| 苜蓿根瘤菌绿色荧光蛋白标记株的构建及对菌株结瘤固氮能力的影响 |
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| 引用本文： | 杨晓玫,周彤,阿芸,师尚礼.苜蓿根瘤菌绿色荧光蛋白标记株的构建及对菌株结瘤固氮能力的影响[J].草原与草坪,2018(1):44-49,56. |
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| 作者姓名： | 杨晓玫 周彤 阿芸 师尚礼 |
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| 作者单位： | 甘肃农业大学草业学院/草业生态系统教育部重点实验室/甘肃省草业工程实验室/中-美草地畜牧业可持续发展研究中心,甘肃兰州,730070 |
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| 摘 要： | 以苜蓿中华根瘤菌Sinorhizobium meliloti12531,Sinorhizobium meliloti343和苜蓿根瘤菌Rhizobium meliloti GN5为受体菌,以Escherichia coli pRK2073为辅助菌及绿色荧光蛋白GFP104为供体菌,采用三亲本杂交法进行结合转导。以Winogradsky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,再对选出的荧光标记菌株以甘农5号紫花苜蓿和WL.343紫花苜蓿为宿主进行回接验证,测定了回接植物的生物量,结瘤数和标记菌的占瘤率等指标。结果表明:(1)三亲本杂交法适用于苜蓿根瘤菌GFP荧光标记菌株的构建,能以S.meliloti 343、R.meliloti GN5和S.meliloti 12531为出发菌株成功构建荧光标记菌株;(2)传代筛选后得到的荧光标记菌株中,S.meliloti 343-gfp2、R.meliloti GN5-gfp4和S.meliloti12531-gfp4遗传稳定性好,荧光表达量高;(3)含抗生素Winogradsky无氮培养基平板筛选与现有含抗生素平板分离筛选法相比,能显著提高荧光标记根瘤菌株的筛选效率;(4)获得的苜蓿根瘤菌荧光标记菌株间无显著差异,对标记菌株形成根瘤的固氮能力,及标记菌株对苜蓿植株生物量的影响进行比较,GFP荧光标记根瘤菌具有较高的遗传稳定性。
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| 关 键 词： | 苜蓿根瘤菌 荧光蛋白 荧光标记菌构建 三亲本杂交 alfalfa rhizobium green fluorescent protein fluorescent marker tri-parental hybridization root nodules |
Establishment and nodular nitrogen fixation effect of green fluorescent protein labeled strains of alfalfa rhizobia |
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| YANG Xiao-mei,ZHOU Tong,Ayun,SHI Shang-li.Establishment and nodular nitrogen fixation effect of green fluorescent protein labeled strains of alfalfa rhizobia[J].Grassland and Turf,2018(1):44-49,56. |
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| Authors: | YANG Xiao-mei ZHOU Tong Ayun SHI Shang-li |
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| Abstract: | Recipient bacteria strains Sinorhizobium meliloti 12531,Sinorhizobium meliloti 343 and Rhizobium meliloti GN5,and assistant bacteria strain Escherichia coli pRK2073 and donor strain E.coli GFP104,which provided green fluorescent protein,were transformed using tri-parental hybridization method in this study.The genetic stability of fluorescence expression and nitrogen fixation of these successful transgenic strains were evaluated in Winogradsky nitrogen-free medium and yeast tryptone agar (TY) medium.The fluorescent strains were inoculated to Medicago sativa cv.No.5 Gannong and Medicago sativa cv.343 to test strain effects.The results indicated that the tri-parental hybridization method was a feasible method for establishing of gfp marked rhizobia stains,which provided gfp labeled strains of S.meliloti 343,R.meliloti GN5 and S.meliloti 12531.The strains of S.meliloti 343-gfp2、R.meliloti GN5-gfp4 and S.meliloti 12531-gfp4 had the highest genetic stability with high fluorescence expression.Compared with the existing antibiotic plate separation and screening method,the screening of antibiotic Winogradsky nitrogen-free medium can significantly improve the screening efficiency of fluorescently labeled rhizobial strains.The difference of growth characteristic was insignificant between different marked alfalfa rhizobia stains.GFP fluorescence labeling rhizobia had a high genetic stability considering nitrogen fixation and plant biomass matter. |
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