摘要
以携带质粒pAM120(Ap^r,Tc^r/Tn916)的大肠杆菌(E.coli CG120)为供体菌株,与受体菌巴西固氮螺菌(Azospirillum brasilense)采用滤膜接合法进行接合转移,在选择平板上得到具较高频率的接合子(10^(-5)/每个供体菌,选择四环素抗性)。从846株四环素抗性接合子中进一步用奈氏法筛选得到氨分泌突变株3株。在无氮培养基上,其氨分泌量可达7.5～14.0mmol/L。用乙炔还原法分析氨分泌突变株在不同浓度氮源上的固氮活力,发现20mmol/L NH Cl的存在不抑制其固氮活性,固氮活力与无氮条件下野生株的活力相差不多。无选择压力下细胞分裂50代后的稳定性实验证明,转座子Tn916在氨分泌突变株中的稳定性在50％～80％之间。以固氮螺菌氨分泌突变株为供体菌株,对E.coli HBX1进行反向接合转移实验,证实Tn916确实存在于氨分泌固氮螺菌接合子中。
Cell conjugation was carried out between the donor E. coli CG120(pAM120:Ap^r, Tc^r/ Tn916) and the recipient Azospirillum brasilense Sp7 by filter mating. The transconjugants were obtained from a selective medium containing tetracycline with high frequency (10^(-5)/ per donor). Three NH_4^+-excreting mutation strains were screened by method of Naishi from 864 Tc^r transconjugants. The quantifies of excreted ammonium by mutants were 7.5～14.0retool/L in nitrogen-free medium. Enzyme activities involved in nitrogen fixation were not affected by the mutation. The acetylene reduction activity was not inhibited in the presence of 20retool/L NH_4Cl. The data suggest that the mutation occured at a site which regulates ammonia transport funtion. The stability experiment in the absence of selective pressure demonstrated that the stability of mutation strains were between 50%～80% after 50 generations.
