A comparison of the activity of three cauliflower mosaic virus 35S promoters in rice seedlings and tobacco (BY-2) protoplasts by analysis of GUS reporter gene transient expression

Abstract

The cauliflower mosaic virus (CaMV) 35S promoter is active in most plant species and has been used for genetic engineering to obtain strong, constitutive expression of numerous, unrelated genes. We examined the transient expression of constructs in which the uidA (GUS) reporter gene was driven by three somewhat different, independently isolated CaMV 35S promoters. These CaMV 35S promoters differed principally in 5–6 bases outside the region containing cis-acting regulatory elements, but they were identical in sequences within known regulatory elements. Plasmid DNA containing the chimeric constructs was delivered to protoplasts derived from cultured BY-2 tobacco cells by electroporation and to rice embryos by DNA uptake during imbibition. GUS enzyme activity was determined by a fluorometric method 72 h after protoplast electroporation. The rice embryos were germinated after DNA uptake during imbibition and GUS activity was determined histochemically and fluorometrically in 7-day-old seedlings. All three CaMV 35S promoters gave similar levels of GUS expression in rice seedlings, both in terms of the pattern of expression in the various seedling tissues and in terms of the total level of GUS activity per seedling. In contrast, there was nearly a 14-fold difference in the activity of the promoters in the tobacco protoplasts. Promoter activity in tobacco appeared to be strongly affected by the sequence near the transcriptional start site where the promoters differed. These results suggest that monocots and dicots may utilize somewhat different signals for transcription initiation.