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We have focused our efforts on cDNA cloning genes which are involved in the synthesis of anthocyanins during grape ripening. Subsequently, these probes can be used to study the hormonal and developmental regulation of gene expression during grape ripening. In addition, since color and phenolic content are quality attributes of both table and wine-making cultivars, respectively, the generated information from this study can be used in the future for the genetic manipulation of grape berries.
Firstly, we have developed a method of isolating intact and active RNA from grape tissues. This method yields undegraded RNA from various grapevine tissues including whole berries, berry skins, leaves, callus and cell cultures. Next, we have constructed a cDNA library from mRNA isolated from berries highly expressing these key phenylpropanoid biosynthetic genes. Parallel with this, another cDNA library was constructed using mRNA isolated from cell cultures of the same grape cultivar. 5X106 and 8X107 recombinants were generated from ripe berries and cell cultures, respectively. In vivo excision of phagemids from single plaques selected at random indicated that cDNA inserts had an average size of 1.3kb and 1.5kb for berries and cell cultures, respectively. The berry and cell cultures cDNA libraries were screened with cDNAs for PAL, CHS and DFR from parsley. A cDNA corresponding to a full-length clone of DFR from cell cultures was isolated and sequenced. Positive recobinents for PAL and CHS are under characterization.
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