Poster: Late and Moved Abstracts

Abs # 1378: Characterization of transgenic tobacco cell culture secreting functional green fluorescent protein

Presenter:	Guan, Peizhu , peizhu@hawaii.edu
Authors	Guan, Peizhu  (A)   Su, Wei-Wen  (A)
Affiliations:	(A): Department of Molecular Bioscience and Bioengineering, University of Hawaii at Manoa

We established tobacco cell cultures (Nicotiana tabacum cv Xanthi) expressing green fluorescent protein (GFP) localized to the cytosol, the endoplasmic reticulum, or the extracellular compartment. An N-terminal Arabidopsis basic chitinase signal sequence was used for ER targeting (SP-GFP), and it was used along with a C-terminal HDEL tetrapeptide to retain GFP on the ER network (SP-GFP-HDEL). Highly efficient secretion of fluorescent GFP from transgenic plant cell culture expressing SP-GFP was observed. Western blot analysis indicated secreted GFP had the correct molecular weight. The fluorescence spectrum of the spent medium mimics that of the pure GFP, indicating GFP as the predominant fluorescent compound in the medium. Over the course of the culture, fluorescence signal of the spent medium linearly correlated with the secreted GFP concentration, which provide a tool to quantitate the GFP product. Upon brefeldin A (BFA) treatment, GFP secretion was substantially reduced, and several bright spots with highly localized GFP accumulation were noted on the reticulate network. Such an intracellular GFP distribution is distinct from that seen in the cells expressing SP-GFP-HDEL. These results provide further evidences that the presence of extracellular GFP in the tobacco cell culture is a result of regulated secretion. The secretory GFP reporter provides a useful tool for studying protein trafficking in plant cells.