Introduction of Maize Glutamine Synthetase (MGS)c DNA into Agrobacterium Tumefaciens
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摘要: 用粘端连接法将构建于Escherichia coli FDB213菌株中的玉米谷氨酰胺合成酶(MGS)c-DNA亚克隆于由农杆菌质粒改造的pBI121载体中。在测定的72个转化菌中有7株(9.2%)插入MGSc-DNA片段。亚克隆后的质粒DNA用HindII和BstxI酶解可确定其c-DNA片段的插入方向。结果显示,P9、P15、P16与P17为正向插入,P2为反向插入的菌株。P16质粒DNA进一步用直接基因转化法——冻融法(Freeze-thaw)导入农杆菌15955菌株。经Southern印迹法与DNA杂交法测定表明,转化菌A11已携带MGSc-DNA片段。Abstract: The maize glutamine synthetase c DNA in Escherichia coli FDB213 was firstly subcloned into pBI121 vector by using sticky end ligation Seven of seventy two colonies tested (9.7%) were identified to be inserted with MGS c DNA The orientation of inserted c DNA fragments was measured by the double digestion of the subcloned plasmid DNAs with Hind III and Bstx I endonucleases Strains P9、P15、P16 and P17 have the insert from 5’ to 3’ (sense) and P2 from 3’ to 5’ (antisense) The subcloned plasmid DNA of P16 was further introduced into Agrobacterium tumefaciens 15955 by using direct gene transformation,freeze thaw method The transformed colonies were preliminarily selected by antibiotic screening method,50 u/ml kanamycin was supplied in LB medium,then the transformation was confirmed by Southern blot analysis A11 was identified to carry MGS c DNA fragment
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[1] Gasser C S and Fraley R T.G enetically engineering plants for cropim provement.Science.1989,244:1293-1299 [2] Mifl in and Lee.A m monia ass imilation:the Biochemis try of plant s.Vol.5.Edited by B.J.Miflin.A cademic Press.1980,169-202 -
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