Construction and Expression in Tobacco of a β-Glucuronidase (GUS) Reporter Gene Containing an Intron Within the Coding Sequence

Abstract

An Intron-GUS reporter gene which contains a modified intron of the castor bean catalase gene within the N-terminal part of the β-glucuronidase (GUS) coding sequence was constructed. The modified 190 bp intron sequence contains a termination codon in the same reading frame as GUS coding sequence near the 3′ splice site so that removal of the intron from the transcript is required for the synthesis of GUS polypeptide. Unlike the original GUS reporter gene, this Intron-GUS reporter gene did not express detectable GUS activity in Agrobacterium tumefaciens cells. However, when the Intron-GUS reporter gene placed downstream of the cauliflower mosaic virus (CaMV) 35S promoter (35S-Intron-GUS) was introduced into protoplasts of tobacco suspension-cultured cells by electroporation, expression of GUS activity was observed suggesting splicing of the intron occured in tobacco cells. The 35S-Intron-GUS fusion gene also expressed GUS activity in various organs of transgenic tobacco plants. The level of GUS activity obtained with the 35S-Intron-GUS fusion gene was slightly higher than those obtained with the 35S-GUS fusion gene in both transient expression assay and stable expression in transgenic plants. However, the presence of intron did not alter the pattern of cell-type-dependent expression of the CaMV 35S promoter in various organs. These results indicate that the Intron-GUS reporter gene is useful to monitor the Agrobacterium-mediated transfer and expression of foreign genes in plant cells, and to study the mechanism of intron-splicing in plants.