The ipt gene of Agrobacterium tumefaciens T-DNA encodes for isopentenyl transferase, which is an enzyme active in cytokinin biosynthesis. While it is known that cytokinins are associated with in vitro promotion of cell division and stimulation of shoot production, little is known about their mode of action. As the first step in localizing cytokinin synthesis, we present a cloning and expression strategy for the ipt gene. The source of the ipt gene was Agrobacterium tumefaciens octopine Ti plasmid 15955. The ipt gene was amplified by the polymerase chain reaction (PCR) and cloned into pMal-c2 (New England Biolabs, Beverly, MA). This construct was transformed into E. coli and the ipt gene was expressed as a fusion protein. The protein was purified by affinity chromatography to serve as an antigen for polyclonal antibody production. These antibodies will be used to localize isopentenyl transferase in plant tissue.
Transformation of Nicotiana alata Link and Otto. with an Autoregulatory Senescence-inhibitor Gene Construct
