当前位置: 首页 > 文章 > 导入外源DNA的大豆根瘤菌22-10基因工程菌株的构建与分析 华中农业大学学报 1993 (3) 255-264
Position: Home > Articles > CONSTRUCTION AND SCREENING OF BRADYRHIZOBIUM JAPONICUM ENGINEERING STRAINS BY THE METHOD OF FOREIGN DNA TRANSFER Journal of Huazhong Agricultural University 1993 (3) 255-264

导入外源DNA的大豆根瘤菌22-10基因工程菌株的构建与分析

作  者:
缪礼鸿;周俊初
单  位:
华中农业大学生物技术中心
关键词:
大豆根瘤菌;转移接合子;质粒
摘  要:
通过三亲本杂交,分别将紫云英根瘤菌7635R的基因文库和慢生型大豆根瘤菌22-10的基因文库引入到慢生型大豆根瘤菌22-10中,获得了大量的含有外源DNA片段的转移接合子。以结瘤试验作选择标准,从中筛选到3株具有较高固氮效率的基因工程菌株HN5-1、HN5-2和HN22-2。采用反向杂交,将接合子HN5-2中的外源重组质粒转回到大肠杆菌中,提取质粒作EcoRI酶切。表明pLAFR1质粒载体上携带有大约20 kb大小的DNA片段。实验还证明,在含有从根瘤菌转移而来的pLAFR1质粒的大肠杆菌细胞中,常伴随有辅助性质粒pRK2013的存在,用四环素和卡那霉素两种选择性抗性可淘汰其中的pRK2013质粒而仅保留有来自根瘤菌的pLAFR1重组质粒。
译  名:
CONSTRUCTION AND SCREENING OF BRADYRHIZOBIUM JAPONICUM ENGINEERING STRAINS BY THE METHOD OF FOREIGN DNA TRANSFER
作  者:
Miao Lihong Zhou Junchu (Bitoechnology Center, Huazhong Agricultural University, Wuhan 430070)
关键词:
Bradyhizobium japonicum, conjugants, plasmid
摘  要:
With the help of pRK2013 the gene libraries of Rhizobium huakuii 7653R and Bradyrhizibum japonicum 22-10 were separetely conjugated enmasse and transfered into the effective recipient strain B. japonicunm 22-10. The conjugants were washed and inoculated into soybean plants grown in pots. Based on the plant growth and acetylene reduction activity of nodules, three superior strains (HN5-1, HN5-2 and HN22-2) were obtained from conjugants of 7653RGB×22-10. A recombiant plasmid, pMR52 from strain HN5-2, was transfered into E. coli and then isolated. Digestion of this plamid with EcoRI created two fragments correspoding to vector pLAFR1 and foreign DNA of approximately 20kb. A new method was reported regarding the mobilization of recombiant plasmid from B. japonicum into E. coli.

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