Isolation of grapevine promoters with special emphasis on the vacuolar pyrophosphatase

[摘要] ENGLISH ABSTRACT: Understanding the complex nature of grapevine molecular biology is of great importance forviticulturists. Progress in the elucidation of key events on a genetic level could provide furtherinsight into the underlying cues responsible for the precise control of physiological andmetabolic changes during a specific condition such as fruit development. The use and analysisof molecular 'tools', such as promoters controlling the site and level of gene activity, couldassist in the understanding of grapevine biology and serve as a platform for the future designand development of recombinant DNA protocols and strategies for Vitis vinifera L.A high-throughput gene expression system, cDNA-AFLPs, was successfully used to analyselarge-scale transcriptional activity during berry ripening. Candidate cDNA fragments wereselected on the basis of desired expression patterns and/or known gene function for subsequentpromoter isolation. From three candidate cDNAs selected, the promoter of a gene encodingvacuolar pyrophosphatase (V-PPase) was isolated for computational and comparative analyses.Promoter activity was evaluated on a transient level using the green fluorescent protein (GFP)reporter gene. Comparative integration has allowed for putative correlation of cis-elements,acting as receptors within promoter regions, to regulate V-PPase gene expression in response todevelopment, environmental stress and tissue-specificity.In this study, integration of genetic data have advanced the understanding and transcriptionalrole of a key enzyme (V-PPase) during grape ripening. Although never a replacement forexperimental verification, this integrative strategy of combining gene expression profiles withbioinformatics and regulatory data will greatly assist in further elucidation of various other keycomponents and regulatory cues associated with grapevine molecular biology. This study hasallowed us to use molecular tools that could assist in gaining further insight into geneticcomplexities and could serve as a platform for a more refined genetic manipulation strategy inVitis vinifera L.

[发布机构] Stellenbosch University