Introduction of a chimeric gene encoding an oryzacystatin-β-glucuronidase fusion protein into rice protoplasts and regeneration of transformed plants.

In order to construct transgenic rice plant with an introduced oryzacystatin (OC)-β-glucuronidase (GUS) fusion gene, we first introduced it into rice protoplasts by electroporation, together with a marker gene conferring hygromycinresistance (pUC-HPH). In a transient assay using the transfected protoplasts, both OC and GUS activities were detected. The GUS activity was higher when the OC-GUS fusion protein was expressed than when only a single GUS protein was expressed. Next, to isolate stable transformants, hygromycin-resistant calli were selected. Forty one out of 116 hygromycin-resistant calli expressed a 2.2 kb mRNA transcribed from the chimeric gene and their extracts exhibited the activities of both OC and GUS. Finally, the transgenic calli were regenerated into rice plants whose tissues (leaves, roots and seeds) exhibited GUS activity probably derived from the fusion protein.