慢生型大豆根瘤菌的结瘤基因转入快生型大豆根瘤菌中

NODULATION GENE OF BRADYRHIZOBIUM JAPONICUM TRANSFERRED INTO RHIZOBIUM FREDⅡ

通过三亲本杂交把慢生型大豆根瘤菌USDA110的基因文库转移至Tn5诱变的不结瘤的快生型大豆根瘤菌321,338中,用链霉素和四环素平板选择大量接合子,接种大豆,通过结瘤基因功能互补,获得7个根瘤,从中分离出的每个菌株都仍具有链霉素和四环素抗性。分离其质粒,发现每个菌株都多了一条较载体质粒pLAFR1分子量大的质粒。将这些质粒转移至大肠杆菌HB101中,分离其质粒,用^(32)P标记的nod探针进行DNA-DNA分子杂交,除载体质粒pLAFR1为阴性反应外,其他重组质粒均为阳性反应,即所获得pLAFR1克隆的DNA片段上确有nod结构基因。回收重组质粒pLAFR1::nod,用限制性内切酶EcoR Ⅰ进行酶切,从其琼脂糖凝胶电泳图上估计pLAFR1上克隆的DNA片段分子量为32kb。

The gene library of Bridyrhizobium japonicum USDA 110 was transferred into non-nodulation mutants (mutated with Tn5) of Rhizobium fredii USDA 191. The conjugants were inoculated in soybean to obtain Nod^+ strains which have the nod gene of USDA 110. Strains isolated from seven nodules are Sm^rand Tc^r and have a new plasmid larger than vector plasmid pLAFR1. Recombinant plasmids were transferred into E. coli HB101 for further study. They showed active reaction when hybridizing with nod probe. Digested with EcoRI, the recombinant plasmids all produced a 25kb fragment and a 7.0 kb fragment apart from the 21.6kb pLAFR1 fragment. So the fragement containing nod gene cloned into pLAFR1 is about 32kb.