Isolation of cDNA clone of key enzymes realting sorbitol synthesis and the analysis of sugar accumulation

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Both sorbito-6-P dehydrogenase (S6PDH) and sotbitol dehydrogenase (SDH) are important as key enzymes to accumulate sugar into fruit tissue of apple. The purpose of this research is to make clear the role of lthese enzymes by purifying and characterizing them, and to clarify the regulation on gene expression of S6PDH by cloning the cDNA.We cucceeded to isolate both enzymes through some purification steps. Especially, the isolation of S6PDH is very worth because this was firstly isolated from plant tissues. From the properties of purified enzymes, S6PDH was certified to react toward S6P synthesis and SDH toward the convertion of sorbitol into fructose in vivo. Further, we succeeded to isolate the cDNA clone of S6PDH using apple seedling. The cDNA is composed of a open reading fram of 930 bases coding 310 mino acid. The molecular weight of S6PDH was 35,000 and corresponded to the molecular weight estimated by SDS-PAGE.Next, the gene expression of S6PDH with germination of apple seeds was studied using this cDNA probe. It was suggested that the gene expression of S6PDH activity acts under some post-transcriptional or translational regulations since m-RNA of S6PDH appeared 2 days ealier than the protein synthesis of S6PDH.Infuture, it will be expected to make clear the detail mechanism of sugar accumulation into fruit throught the study of gene expression of SDH by cloning the cDNA.