Abstract

Expressed sequence tags (ESTs) are partial DNA sequences generated from either the 5 ' or the 3 ' end of cDNA clones. Many large scale cDNA sequencing projects generating thousands of ESTs have been performed. Such EST collections can reflect a substantial proportion of the expressed genes of a species under a given set of conditions. Two potato cDNA libraries derived from pathogen challenged tissue were enriched by virtual subtraction and single pass sequencing of selected clones resulted in 4,795 EST sequences. 4,184 unigenes (3,305 singletons + 879 contigs) were discovered from grouping the ESTs into clusters and contigs. The virtual subtraction enrichment before sequencing of the cDNA libraries was found to be highly effective at reducing EST redundancy and enriching for ESTs of genes expressed at low levels, namely transcription factor genes. As much as 30 fold decreases in the numbers of ESTs representing certain highly expressed genes were observed while some transcription factor ESTs were up to 10 times more abundant than in public EST sets developed from randomly selected clones. In addition, the EST collection was analyzed for percent full length cDNA composition corresponding to genes of different lengths. A potato microarray was constructed from the enriched set of cDNA clones. The B2 gene of potato, a gene believed to be involved in its disease response, was cloned into the gene overexpression vector pRD526 and into the RNAi gene silencing vector pDARTHVECTOR. The constructs were each transformed into Agrobacterium tumefaciens . Future studies conducted using transgenic plants made from these constructs and the microarray will enable the elucidation of the function of B2 through the discovery of interactions between B2 and other disease response genes.