Novakova M, Mackova M, Chrastilova Z, Viktorova J, Szekeres M, Demnerova K, Macek T
ICT Prague, Faculty of Food and Biochemical Technology, Department of Biochemistry and Microbiology, Technicka 3, 16628 Prague, Czech Republic.
Biotechnol Bioeng. 2009 Jan 1;102(1):29-37. doi: 10.1002/bit.22038.
The aim of this work is to increase the efficiency of the biodegradation of polychlorinated biphenyls (PCBs) by the introduction of bacterial genes into the plant genome. For this purpose, we selected the bphC gene encoding 2,3-dihydroxybiphenyl-1,2-dioxygenase from Pseudomonas testosteroni B-356 to be cloned into tobacco plants. The dihydroxybiphenyldioxygenase enzyme is the third enzyme in the biphenyl degradation pathway, and its unique function is the cleavage of biphenyl. Three different constructs were designed and prepared in E. coli: the bphC gene being fused with the beta-glucuronidase (GUS) gene, with the luciferase (LUC) gene, and with histidine tail in three separate plant cloning vectors. The GUS and LUC genes were chosen because they can be used as markers for the easy detection of transgenic plants, while histidine tail better enables the isolation of protein expressed in plant tissue. The prepared vectors were then introduced into cells of Agrobacterium tumefaciens. The transient expression of the prepared genes was first studied in cells of Nicotiana tabacum. Once this ability had been established, model tobacco plants were transformed by agrobacterial infection with the bphC/GUS, bphC/LUC, and bphC/His genes. The transformed regenerants were selected on media using a selective antibiotic, and the presence of transgenes and mRNA was determined by PCR and RT-PCR. The expression of the fused proteins BphC/GUS and BphC/LUC was confirmed histochemically by analysis of the expression of their detection markers. Western blot analysis was performed to detect the presence of the BphC/His protein immunochemically using a mouse anti-His antibody. Growth and viability of transgenic plants in the presence of PCBs was compared with control plants.
这项工作的目的是通过将细菌基因导入植物基因组来提高多氯联苯（PCBs）的生物降解效率。为此，我们选择了来自睾丸酮假单胞菌B - 356的编码2,3 - 二羟基联苯 - 1,2 - 双加氧酶的bphC基因，将其克隆到烟草植物中。二羟基联苯双加氧酶是联苯降解途径中的第三种酶，其独特功能是裂解联苯。在大肠杆菌中设计并制备了三种不同的构建体：bphC基因分别与β - 葡萄糖醛酸酶（GUS）基因、荧光素酶（LUC）基因以及组氨酸尾在三种不同的植物克隆载体中融合。选择GUS和LUC基因是因为它们可作为标记用于轻松检测转基因植物，而组氨酸尾更便于分离植物组织中表达的蛋白质。然后将制备好的载体导入根癌农杆菌细胞。首先在烟草细胞中研究制备基因的瞬时表达。一旦确定了这种能力，就通过农杆菌感染用bphC/GUS、bphC/LUC和bphC/His基因对模式烟草植物进行转化。在含有选择性抗生素的培养基上选择转化后的再生植株，并通过PCR和RT - PCR确定转基因和mRNA的存在。通过分析其检测标记的表达，组织化学法证实了融合蛋白BphC/GUS和BphC/LUC的表达。使用小鼠抗组氨酸抗体进行蛋白质免疫印迹分析，以免疫化学方法检测BphC/His蛋白的存在。将转基因植物在多氯联苯存在下的生长和活力与对照植物进行比较。
