CLONING AND EXPRESSION OF THE IPT GENE

Abstract

Cytokinins are involved in in vitro shoot initiation, although 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 isopentenyl transferase (ipt) gene. The source of the Agrobacterium tumefaciens ipt gene was a 7.2 kb Eco RI fragment isolated from pBREF7 (Dr. Ann Smigocki, USDA, Beltsville, MD). The ipt gene was amplified by polymerase chain reaction (PCR) and cloned into pMal-cRI (New England Biolabs, Beverly, MA). Using the pMAL-cRI expression and purification system, the isopentenyl transferase protein will be purified for antibody production. These antibodies will be used in future work to localize the isopentenyl transferase enzyme.