Cloning the Bacterial bphC Gene Into Nicotiana tabacum to Improve the Efficiency of PCB-Phytoremediation
Cloning the Bacterial bphC Gene Into Nicotiana tabacum to Improve the Efficiency of PCB-Phytoremediation
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DOI：
10.1002/bit.22038
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发表时间：
2009-01-01
影响因子：
3.8
通讯作者：
Macek, T.
中科院分区：
文献类型：
--
作者：
Novakova, M.;Mackova, M.;Macek, T.
为了提高植物基因组中多氯联苯（PCBs）的生物降解效率，我们从睾丸酮假单胞菌（Pseudomonastestosteroni）B-356中筛选出编码2，3-二羟基联苯-1，2-双加氧酶的bphC基因，并将其克隆到烟草中。二羟基联苯双加氧酶是联苯降解途径中的第三种酶，其独特的功能是裂解联苯。设计并在E.大肠杆菌：bphC基因与β-葡糖醛酸酶（GUS）基因、荧光素酶（LUC）基因和组氨酸尾融合在三个单独的植物克隆载体中。选择GUS和LUC基因是因为它们可以用作标记以容易地检测转基因植物，而组氨酸尾更好地使得能够分离在植物组织中表达的蛋白质。然后将制备的载体导入根癌农杆菌细胞中。首先在烟草细胞中研究了所制备的基因的瞬时表达。一旦建立了这种能力，通过农杆菌感染用bphC/GUS、bphC/LUC和bphC/His基因转化模型烟草植物。使用选择性抗生素在培养基上选择转化的再生体，并通过PCR和RT-PCR确定转基因和mRNA的存在。融合蛋白BphC/GUS和BphC/LUC的表达通过其检测标记的表达分析进行了组织化学证实。进行Western Not分析以使用小鼠抗His抗体免疫化学地检测BphC/His蛋白的存在。在多氯联苯的存在下，转基因植物的生长和生存能力进行了比较，与对照植物。Biotechnol. Bioeng. 2009;102：29-37. (C)2008 Wiley Periodicals，Inc.
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 P-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 Not 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. Biotechnol. Bioeng. 2009;102: 29-37. (C) 2008 Wiley Periodicals, Inc.