摘要：白藜芦醇（Resveratrol）是一种具有多种医疗保健功能的非黄酮类多酚化合物，它对人体有多种有益作用，红葡萄酒是白藜芦醇的主要饮食来源，但人们从葡萄酒中可以获得白藜芦醇的量是有限的，天然植物中含有的白藜芦醇极低。现阶段可以用化学合成法提取白藜芦醇，但需要使用有毒有机溶剂并且会产生大量副产品。本文分别从拟南芥和桑葚中扩增了4CL基因以及RS2基因，并与大肠杆菌表达载体pETDuet-1连接后转入大肠杆菌（Escherichiacoli，简称E.coli）BL21中。通过发酵及优化希望使白藜芦醇的产量得到进一步的提高。取构建好的大肠杆菌进行发酵实验,最开始发酵时，当加入0.5mM底物及温度控制在30℃时，待菌体生长12h后，加入诱导剂。用HPLC检测发酵液时并未发现产物，随后又通过聚丙烯酰胺凝胶电泳(SDS-PAGE)进行检测酶是否产生，但经过多次检测后发现，条带较暗，浓度较低。可能是酶未达到最适温度，导致表达受到阻碍，或发酵条件受限，所以导致白藜芦醇未产生。综上所述，重组大肠杆菌的成功构建为后续发酵等实验提供了基础，而发酵实验以及诱导实验中还需改变一定的条件进行优化。
关键词：大肠杆菌;白藜芦醇;桑葚
Abstract：Resveratrol is a non-flavonoid polyphenolic compounds with lots of medical and health functions. It has many beneficial effects to the human body. Red wine is the main source of dietary resveratrol, but the quantity of resveratrol that can be obtained from the wine is limited, and the content of resveratrol in the natural plant is very low . At this stage, resveratrol can be extracted by the means of chemical synthesis. However, this means will produce a large number of by-products when use toxic organic solvents. In this paper, 4CL and RS2 gene were amplified from Arabidopsis thaliana and mulberry, and then ligated with the expression vector pETDuet-1 into Escherichia coli BL21. It is hoped that the yield of resveratrol will be further improved by fermentation method. We established a biotechnological method for resveratrol through the engineered E. coli strain by fermentation method. At the beginning of the fermentation, when 0.5 mM substrate was added with the temperature at 30 ℃, the cells were incubated for 12 h and the inducer (IPTG) was added. The product of resveratrol was not detected by HPLC, and then the enzyme was detected by polyacrylamide gel electrophoresis. However, after repeated tests, it was found that the band was dark. It is possible that the enzyme does not reach the optimum temperature, causing the expression to be hampered, or the fermentation conditions are limited. In summary, the successful construction of recombinant E. coli provides the basis for the subsequent fermentation experiments, and the fermentation experiments and induction experiments need to change certain conditions to optimize.
Key words: Escherichia coli, Resveratrol, Mulberry
目录
第一章绪论 1
1.1白藜芦醇的概述 1
1.1.1白藜芦醇的结构与性质 1
1.1.2白藜芦醇的生理活性 1
1.2白藜芦醇的生产研究进展 4
1.2.1白藜芦醇在植物和微生物中的生物合成 4
1.2.2白藜芦醇从植物中的提取方法 5
1.4本课题研究的内容和意义 6
1.4.1课题主要研究内容 6
1.4.2课题研究意义 桑葚白藜芦醇合成酶RS2在大肠杆菌中的异源表达:http://www.youerw.com/shengwu/lunwen_203697.html