Abstract
We have previously prepared functional single-chain variable fragments (scFvs) against bioactive gibberellins (GAs) from two independent hybridoma cell lines 8/E9 and 21/D13. In the present study, these scFv genes were used for immunomodulational study of GAs, i.e., the scFvs were produced in tobacco and Arabidopsis plants to disturb normal function of GAs. The expression vectors were designed to localize scFvs in various subcellular compartments, cytosol (CY), apoplastic space (AP), endoplasmic reticulum (ER), and outer leaflet of plasma membrane as glycosylphosphatidylinositol (GPI)-anchored proteins (GPI). The scFvs were expressed as fusion proteins with GFP, enabling direct detection of the expressed protein in the expected compartments. In both tobacco and Arabidopsis plants, more than several independent transgenic lines were obtained for each construct. In the first generation of tobacco transformants, production of scFv-GFP was confirmed by ELISA for all the constructs except for 8E/9CY. There was a tendency that the ER lines accumulate more scFv-GFP proteins than other lines (CY, AP, GPI). The results with ELISA were approximately consistent with the results of western analysis and GFP fluorescence insensity. Although CY, ER and AP lines showed expected localization, the compartment of GPI-anchored scFvs have not been determined. In the first and second generations of Arabidopsis, some of the ER lines are showing short stature than wild-type plants.
