摘    要莴苣是植物类群中含天然的倍半萜类化合物较高的种属之一，其产生的中间产物吉马烯A合成酶属于植物萜类合酶，这类酶是萜类物质β-榄香烯合成途径中的关键限速酶，它可以催化FPP合成吉马烯A，吉马烯A可在高温条件下进行内部分子重排生成β-榄香烯。【目的】 针对吉马烯A合成酶的保守模块和活性中心疏水区域边缘关键氨基酸DE（莴苣吉马烯A合酶的第二个DDXXD/T基序的删除）、Y530F和T412S进行改造，并研究这三个突变的氨基酸位点在吉马烯A合酶催化反应中的作用。【方法】检测野生型莴苣吉马烯A合酶的酶活性，并通过改变单一变量温度和PH值探索酶促反应的最适反应条件。参考相关文献确定待突变的三个氨基酸位点。以大肠杆菌BL21为宿主菌,提取PET-28a莴苣吉马烯A合酶质粒DNA作为模板。采用定点突变技术对质粒进行扩增，得到突变后的吉马烯A合酶，然后以FPP为底物进行酶促反应，用GC-MS检测吉马烯A合酶突变前后的活力，以及最终产物β-榄香烯的含量。【结果】待突变的三个氨基酸位点均突变PCR成功，得到了突变后的吉马烯A合酶。根据气相检测结果，突变后吉马烯A合酶的含量和活性都要远远低于野生型莴苣吉马烯A合酶，β-榄香烯的产量也较突变前下降很多。【结论】在对吉马烯A合酶进行定点突变的过程中，三个突变氨基酸位点保守模块的催化性质可能受到抑制，从而降低了突变后吉马烯A合酶的催化活性和含量，本次研究为进一步利用生物合成技术提取β-榄香烯根提供了基础。89306

Abstract Lettuce is one of the higher species of natural sesquiterpenoids in plant groups。 The intermediate products produced by it are the terpene synthases belonging to the plant terpene synthase, which is a terpenoid The critical rate-limiting enzyme in the Synesis pathway, which catalyzes the synthesis of carbamene A by FPP, and the formation of β-elemene by internal molecular rearrangement at high temperature conditions。 【Objective】 To modify the conserved modules of the germacrene A synthase and the secondary amino acid DE at the edge of the hydrophobic region of the active center (the second DDXXD / T motif of the lettuce unit), Y530F and T412S were modified and To study the role of these three mutated amino acid sites in the catalytic reaction of  germacrene  A synthase。 【Method】 The enzymatic activity of wild-type lettuce  germacrene  A synthase synthase was detected and the optimum reaction conditions were explored by changing the temperature and pH of single variable。 Reference to the relevant literature to determine the three amino acid sites to be mutated。 Using E。 coli BL21 as host strain, PET-28a lettuce germacrene  A synthase plasmid DNA was extracted as template。 The plasmid was amplified by site-directed mutagenesis, and the mutant  germacrene  A synthase was obtained。 The enzyme was subjected to enzymatic reaction with FPP as the substrate。 The activity of the prokimene A synthase mutation was detected by GC-MS, The content of the product β-elemene。【Result】The three amino acid sites to be mutated were mutated successfully, and the mutated germacrene A synthase was obtained。 According to the results of gas phase detection, the content and activity of SYMA synthase were much lower than that of wild type lettuce  germacrene  A synthase。 The yield of β-elemene was also decreased before the mutation。 【Conclusion】The catalytic properties of the three mutant amino acid sites may be inhibited during the site-directed mutagenesis of the  germacrene A synthase, thereby reducing the catalytic activity and content of the mutant germacrene A synthase, This study provides a basis for further extraction of β-elemene by biosynthesis。

毕业论文关键词：吉马烯A合酶; β-榄香烯; 定点饱和突变; 气相色谱-质谱联用仪