Glucocorticoid-inducible transgene expression in loblolly pine ( Pinus taeda L.) cell suspension cultures

Abstract

A dexamethasone-inducible transgene expression system has been developed in transgenic loblolly pine ( Pinus taeda L.) cells. This system uses green fluorescent protein ( gfp) gene as a reporter gene and is based on the chimaeric transcriptional activator GVG, which is originally developed by T. Aoyama and N.-H. Chua [Plant J. 11 (1997) 605] and improved by P.B.F. Ouwerkerk et al. [Planta 213 (2003) 370]. We have inserted a fragment of m-gfp5 -ER into an optimized binary vector pINDEX3 and used this vector to produce transgenic loblolly pine cell lines via Agrobacterium-mediated transformation. Three transgenic cell lines were used to test the function of inducer. Though there are differences in transgene expression among transgenic cell lines, the highest level of green fluorescent protein expression was observed 48 h after addition of the glucocorticoid hormone dexamethasone and by the concentration of 5 mg/l for all three transgenic cell lines. Differential expressions of gfp at 24, 48, and 72 h after treatment of 0.5, 1, 5, and 10 mg/l dexamethasone, respectively, were confirmed by northern blot analysis and by quantitative fluorescence analyses of confocal images taken by a laser scanning microscope (LSM 510). This investigation has demonstrated that the gfp expressions in transgenic cell lines were tightly controlled by inducer and the inducible gene expression system could be useful in molecular physiology and functional identification of novel genes in cultured pine cells.