百菌清水解脱氯酶（Chd）在枯草芽孢杆菌WB800染色体上整合表达和液态发酵
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