Introduction and expression of foreign DNA in isolated spinach chloroplasts by electroporation.
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 beta-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.
To
K Y
KY
Institute of Botany, Academia Sinica, Nankang, Taipei, Taiwan.
Cheng
M C
MC
Chen
L F
LF
Chen
S C
SC
D002458:Cell Fractionation; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D002736:Chloroplasts / Q000235:genetics*; D018274:Electroporation / Q000379:methods*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D005966:Glucuronidase / Q000096:biosynthesis / Q000235:genetics; D007118:Immunoassay; D010957:Plasmids; D018724:Spinacia oleracea / Q000235:genetics
D015500:Chloramphenicol O-Acetyltransferase; D005966:Glucuronidase