Large-scale characterization of promoters from grapevine (Vitis spp.) using quantitative anthocyanin and GUS assay systems

Abstract

Successful implementation of cisgenic/intragenic/ingenic technology for crop improvement necessitates a better understanding of the function of native promoters for driving desired gene expression in host plant. Although the genome of grapevine (Vitis vinifera) has been determined, efforts to explore promoter resources for the development of cisgenics are still lacking. Particularly, there is a shortage of constitutive promoters for marker and/or target gene expression in this species. In this work, we utilized an anthocyanin-based color histogram analysis method to evaluate quantitatively a large number of promoters for their ability to activate transgene expression. Promoter fragments corresponding to known genes were amplified from various genotypes and used to drive the VvMybA1 gene of 'Merlot' for anthocyanin production in non-pigmented somatic embryo (SE) explants to infer transcriptional activity. Results revealed that among 15 tested promoters belonging to seven ubiquitin genes, at least three promoters generated constitutive activities reaching up to 100% value of the d35S promoter. In particular, the high activity levels of VvUb6-1 and VvUb7-2 promoters were verified by transient GUS quantitative assay as well as stable anthocyanin expression in sepal and corolla of transgenic tobacco. Variations in promoter activity of different ubiquitin genes in grapevine did not correlate with the presence and sizes of 5' UTR intron, but seemed to be related positively and negatively to the number of positive cis-acting elements and root-specific elements respectively. In addition, several of the 13 promoters derived from a PR1 gene and a PAL gene produced a higher basal activity as compared to previously reported inducible promoters and might be useful for further identification of strong inducible promoters. Our study contributed invaluable information on transcriptional activity of many previously uncharacterized native promoters that could be used for genetic engineering of grapevine.