摘要：白藜芦醇属于多酚类化合物，对人体有很多好处。包括心脏保护，抗氧化，抗 炎，抗肿瘤，抗衰老等作用。由于这些健康益处，白藜芦醇已经吸引了保健食品， 化妆品和制药等行业的广泛关注。然而人们对于白藜芦醇的获取量是有限的，高昂 的提取代价和污染阻碍了白藜芦醇的广泛应用。为了降低成本，同时提高产量，通 过生物技术的途径构建营养缺陷型酿酒酵母来发酵产生目的产物。酿酒酵母代谢背 景清晰，营养要求低，易于培养，同时具有良好的耐酸性，最重要的是完整的基因 组已知，这为本实验的研究打下良好的基础。

基于桑葚中白藜芦醇基因，提取总 RNA 后反转录 cDNA，设计引物进行 PCR 扩增 RS3 基因，经基因测序和分析可知 RS3 基因片段长度约 1200 bp。经双酶切、 片段回收、连接以及转化等实验步骤构建重组表达载体，以 pESC-Trp 作为基因载 体，将重组质粒利用 liAc/PEG 化学转化法将重组质粒转化进入活化的酿酒酵母菌 株 CEN.PK1-2c 中，对照组为空质粒 pESC-Trp 酿酒酵母。通过摇瓶培养最终扩大培 养并进行发酵，半乳糖诱导并加入底物对香豆酸后最终提取发酵产物进行高效液相 色谱测定，绘制产物白藜芦醇的标准曲线并通过定量的方法测取白藜芦醇产量，当 底物对香豆酸浓度为 0.5，1.0，1.5，2.0 mM 时，产量分别时 0.00648，0.00613，0.00225， 0.00070 mg/L。根据同一时间点峰值的比较检测导入重组质粒的工程菌产生的白藜 芦醇的含量。

关键字：白藜芦醇，酿酒酵母，桑葚

Abstract：Resveratrol belongs to the polyphenols and has many advantages to the human body, including heart protection, anti-oxidation, anti-inflammatory, anti-tumor, anti-aging effects. Because of these health benefits, resveratrol has attracted widespread attention in health food, cosmetics and pharmaceutical industries. However, the amount of resveratrol is limited, and the high cost of extraction and contamination hampered the widespread usage of resveratrol. In order to reduce costs, biotechnology approach has been used for producing the desired product. Saccharomyces cerevisiae have many advantages, such as clear background, low nutritional requirements, easy cultivation,  and  good acid resistance etc. The most important is the complete genome known, which lay a good foundation for this study.

Based on the Resveratrol synthase gene (RS3) in mulberry, the total RNA of mulberry was extracted and then reverse transcribed to cDNA. The primers  were designed for PCR amplification of RS3. The gene sequencing and analysis showed that the length of RS3 gene fragments was about 1200 bp. Recombinant expression plasmid was constructed by double digestion, fragment recovery, ligation and transformation. The recombinant plasmid was transformed into S. cerevisae CEN.PK1-2c by LiAc/PEG method (chemical conversion) or electrochemical conversion. The final production of resveratrol was extracted by shrimp culture after 96 h fermentation (added galactose and p-coumaric acid ). The final product was extracted with ethyl acetate after concentrating. The standard curve of resveratrol was plotted and quantified by quantitative method. When the concentration of the substrate on the p-coumaric acid concentration of 0.5, 1.0, 1.5,  2.0 mM/L, the output were 0.00648, 0.00613, 0.00225, 0.00070 mg/L. The content of resveratrol produced by engineered strain were determined according to the comparison of the HPLC standard curve.

Keywords: Resveratrol, Saccharomyces cerevisiae, Mulberry

目录 

第一章 绪 论 1

1.1  引言 1

1.1.1  白藜芦醇概述 1

1.1.2  白藜芦醇的生物活性