Expression analysis of the 35S CaMV promoter and its derivatives in transgenic hairy root cultures of cucumber (Cucumis sativus) generated by Agrobacterium rhizogenes infection

Abstract

The cauliflower mosaic virus (CaMV) 35S promoter is the most commonly used viral-based promoter to drive transgene expression in plants. Although, many studies have demonstrated the constitutive nature of this promoter, some reports have suggested varied expression levels in different parts of the plant. Therefore, our aim was to study the activity of the CaMV 35S promoter in the hairy root system. The CaMV 35S promoter, the duplicate CaMV 35S promoter (designated CaMV 35ST) and the duplicate CaMV 35S promoter containing a 5’- untranslated leader sequence from the alfalfa mosaic virus RNA4 promoter (designated CaMV 35ST/AMV) were compared to evaluate their effects on the expression of the gus reporter gene in transgenic hairy roots, which was mediated using the Agrobacterium rhizogenes A4 transformation system. The integration of T-DNA containing a gus reporter gene in hairy root lines was confirmed at low copy numbers ranging from 1 to 4 copies using quantitative real-time PCR. Histochemical staining of cucumber hairy roots showed over-expression of the gus gene when driven with the CaMV 35S promoter. The expression of the gus gene when driven with the CaMV 35ST promoter showed a lower expression than that driven by the CaMV 35S promoter. However, the expression of the gus gene driven by the CaMV 35ST/AMV promoter was slightly higher than that driven by the CaMV 35ST promoter. In this study, the reduced activity of the CaMV 35ST promoter was observed for the first time. Further investigation is required to elucidate the factors that mediate the decline in promoter activity.

Key words: Cucumis sativus L, hairy root, Agrobacterium rhizogenes, promoter 35S cauliflower mosaic virus (CaMV), β-glucuronidase (GUS), 5’UTR AMV.