摘要
荧光标记根瘤菌在研究根瘤菌侵染宿主形成结瘤时具有良好的示踪效果。本研究以辅助菌株Escherichia coli pRK2073,受体菌苜蓿中华根瘤菌Sinorhizobium meliloti 12531和苜蓿根瘤菌Rhizobium meliloti GN5,及cfp青色荧光基因供体菌E.coli pMP45179为供试菌株,以三亲本杂交法进行结合转导,并以无氮培养基和TY培养基对标记菌株进行荧光表达及固氮特性的遗传稳定性检测,再对初选菌株以甘农5号紫花苜蓿为宿主进行回接验证,测定了回接植株的生物量,结瘤数和标记菌的占瘤率等指标。结果表明,1)三亲本杂交转导法适用于苜蓿根瘤菌cfp标记菌株的构建,获得大量S.meliloti 12531和R.meliloti GN5的荧光标记株;2)经逐层筛选获得的荧光标记菌中,S.meliloti 12531-cfp6和R.meliloti GNf-cfp5遗传稳定性好,荧光表达量高,结瘤促生能力强;3)与现有抗生素平板分离筛选相比,无氮培养基结合耐药平板筛选能显著提高荧光标记根瘤菌株的甄别筛选效率;4)三亲本杂交法获得的苜蓿根瘤菌荧光标记株个体间差异较大,对标记株固氮及荧光表达能力遗传稳定性的验证和对宿主植物的结瘤促生能力的检测是cfp荧光标记根瘤菌筛选的必要手段。
Fluorescent protein labeled strains of rhizobia play an important role in the research about the progress of strain infecting plant and forming nodules,as well as determining strain competitive capability.In the present study,assistant bacteria strain Escherichia coli pRK2073,recipient bacteria strains Sinorhizobium meliloti 12531 and Rhizobium meliloti GN5,and donor bacteria strain E.coli pMP45179 which provided cyan fluorescent protein were transformed based on tri-parental hybridization technology.The genetic stability of fluorescence expression and nitrogen fixation of these successful transgenic strains were evaluated on N-free and TY medium.Then,the preliminarily screened stains were inoculated to alfalfa cv.Gannong No.5to study their effects.The results indicated that tri-parental hybridization method was feasible for construction of cfp labeled rhizobia stains which provided abundant cfp labeled strains of S.meliloti 12531 and R.meliloti GN5.The strains of S.meliloti 12531-cfp6 and R.meliloti GNf-cfp5 had the best genetic stability with more fluorescence expression and better nodulation effects.N-free medium with antibiotics have higher screening efficiency than the current medium with antibiotics.The large variations existed among these labeled strains which suggested that it was necessary to confirm the genetic stability of fluorescence expression,N-fixation and the effects on growth for cfp labeled strains selection.
出处
《草业科学》
CAS
CSCD
北大核心
2015年第5期711-718,共8页
Pratacultural Science
基金
国家自然科学基金项目(31300389)
贵州省科学技术基金项目(黔科合J字[2014]2136号)
贵州师范学院博士项目(14BS020)
贵州环境科学省级特色重点学科专项基金
关键词
苜蓿根瘤菌
荧光标记
三亲本杂交
菌种筛选
根瘤
Rhizobium meliloti
fluorescent marker
triparental hybridization
strains screening
root nodules
