地衣芽孢杆菌耐高温α-淀粉酶基因的克隆、烟草瞬时表达及转化拟南芥的研究

从地衣芽孢杆菌(Bacillus licheniformis)中克隆到耐高温α-淀粉酶基因全长, 构建了原核表达载体, 转入大肠杆菌(Escherichia coli)中, 使用IPTG于28°C诱导6小时后, 通过SDS-PAGE检测到目的蛋白, 分子量约为55 kDa, 并通过酶活力检测实验证明该蛋白具有耐高温α-淀粉酶活性。同时构建了该基因融合GFP的植物表达载体, 通过农杆菌(Agro- bacterium tumefaciens)介导瞬时转化烟草(Nicotiana tabacum)下表皮细胞并在荧光显微镜下观察, 发现在烟草下表皮细胞的细胞质和液泡中均有绿色荧光。使用I₂-KI溶液对乙醇脱色后的烟草叶片进行染色, 显色反应表明在烟草中表达的耐高温α-淀粉酶具有酶活性。最后, 采用农杆菌介导的花蕾浸泡法将重组载体转化到拟南芥(Arabidopsis thaliana)中, 筛选到稳定遗传的耐高温α-淀粉酶基因的拟南芥纯合子。研究结果为后期开展表达耐高温α-淀粉酶的转基因植物的相关研究奠定了实验基础。

Studies of the Transient Expression and Transformation of Cloned Thermostable α-Amylase Genes from Bacillus licheniformis in Tobacco and Arabidopsis

The full-length thermostable α-amylase gene from a Bacillus licheniformis strain was cloned in a prokaryotic expression vector and a plant expression vector with GFP as reporter gene. Thermostable α-amylase was overexpressed in Escherichia coli by IPTG induction for 6 h at 28°C, protein bands at 55 kDa were detected with SDS-PAGE, and activity of the enzyme was confirmed by amylolysis experiments. The plant expression vector was transformed into tobacco via Agrobacterium tumefaciens mediated transformation. GFP expression was observed both in cytoplasm and vacuoles of the lower epidermis of tobacco by fluorescence microscopy. Staining of the tobacco leaf by I₂-KI revealed that the thermostable α-amylase expressed in tobacco leaf had enzyme activity. Finally, the gene was introduced into Arabidopsis by the floral dip method and homozygous transgenic plants expressing α-amylase gene were identified. The results of this research provide further information for overexpression of thermostable α-amylase in transgenic plants.