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摘要：百菌清（四氯间苯二腈）是一种广谱氯代苯腈类杀菌剂，在农业生产中已使用30年之久，是世界上使用最广泛的杀真菌剂之一，在环境中较稳定、残效期长，且具有明显的蓄积毒性。因此，百菌清的降解变的越来越重要。本论文将含转录终止子、P43启动子、npr B信号肽并在末端加入组氨酸标签的百菌清水解脱氯酶（Chd）基因片段，插入到pDG1730H质粒上。之后又构建了两拷贝和三拷贝的chd基因的整合质粒。然后将含有单拷贝、两拷贝、三拷贝chd基因的整合质粒整合到枯草芽孢杆菌WB800染色体上，使chd基因在枯草芽孢杆菌体内稳定表达和遗传。我们将带有pP43质粒的菌株与整合到染色体上单拷贝、两拷贝、三拷贝chd基因的菌株的Chd酶活进行比较。实验结果表明，在低拷贝数情况下，拷贝数越高，酶活越高；pP43质粒的酶活介于单拷贝和两拷贝之间。30819
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毕业论文关键词：百菌清；整合；表达；酶活
Chromosomal Integration and Expression of Chlorothalonil Hydrolytic Dehalogenase in Bacillus Subtilis WB800
Abstract: Chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile, TPN), a broad-spectrum fungicide, has been used in agricultural production for 30 years and has become one of the most widely used agricultural fungicides in the world, featuring better environmental stability, longer residual time and obvious accumulation of toxicity. Therefore, the degradation of chlorothalonil is becoming more and more important. In this paper, a transcriptional terminator, P43 promoter, npr B signal peptide and a histidine-tagged chlorothalonil hydrolytic dehalogenase (Chd) gene fragment were inserted into pDG1730H plasmid, and then two and three copies of the chd gene of the integrated plasmid was built up. After that, we integrated single copy, two copies and three copies of chd gene fragment into the Bacillus Subtilis WB800 Chromosome so as to make the chd gene stably express and inherit in the Bacillus Subtilis. The Chd activity of the strain with the pP43 plasmid and the aforementioned integrated strains was compared. The experimental results show that, the larger the copy number, the higher the activity of the enzyme when the copy number is low and enzymatic activity of the pP43 plasmid is between the single copy and the two copies.
Key words: chlorothalonil; integrate; expression; enzymatic activity
目录
摘要 1
关键词 1
Abstract 1
Key words 1
引言 1
1 材料与方法 2
1.1 材料 2
1.1.1 菌株和质粒 2
1.1.2 培养基 2
1.2 实验方法 2
1.2.1 枯草杆菌感受态细胞的转化 3
1.2.2 体外单拷贝质粒的构建 3
1.2.3 体外多拷贝质粒的构建 3
1.2.4 将质粒上的pP43-chd序列整合到染色体上 4
1.2.5 标准曲线的绘制 4
1.2.6 Chd酶活定义及测定方法 4
1.2.7 重组枯草芽孢杆菌WB800的液态发酵 4
2 结果与分析 4
2.1 构建整合质粒pDG1730H-chd 4
2.2 百菌清水解脱氯酶（Chd）在整合质粒上的验证 5
2.3 百菌清水解脱氯酶（Chd）在枯草芽孢杆菌WB800染色体上单拷贝整合表达及验证 5
2.4 液态发酵 7
2.4.1百菌清标准曲线的绘制及发酵过程百菌清水解脱氯酶的变化曲线 7
2.4.2发酵过程残糖量和生物量的变化曲线 7
2.5 百菌清水解脱氯酶（Chd）在枯草芽孢杆菌WB800染色体上两拷贝整合表达及验证 8