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摘要 随着化石能源对全球经济、环境的制约而迫切需要需找一种可代替化石能源的可再生的绿色能源物质，丁醇作为一种大宗化合物质，也是继乙醇后又一种新型的生物燃料，在生物发酵丁醇中，拜氏梭菌具有良好的水解液耐受性而作为首选菌株，粮食丁醇成本占75%，利用木制纤文素代替粮食原料作为生物丁醇发酵底物具有良好的发展前景，木糖做为木质纤文素原料中除葡萄糖外的第二大糖类物质具有很大的发展前景，然而木糖在拜氏梭菌中利用速度慢。
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本实验拟采用拜氏梭菌作为出发菌株，我们在预测的拜氏梭菌木糖转运基因簇中预测木糖转运基因xylFGH为木糖转运ABC transporters，而xylFII（Cbei2377）与xylF功能相同，并通过体外功能互补、双杂交、ITC、发酵等试验鉴定木糖转运基因尤其是xylFII的功能。试验分过表达和互补两部分进行，过表达试验发现xylFGH为木糖转运蛋白而xylFII不参与木糖转运，且xylFII与LytS-YesN在同一信号通路上，而猜测可能与LytS-YesN一起参与了木糖信号的接受，试验为提高木糖在拜氏梭菌中的代谢速率奠定了基础。8641
关键词：拜氏梭菌，丁醇，发酵，木糖转运，PCR
Identify of gene function in Clostridium beijerinckii xylose metabolism
Abstract: Urgent need to find a kind of renewable green energy can replace fossil energy As the fossil energy to the global economic and environmental constraints.butanol is not only an important bulk chemical but also an new excellent transportation fuel After ethanol ,Clostridium beijerinckii as the preferred strains with hydrolyzed good tolerance in the biological fermentation butanol,cost of butanol fermentation food for 75%,the use of wood cellulose instead of food raw materials as bio-butanol fermentation substrates with good the development prospects of xylose as lignocellulosic feedstock second largest outside than glucose sugars has great prospects for development, however xylose utilization in Clostridium beijerinckii slow.
We take Clostridium beijerinckii as the starting strain in this study, We focus on xylose transfer gene cluster in Clostridium beijerinckii, forecast xylose transfer genes xylFGH as well as ABC transporters of xylose transport,and forecast xylFII (Cbei2377) as same as xylF function , and with experiment such as complementary functions in vitro , double hybrid, ITC, fermentation test to identify the function of xylose transfer gene especially xylFII. Overexpression and complementary test of two parts, over-expression of trials found xylFGH as xylose transport proteins and xylFII not involved in xylose transport and xylFII with LytS-YesN in the same signaling pathway, while speculation may LytS-YesN join the xylose signal is received, the test in order to improve xylose Clostridium beijerinckii metabolic rate in the foundation.
Key words: Clostridium beijerinckii, butanol, fermentation, xylose transport, PCR
目录 3
1. 前言 1
1.1丁醇国内外研究现状概述 1
1.1.1丁醇性质及用途 1
1.1.2生物丁醇生产 1
1.2 梭菌生物发酵 2
1.2.1 拜氏梭菌选择的意义 2
1.2.2 研究木糖发酵丁醇的意义 3
1.2.3 拜氏梭菌中木糖代谢途径 4
1.3 课题研究目的及意义 5
2. 材料与方法 7
2.1实验材料 7
2.1.1 主要试剂 7
2. 1.2主要仪器与设备 8
2.1.3 菌种 8
2.1.4 培养基及溶液配制 8
2.2 试验方法 10
2.3 测定方法 11
3. 结果与讨论 12
3.1 质粒的构建 12