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

作　　者：农广[1] 张忠明[1] 胡福荣[1] 陈华癸[1] 

机构地区：[1]中山大学生物防治国家重点实验室,农业部华中农业大学农业微生物重点开放实验室

出　　处：《中山大学学报（自然科学版）》1998年第1期73-77,共5页Acta Scientiarum Naturalium Universitatis Sunyatseni

基　　金：国家攀登计划"共生体系中最佳结瘤固氮模型控制的研究"资助项目

摘　　要：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.

关 键 词：紫云英根瘤菌 nod基因 分子杂交 结瘤基因 克隆 

分 类 号：Q939.114[生物学—微生物学]