Introduction of Maize Glutamine Synthetase (MGS)c DNA into Agrobacterium Tumefaciens

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