PJB-2001-6
UTILIZING HETEROLOGOUS PROMOTERS TO EXPRESS GREEN FLUORESCENT PROTEIN FROM JELLYFISH IN TOBACCO CHLOROPLASTS
MUHAMMAD SARWAR KHAN
Abstract
The green fluorescent protein (GFP) from the jellyfish (Aequorea victoria) has become a vital reporter not only to identify and screen transformed organisms including bacteria, animals and plants but also to study gene expression. A modified form of the green fluorescent protein was expressed in tobacco (Nicotiana tabacum var. Samsun) chloroplasts using both the bacterial as well as chloroplast specific promoters. A number of species-specific promoters have been used to express foreign DNA in chloroplasts, but there is no such report where DNA has been expressed in chloroplasts from bacterial promoters. This is the first report of stable expression of reporter gene (gfp) in chloroplasts using bacterial promoter. The GFP fluorescence was detected only in transformants where the trc promoter used to regulate gfp. In transformants where gfp was under the control of the chloroplast rrn promoter, fluorescence was comparable to controls without an introduced gfp gene. The transformed seedlings gave a green fluorescence after illumination with long-wave UV light. Fluorescence excitation and emission spectra of leaf extracts from the transformed plants confirmed the presence of GFP. Analysis of high expressing lines was carried out using Confocal Laser Scanning Microscopy. Gfp was found as a versatile and sensitive reporter and can be used to study promoters. The bacterial trc promoter appeared to be stronger than the chloroplast rrn promoter in E. coli as well as in chloroplasts of tobacco.
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