Evaluation of Constitutive Cestrum Yellow Leaf Curling Virus Promoter in Maize and Tomato

Abstract

We have cloned and evaluated two versions of a novel, strong and constitutive promoter from Cestrum yellow leaf-curling virus (CmYLCV) called CmpC (short- 346bp) and CmpS (longer- 400bp), which can be used for regulating transgene expression in a wide variety of plant species. CmYLCV belongs to the Caulimoviridae family and was first reported in Cestrum parqui from the Solanaceae by Ragozzino (1974). Recently, CmYLCV was cloned and seven open reading frames were identified in the genomic sequence (Hohn et al., 2001). To evaluate the utility of the CmYLCV promoter to drive expression of heterologous genes in plants, two versions of the full-length transcript promoter were cloned in front of the GUS, CAT and FP reporter genes and tested in transient assays in Nicotiana plumbaginifolia, Orichophragmus violaceus and Oriza sativa protoplasts as well as in stably transformed Zea mays and Lycopersicon esculentum. The transient expression experiments show that, depending on the plant system used, the expression level of CmpC and CmpS promoter fragments are higher than the expression level of the widely used 35S promoter from Cauliflower Mosaic Virus (Hohn et al., 2001) and that the longer promoter fragment is the weaker one. Expression analysis of CmpC and CmpS promoter fragments in stably transformed maize (Figurel) have shown that both fragments are on average ten times greater than the strong constitutive Ubi 1 promoter from Z. mays (Christensen et al., 1992) and that the expression levels in tomato (Figure 2) are comparable with the strong, constitutive SMAS promoter (Ni et al., 1994). Moreover, the spatial expression analysis has shown that both promoters express in various tissue types, except pollen in both maize and tomato and that both promoter fragments retain their high expression levels through at least two generations. We limited our analysis to the single copy events only (Ingham et al., 2001) since it is a widely accepted idea that the copy number of a transgene affects the expression level in transgenic plants.