IN-SITU TRANSIENT EXPRESSION OF THE ESCHERICHIA-COLI BETA-GLUCURONIDASE MODIFIED AT THE GLYCOSYLATION SITE

The beta-glucuronidase (GUS) gene from Escherichia coli has been used as a reporter for gene fusion studies in E. coil, yeast and plants. Es pecially in a wide variety of plants, it has been extensively used to examine the promoter strength, tissue- and stage-specific expression o f many genes. However, it can not be used as a reporter for secretion and targeting of proteins since it loses its activity by glycosylation in the ER. To overcome this problem, I have destroyed the glycosylati on site in the GUS gene using site directed mutagenesis. The asparagin e residue at the glycosylation site was substituted by glutamine, lysi ne and serine, respectively. After being fused to a strong promoter ei ther with or without sequences coding a signal peptide, modified GUS g enes were separately introduced into the aleurone layer of rice seeds using a particle gun. The enzymatic activity of transiently expressed GUS was assayed in situ. The results revealed that only serine-substit uted GUS is active and the others lost their activity due to mutations . Moreover, the in situ expression assay of GUS with a signal peptide revealed that serine-substituted GUS retains activity even when sent t o ER, by which the wild type loses its activity, The result clearly sh ows that modified GUS is suitable as a reporter in gene fusion studies for secretion and targeting of proteins.