阔叶猕猴桃GalDH基因植物表达载体的构建及转化番茄的研究

Plant Expression Vector of Kiwi Fruit GalDH Gene and Its Transformation into Tomato

L-半乳糖脱氢酶(GalDH)是L-半乳糖途径合成抗坏血酸(ASA)的关键酶之一,为了研究GalDH基因的功能,获得转基因植株,用限制性内切酶BamHⅠ和KpnⅠ将已克隆的阔叶猕猴桃GalDH基因从克隆载体pMD-19T切下,定向插入到植物表达载体pCSB,成功构建阔叶猕猴桃GalDH基因植物表达载体pCSB-GalDH及工程农杆菌,以Micro-Tom番茄叶片及茎段为受体,通过农杆菌介导法转化番茄,PCR检测、GUS染色和Real-time PCR检测表明,阔叶猕猴桃GalDH基因已整合到番茄基因组中。

L-Galactose dehydrogenase（GalDH） is one of the key enzymes in the L-galactose pathway of ascorbic acid biosynthesis.In order to study the gene function of kiwi fruit GalDH and obtain the transgenic plants,the plant expression vector pCSB-GalDH was constructed by inserting the GalDH gene coding fragment from cloning vector pMD-19T by BamHⅠ and KpnⅠ sites into pCSB.PCR and enzyme-digestion results showed that the plant expression vector pCSB-GalDH was constructed and transformed into Agrobacterium tumefaciems successfully.Subsequently,the GalDH gene was transformed into Micro-Tom tomato leaves and shoot fragments mediated by Agrobacterium tumerfaciens.PCR and GUS staining proved that the GalDH gene had integrated into tomato genome.