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Introduction and expression of foreign DNA in isolated spinach chloroplasts by electroporation
Author(s) -
To KinYing,
Cheng MingChih,
Chen LongFang Oliver,
Chen ShuChen Grace
Publication year - 1996
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.1996.10040737.x
Subject(s) - chloroplast , electroporation , reporter gene , biology , microbiology and biotechnology , chloramphenicol acetyltransferase , expression vector , gene expression , glucuronidase , gus reporter system , gene , spinach , chloroplast dna , chimeric gene , genetics , recombinant dna , biochemistry
An electroporation‐mediated method for the study of foreign gene expression within chloroplasts has been developed. The chloroplast expression vector pHD203‐GUS, which consists of coding regions for β‐glucuronidase (GUS) and chloramphenicol acetyltransferase (CAT) separated by a double psbA promoter fragment from pea (in opposite orientation) was electroporated into spinach chloroplasts and the transient gene expression was examined. Conditions for the expression of the reporter genes have been optimized. Both CAT and GUS activities were detected in chloroplasts electroporated with pHD203‐GUS, but not with nuclear expression vector pBI221 or negative control pUC18. No GUS activity was detected when pHD203‐GUS was electroporated into spinach protoplasts. Dot immunoblot analysis using anti‐GUS antibody confirmed the existence of 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 protein in treated chloroplasts was further confirmed by Western blot analysis.