紫云英根瘤菌结瘤基因调控片段克隆和分析

Cloning and Analysis of the Regulatory DNA Fragment of Symbiotic Genes in Rhizobium huakuii- Strain 7653R

通过对紫云英根瘤菌７６５３Ｒ及其Ｎｏｄ－突变株７６５３Ｒ－１侵染根毛试验证实，７６５３Ｒ菌株３１８×１０３ｂｐ大质粒决定早期结瘤功能，分子杂交结果显示，其ｎｏｄＤＡＢＣ定位于该质粒上．将７６５３Ｒ菌株的大质粒ＤＮＡ进行酶切并克隆到表达载体ｐＭＰ２２０上，构建ｌａｃＺ重组子文库，将ｌａｃＺ重组子文库导入７６５３Ｒ菌株，在加有Ｘ－ｇａｌ和种子浸提物的根瘤菌培养基上选择蓝色菌落．通过Ｍｉｌｅｒ试验测定它们的β－半乳糖苷酶活性，获得１株种子浸提物对其有诱导效应的菌株ＨＮ１８，对该菌株所含的重组质粒ｐＨＮ１８进行ｎｏｄＤＡＢＣ探针杂交，发现有１条１．７×１０３ｂｐ的阳性杂交带。

The experiment of rhizobia inoculation with the Rhizobium huakuii- strain 7653R and its mutant Nod- strain 7653R1 showed that the 318×105 bp large plasmid in strain 7653R was responsible for early nodulation. The Nod- strain 7653R1 was cured of the 318×105 bp plasmid and unabled to cause root hair curling, which means that the rhizobialegume interaction stops before or at the Hac stage. Hybridization of probe nodDABC showed that the tested genes locate on the 318×105 bp plasmid of the strain 7653R. Hence, the 318×105 bp plasmid was identified as the symbiotic plasmid in strain 7653R. The large plasmids of strain 7653R was digested with restriction enzyme EcoRI and cloned into the expression vector pMP220. A gene library was construted,containing different recombinant plasmids harbouring LacZ gene,which were transferred into the recipient strain 7653R. Tritransconjugants were selected onto plates containing Xgal and seed extract. Five blue colonies were assayed of their βgalactosidase activity after incubation with or without seed extract. A positive inducable strain HN18 was obtained. Hybridization between nodDABC probe and recombinant plasmid pHN18 showed a 17×103 bp positive band, which means that the early nodulation genes have been partially cloned. The evidence suggests that the pHN18 contains a promoter of nod operon.