华癸中生根瘤菌共生质粒的定向标记、消除与结瘤功能的鉴定

采用Tn5-mob-sacB转座子对华癸中生根瘤菌(Mesorhizobium huakuii)菌株7653R的共生质粒进行定向标记,获得该质粒标记菌株7653RT14.利用sacB基因对蔗糖的敏感性,对标记质粒进行消除实验,获得7653R的共生质粒消除突变株7653R-1.测得Tn5-mob-sacB转座频率高于10-5.突变株的培养特征与出发菌株基本一致.采用琼脂管法对7653RT14和7653R-1进行回接实验,结果显示7653RT14能正常结瘤固氮,表明Tn5的插入并未影响其共生能力,但失去共生质粒的7653R-1则为不结瘤或只结个别小瘤.稳定性实验结果表明供试菌株的标记质粒在本实验条件下是稳定的,可以作为共生质粒转移的供体菌.

Labeling, Curing and Identification of Nodulation Ability of Symbiotic Plasmids in Mesorhizobium huakuii

The symbiotic plasmid pMh7653Rb of Mesorhizobium huakuii strain 7653R was labeled by Tn5-mob-sacB insertion, the mutant strain 7653RT14 labeled its symbiotic plasmid was obtained . Plasmid curing experiment was carried out using the sacB positive selection method, the derivative 7653R-1 of symbiotic plasmid cured was obtained. Transpositional frequency of transposon Tn5-mob-sacB was higher than 10-5. Cultural characteristics of the mutant derivatives were similar to wild type strain 7653R. The nodulation tests presented that 7653RT14 could form pink effective nodules, the result showed that pMh7653Rb inserted by Tn5-mob-sacB did not lost nodulation ability. However, 7653R-1 lost nodulation ability or only formed a few small null nodules. The results of stability tests indicated that 7653RT14 carrying symbiotic plasmid inserted by Tn5-mob-sacB was stably inherited under the conditions tested, and could be used as donor strain of symbiotic plasmid transfer.