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

本课题的目的是通过转山梨醇-6-磷酸脱氢酶（S6 PDH）基因的方法建立柑橘山梨醇高产菌株。S6 PDH基因在桃和苹果中的表达：在叶片和子叶中检测到S6 PDH活性和蛋白，而在根和轴中未检测到。研究了S6 PDH在不同叶位上的差异，结果表明，在折叠叶中S6 PDH活性很弱，随着叶片的发育而增强，其活性变化与其蛋白质和mRNA含量变化相一致。在S6 PDH基因表达的季节性变化上，其活性的变化是由翻译和转录引起的。结果表明，成熟前的高活性主要是为了运输糖的供应，而成熟后的高活性主要是为了碳水化合物在树干中的储存。S6 PDH cDNA嵌合基因的构建：将CaMV 35 S启动子、S6 PDH编码序列和Nos终止子插入SAori载体的克隆位点，构建pSA-S6 PDH嵌合基因。此外，pBI 112-S6 PDH嵌合基因也由商业载体形成。通过三亲法将两个嵌合基因的载体转入农杆菌中，PCR检测证实S6 PDH编码区已插入，嵌合基因的转移及转基因柑橘的形成：通过与农杆菌共培养，将S6 PDH嵌合基因转入柑橘愈伤组织中，并对甜橙子种胚性愈伤组织的转化菌落进行卡那霉素筛选。S6 PDH基因在转基因柑橘中的表达：通过北方杂交，即用S6 PDH基因探针与柑橘总RNA杂交，证明S6 PDH基因在转基因柑橘中表达。

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.