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Free keywords:
EST; cluster analysis; macroarray; expression profiling; sugar beet; root-expressed genes
Abstract:
An integrated approach involving EST sequencing, data mining and multiparallel expression profiling by macroarray analysis was established to classify sugar beet gene products with respect to their expression in three different organs. The DNA sequences of 2996 ESTs derived from young sugar beet plants specified 2048 unique gene products with putative functions in primary and secondary metabolism (651), transport processes (136), signal transduction (78) and cellular organization (39). The cDNA clone collection was the basis for the generation of a macroarray. Sensitivity and reproducibility of our macroarray hybridization procedure were estimated first. The detection limit was found to correspond to 10-50 copies of single transcripts per cell. Within an interval of two-fold variation in signal intensities, reproducibility between spots on the same filter was determined to be 98.9%, between spots on different filters 89.8%, and reproducibility after hybridization with two probes synthesized from the same poly(A)(+)RNA sample was 97.6%. Expression profiles from roots, leaves and inflorescences of field-grown plant material were generated. Two different samples of each organ were analysed to reduce sampling effects, which accounted on average for 30.3% of spots with at least two-fold deviation. Expression values for each organ were determined by a stringent statistical evaluation of eight hybridizations for each clone. Macroarray expression data were confirmed by Northern blot analysis and quantitative RT-PCR experiments concerning eleven cDNAs. The analysis was then focused on 76 unique cDNAs, for which the amount of detected transcript in roots was at least twice as high as in the other organs tested. Functions of preferentially root-expressed candidate genes in taproot morphology and physiology are discussed.
