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

1)华癸中慢生根瘤菌大共生质粒的物理图谱。rengei：从华癸支原体（M.huakuii subsp.）Rengei B_3是从紫云英（Astragalus sinicus）中分离得到的一种多糖。用超粘粒载体从质粒的DNA片段构建基因文库。2）固氮和固氮相关基因的核苷酸序列测定：共测定了共生质粒pRhYM约80 kb的核苷酸序列。3）华癸木霉转化体系的建立：以pBBR质粒载体为载体，将华癸木霉亚属（M.huakuji subsp.）4）用转基因根瘤菌对人参进行转化，转化后的菌株华癸根瘤菌（M.huakuii subsp.）使用Rengei感染Renge-sou种子。从根瘤中分离出质粒pBBR，以根瘤菌为模式菌，以人参植株为研究对象，开展了共生工程研究。

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.