用Tn5标记带发光酶基因的华癸根瘤菌(Mesorhizobium huakui)7653R质粒

在ｐＲＫ２０７３的辅助作用下，通过三亲本接合转移，将Ｔｎ５（ｓａｃＢ－ｌｕｘＡＢ）插入华癸根瘤菌７６５３Ｒ菌株基因组中。将３２００个Ｔｎ５标记菌株影印到含有８％蔗糖的ＹＭＡ平板，检测这些菌株的发光酶活性和新霉素抗性。共获得８个菌株，其选择标记消失。质粒检测发现其共生质粒有不同程度的缺失甚至消除。结瘤实验证明，所有共生质粒部分缺失（有的缺失达１／３）的菌株依然结瘤。经用ｌｕｘＡＢ探针的分子杂交证实，８个菌株中有３个对应的标记菌株其Ｔｎ５插在共生质粒上。

Mesorhizobium huakuii 7653R HARBOURING PLASMIDS ESSENTIAL TO \=SYMBIOSIS MARKED BY Tn5luxABsacB

With the helper pRK2073,the genome of Mesorhiobium huakuii 7653R was inserted randomly with Tn5sacBluxAB by triconjugation. 3200 transconjugants were obtained and replicated to YMA plate with 8% sucrose.8 colonies (neo-luxAB-sacB-) were selected with the examination of the activities of sacB and luxAB. The plasmid profiles were checked by the modified Eckhardt's method and partial deletions on the pSym and the whole pSym deletion were discovered .However the mutational colonies except one without pSym are able to nodulate on Astragalus sinicus L. even though approximate 1/3 part of Sym plasmid were deleted. Hybridization of the plasmid DNA of Tn5marked colonies corresponding to the mutational colonies with luxAB probe confirms 3 colonies with the Tn5 insertion on the symbiotic plasmid.