INTRODUCTION AND EXPRESSION OF FOREIGN DNA IN ISOLATED SPINACH-CHLOROPLASTS BY ELECTROPORATION

An electroporation-mediated method for the study of foreign gene expre ssion within chloroplasts has been developed. The chloroplast expressi on vector pHD203-GUS, which consists of coding regions for beta-glucur onidase (GUS) and chloramphenicol acetyltransferase (CAT) separated by a double psbA promoter fragment from pea (in opposite orientation) wa s electroporated into spinach chloroplasts and the transient gene expr ession was examined. Conditions for the expression of the reporter gen es have been optimized. Both CAT and GUS activities were detected in c hloroplasts electroporated with pHD203-GUS, but not with nuclear expre ssion vector pBI221 or negative control pUC18. No GUS activity was det ected when pHD203-GUS was electroporated into spinach protoplasts. Dot immunoblot analysis using anti-GUS antibody confirmed the existence o f GUS protein in chloroplasts electroporated with chloroplast-specific vector but not the negative controls, excluding the possibilities of endogenous GUS or bacterial contamination. The expression of GUS prote in in treated chloroplasts was further confirmed by Western blot analy sis.