利用基因突变的方法分析青蒿倍半萜合酶催化特异性决定区域-analysis of catalytic specificity determining region of artemisia annua sesquiterpene synthase by gene mutation method.docxVIP

摘 要 摘 要 萜类是是指具有(C5H8)n 通式的烃类及其含氧衍生物，可以看成是由异戊二烯或者异 戊烷以各种不同方式连结而成的一类天然化合物。迄今为止，人们已经从自然界中各种 生物里分离鉴定了 55,000 多种萜类化合物。 本实验对青蒿（ Artemisia annua L. ） 两 种 倍 半 萜 合 酶 ： 紫 穗 槐 二 烯 合 酶 (Amorpha-4,11-diene Synthase, ADS)和柏木脑合酶(Epi-cedrol Synthase, ECS)的 C 末端结 构通过重叠延伸 PCR 法进行互换，构建出杂交分子，得到的产物失去了催化活性。说 明 C 末端结构对酶活性有重要作用。 对青蒿紫穗槐二烯合酶通过快速定点突变法引入突变，研究酶的催化特异性变化。 此方法所表达的蛋白失去了催化紫穗槐二烯合酶的活性，得到了催化底物产生红没药烯 （β-Bisabolene）的新活性。突变的氨基酸残基与原来的氨基酸具有不同的性质，可使酶 的催化活性发生改变。可为进一步研究紫穗槐二烯合酶的催化特异性决定位点奠定基 础。 利用反转录方法得到青蒿大根香叶烯合酶移码突变的 cDNA 插入到原核表达载体 pET-21 中，构建带有 His 标签的表达载体 pETG。将 pETG 转化入大肠杆菌 Rosett（a DE3） 中诱导表达蛋白，表达产物经镍琼脂糖柱纯化。纯化产物蛋白加入酶促反应体系（FPP）。 经 GC-MS 分析显示酶有催化 FPP 形成 farnesol 的活性。 关键词 青蒿 倍半萜合酶 原核表达 功能鉴定 定点突变 I Abstract Abstract The terpene refers to the hydrocarbons and their oxygenated derivatives which have the general formula of (C5H8)n, that can be considered as a class of natural compounds that linked by the isoprene or isopentane in various ways. So far, 55,000 a variety of terpene compounds has been isolated and identified from the nature organism. With a large number of valuable terpene was found, the biosynthesis and regulation of terpene synthase is becoming the focus of the research. Using the method of overlap extension PCR, we interchanged the the C-terminal structure in Amorpha-4,11-diene Synthase and Epi-cedrol Synthase which of the two kind of sesquiterpene synthase in Artemisia annua L. The hybrid molecule we obtained has lost its catalytic activity. This proves the important role of the C-terminal structure on the enzyme activity. Using the site-directed mutagenesis to introduce mutations to Amorpha-4,11-diene Synthase, we studied the catalytic specificity changes of the enzyme. The protein expressed by this method obtain a new activity to produce β-Bisabolene. The mutation of amino acid residues has a different nature, can make the enzyme changes its catalytic activity. This research can provide a basis for further research. Using the method of reverse transcription, we constructed the