四氢叶酸依赖型的麦草畏脱甲基酶基因表达纯化和特性研究
毕业论文关键词：麦草畏降解；四氢叶酸依赖型脱甲基酶基因；基因外源表达；酶学特性
Expression, purification and characteristic of tetrahydrofolate(THF)-dependent dicamba demethylase
Abstract: Herbicide dicamba (3, 6-dichloro-2-methoxybenzoic acid) is used to control more than 200 kinds of broadleaf weeds worldwide. Furthermore, dicamba is considered to be a suitable target herbicide for engineering of herbicide-resistant transgenic crops. Therefore, dicamba degradation or detoxification gene and enzyme have potential applications in the construction of herbicide-resistant transgenic crops and bioremediation of dicamba residue-containment soil. In previous study, an aerobic dicamba-degrading bacterium Sphingomonas sp. Ndbn-20 was isolated from compost sample. The degradation of dicamba in strain Ndbn-20 is initiated by O-demethylation to generate 3,6-dichlorosalicylic acid (DCSA), a compound without herbicidal activity, two novel THF-dependent dicamba O-demethylases gene dmt50 and dmt66 ware identified in the strain. In this study, the dicamba demethylases gene dmt66 was cloned into pET 29a(+) and expressed in E. coli BL21(DE3), the recombinant protein was purified by Ni-affinity chromatography. The purified His6-Dmt66 catalyze the transformation of dicama to DCSA. Demethylase activity was detected ranging from 4 to 60°C and at pH values ranging from 4.3 to 10.2, with the highest activity being detected at 30°C and pH 7.0. Dmt66 was severely inhibited by the heavy metal ions Ni2+, Zn2+ and Ag+, moderate inhibited by Mn2+, Ca2+ and Cr2+, but not obviously affected by Na+, K+, Mg2+, Fe2+ and Li+. The palent cation-chelating agent EDTA did not inhibit the demethylase activity.
Key words: Dicamba degradation; Tetrahydrofolate-dependent demethylase gene; Gene exogenous expression; Enzymatic properties
目 录
摘要3
关键词3
Abstract3
Key words3
引言3
1 材料与方法4
1.1实验材料 4
1.1．1试剂与培养基 4
1.1.2菌株、质粒和引物4
1.2.1脱甲基酶Dmt66表达载体的构建4
1.2.1 dmt66的PCR扩增 5
1.2.2PCR产物和pET 29a(+)的双酶切、纯化和同源重组平末端连接6
1.2.3平末端连接产物转化和阳性转化子的筛选6
1．3 目标蛋白Dmt66的表达、纯化6
1.3.1粗酶样品的制备 6
1.3.2目的蛋白的纯化 7
1.3.3变性聚丙烯酰胺凝胶电泳（SDS-PAGE） 7
1.3.3.1样品 7
1.3.3.2蛋白凝胶电泳 7
1.3.3.3蛋白凝胶的染色与脱色 7
1. 3．4 蛋白透析与保存 8
1. 3．5 目的蛋白含量的测定 8
1. 4 酶的功能鉴定8
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