Grapevine promoter directs gene expression in the nectaries of transgenic tobacco

Abstract

A major obstacle in the genetic manipulation of fruit ripening in grapevine (Vitis vinifera L.) remains the lack of regulatory elements (promoters) to regulate tissue- and ripening-specific expression of the transgene in the grape berry. In this study, a cDNA previously shown to identify a fruit- and ripening-specific proline-rich protein from grapevine (mrip1) was used to isolate a fruit- and ripening-specific promoter. Sequence analysis of the 5.5-kb region directly upstream of the mrip1 transcription initiation site (TIS) revealed a 2.8-kb AT-rich region directly upstream of the mrip1 TIS and the presence of the coding and the 5′ untranscribed flanking region (UTR) of a proline-rich protein further upstream. In silico analysis of the 2.8 kb directly upstream of the mrip1 TIS revealed several putative cis-acting regulatory elements. These include a spectrum of hormone-, light-, phytochrome-, sugar- and stress-responsive elements. Based on the sugar-responsive 2.2 kb upstream of the mrip1 TIS, a 2.2-kb fragment of the mrip1 5′ UTR flanking region was selected for functional analysis of the promoter. Functionality of mrip1:sgfpS65T fusion gene was confirmed by transient expression in green pepper pericarp tissue, before introduction into tobacco by Agrobacterium-mediated transformation. In transgenic tobacco, transcription of the mrip1:sgfpS65T fusion was developmentally regulated and specific to the ovary and nectary-tissue specific of the developing flower. While low in immature flowers, green fluorescent protein (GFP) rapidly accumulated to the high level of expression visualized in the flower in full-bloom, followed by a decrease in the final stages of ovary development. These observations suggest that the 2.2 kb mrip1 promoter is functional and that this promoter region harbours cis-elements necessary for tissue- and developmental-specific regulation of GFP expression. It furthermore suggests that the transcriptional activation of mrip1 is mediated by developmental signals present in both grapevine berries and tobacco flowers. Copyright © Physiologia Plantarum 2006.