Symbiotic engineering with green manure leguminous plant and nitrogen-fixing rhizobia

1)华葵中根菌一个大型共生质粒的物理图谱。rengei：从M.huakuii亚种制备了一个大质粒pRhYM。利用脉冲场电泳技术对从黄芪中分离得到的菱革B3进行了分离。基因文库是由质粒的DNA片段与一个超级宇宙载体构建而成的。通过杂交分析得到的文库长度约为300 kb。2)固氮结瘤相关基因的核苷酸序列：共确定了共生质粒pRhYM约80kb的核苷酸序列。在该序列上，定位到了nif和nod基因的几个簇。3)利用pBBR质粒载体，建立华氏菌转化体系。用转基因根瘤菌转化仁革菇，在仁革菇上形成根瘤的效率为10^5.4)。仁革被用来感染仁革苏种子。根形成根瘤，从根瘤中分离到质粒pBBR。因此，共生工程的发展目前正在以根瘤菌为模型的连翘植物和根瘤菌两方面取得进展。

1) Physical map of a large symbiotic plasmid in Mesorhizobium huakuii subsp. rengei :A large plasmid, pRhYM was prepared from M.huakuii subsp. rengei B3, which was isolated from renge-sou (Astragalus sinicus), by pulse field electrophoresis. The gene libraries were constructed from DNA fragments of the plasmid with a super cosmid vector. The libraries were analyzed by hybridization and made contig of about 300 kb.2) Nucleotide sequence of genes involved in nitrogen fixation and nodulation :A total about 80 kb nucleotide sequence of the symbiotic plasmid pRhYM was determined. On this sequence, several clusters of the nif and nod genes were located.3) Development of transformation system of M.huakuii :By using pBBR plasmid vector, M.huakuji subsp. rengei was transformed at the efficiency of 10^5.4) Nodule formation on renge-sou with the transgenic Rhizobium :The transformed strain of M.huakuii subsp. rengei was used to infect renge-sou seeds. The roots formed nodules, and the plasmid pBBR was isolated from the nodules.Thus, development of symbiotic engineering is now progress both in renge-sou plant and Rhizobium as a model.