Expression and transfer of gene of sorbitol which works as a functional and stress-tolerant substance

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The purpose of this project is to form Citrus producing sorbitol by gene transter of sorbitol-6-P dehydrogenase (S6PDH). The expression of S6PDH gene in peach or apple : S6PDH activity and protein were clearly detected in leaves and cotyledons, but not detected in root and axis. When we examined the difference of S6PDH among positions of leaf, the activity was very weak in folded leaves and increased with the development of leaves, and the changes in the activity corresponded to those in its protein and mRNA amounts. On the seasonal change of gene expression of S6PDH,the changes of activity was caused by translation and transcription. It was suggested that the high activity before maturation is for the supply of translocating sugar and the high activity after haravest is for the storage of carbohydrate in tree trank. Formation of chimeric gene of S6PDH cDNA : pSA-S6PDH chimeric gene was formed by inserting CaMV35S promoter, S6PDH coding sequence and Nos-terminator into the cloning site of SAori vector. Further, pBIi12-S6PDH chimeric gene waqs also formed from the commercial vector. The vector of two chimeric gene was transferred into Agrobacterium by tri-parent method, and the insertion of S6PDH coding regions was confirmed by PCR.Transfer of chimeric gene and formation of transgenic citrus : S6PDH chimeric gene was transferred into citrus callus through the co-culture with Agrobacterium, and the transformed colonies of embryogenic callus from sweet orange species wwere selected by Kanamaycin. After extracting DNA from leaves of this transgenic citrus, the insersion of S6PDH gene in citrus was confired by PCR.Expression of S6PDH in transgenic citrus : The expression of S6PDH mRNA in transgenic citrus was certified by Northern blotting, that is, the hybridization of probe (S6PDH gene) to total RNA.