| Pak. J. Bot., 33(1): 43-52, 2001. | Back to Contents | ||||
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Updated: 01-12-10 | ||||
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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. National Institute for Biotechnology & Genetic Engineering (NIBGE), P.O. Box 577, Jhang Road, Faisalabad, Pakistan. |
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