK-II) have higher nitrogenase activity than that of wild type Yu62. In contrast, Yu62-Z (containing pVK-Z) and draT-Z (containing pVK-Z) has no effect on nitrogenase activity. The nifA(-)-II (containg pVK-II) and nifA(-)-Z (containing pVK-Z) still have no nitrogenase activity.
巴西固氮螺菌的glnB和glnZ基因在核苷酸序列上具有70%的同源性。glnB位于一个3.7 kb的Eco RI + PstI片段中,glnZ位于一个3.7 kb的SalI片段中。glnB和glnZ基因均通过卡那霉素抗性盒插入进行诱变,并获得了glnB -和glnZ -突变体。glnB -突变体没有任何固氮酶活性,而glnZ -突变体仍具有固氮酶活性。glnB和glnZ的编码区分别克隆到pVK100载体中,分别获得重组质粒pVK-II和pVK-Z。将重组质粒pVK-II和pVK-Z分别导入glnB -和glnZ -中,产生C-glnB和C-glnZ。C-glnB可以恢复固氮酶活性,而C-glnZ对固氮酶活性没有影响。当将pVK-II和pVK-Z分别导入巴西固氮螺菌Yu62和draT -中时,Yu62-II(含有pVK-II)和draT-II(含有pVK-II)的固氮酶活性高于野生型Yu62。相反,Yu62-Z(含有pVK-Z)和draT-Z(含有pVK-Z)对固氮酶活性没有影响。nifA(-)-II(含有pVK-II)和nifA(-)-Z(含有pVK-Z)仍然没有固氮酶活性。