Analysis of grape EST’s: investigation of gene function in Arabidopsis

The Australian Agriculture Research Institute is embarking on a large-scale grape EST analysis project. cDNA libraries are being prepared from a range of tissues, developmental stages and cultivars with the aim of producing a data base of up to 50,000 sequences, covering a high proportion of grape genes. 2,466 sequences have been obtained from a leaf cDNA library, and 2,438 from a berry library. Analysis of the leaf and berry EST’s indicate 59% of clones have significant homology with known plant genes, 16% with more distant organisms and 25% may represent novel genes (no BLAST 2 match or match to EST’s of unknown function). Redundancy levels in the leaf library are about 42%, and 39% in the berry library. Overall both libraries may yield more than 3,000 unique sequences. We are using expression in Arabidopsis to investigate the function of 58 EST’s that match to 26 different transcription factors with unknown function in plants, and 330 EST’s with no homology to known genes or other plant EST’s. The inserts from the cDNA clones of interest are being cloned into pKMB and pSMB (1). Arabidopsis plants will be transformed with each construct to either over-express or knock out the cloned grape gene of interest. Transformed seeds will be germinated and the plants studied to determine phenotypic changes produced by the altered expression of the grape gene. The functional analysis in Arabidopsis of novel grape genes identified as EST’s will provide a powerful new resource for discovering genes with the potential to improve table and wine grapes. (1) Mylne, J., and Botella, J.R. (1998) Binary Vectors for sense and antisense expression of Arabidopsis EST’s. Plant Mol. Biol. Reporter 16, 257-262