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摘要：麦草畏（3,6-二氯-2-甲氧基苯甲酸）是一种在全球范围内得到广泛应用的除草剂，能防除200多种阔叶杂草。并且，麦草畏因其除草谱广、除草活性高及杂草抗性产生慢等优点，是除草剂抗性转基因作物工程理想的靶标除草剂。因此，麦草畏降解或解毒基因和酶在构建除草剂抗性转基因作物和麦草畏抑制土壤的生物修复方面具有潜在应用价值。Sphingomonas sp. Ndbn-20 是本课题组从堆肥样品中分离的高效麦草畏降解菌。菌株Ndbn-20降解麦草畏脱甲基生成无除草活性的3,6-二氯水杨酸（DCSA），并从该菌中克隆到两个新的THF依赖性麦草畏脱甲氧基酶基因dmt50和dmt66。本论文将麦草畏脱甲基酶基因dmt66克隆到pET29a（+）中，并在E. coli BL21(DE3)中表达，利用镍亲和层析纯化获得纯的重组蛋白。纯化的后的His6-Dmt66能催化麦草畏转化为DCSA。His6-Dmt66在温度为4至60℃，pH值范围为4.3至10.2时检测到麦草畏降解活性，最佳反应温度为30℃，最佳pH 为7.0。重金属离子Ni2+，Zn2+和Ag+严重抑制His6-Dmt66活性，Mn2+，Ca2+和Cr2+具有中度抑制作用，但Na+、K+、Mg2+、Fe2+、Li +和二价阳离子螯合剂EDTA对His6-Dmt66活性基本没有影响。30388
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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