摘    要青蒿素（Artemisinin）是从著名的草药植物黄花蒿（Artemisia annua L。）的茎和叶中加工获得的倍半萜内酯化合物。青蒿素联合治疗（Artemisinin-based combinationtherapies，ACTs）对疟疾具有显著疗效。近年来研究发现青蒿素生物合成途径下游两个关键酶基因DBR2（蒿醛Δ11，13双键还原酶 2）和ALDH（醛脱氧酶）对于青蒿素合成有着非常关键的作用。DBR2属于烯醇还原酶小家族中的一种，功能为催化青蒿醛Δ11，13双键生成二氢青蒿醛。ALDH能催化二氢青蒿醛转化为二氢青蒿酸。二氢青蒿酸通过单重态氧Ene反应以及Hock断裂后生成的中间产物在温和条件三线态氧的作用下生成过氧化氢中间产物，之后脱水形成过氧桥和内脂结构，生成青蒿素。但是，DBR2和ALDH的功能仅在体外水平上验证，其在青蒿中的生物学功能仍是未知的。【目的】建立青蒿无菌苗培养及农杆菌侵染方法,导入“ALDH”、“DBR2”以及“DBR2-ALDH”酶基因建立毛状根转基因体系。利用毛状根生长快，易培养的特性培养青蒿转基因毛状根，运用气相色谱分析，对青蒿素标品制作浓度对峰面积的标准曲线，进而测定转基因毛状根青蒿素含量，同时测定青蒿素合成途径上的几种中间体：青蒿素B，青蒿酸，二氢青蒿酸等，确定DBR2和ALDH在青蒿素生物合成途径上的功能。【方法】运用农杆菌转化法构建过表达目的基因的青蒿毛状根，并使用PCR电泳方法验证是否成功导入目的基因。取目的毛状根进行摇瓶振荡培养，提取后运用气相色谱技术检测青蒿素，二氢青蒿酸，青蒿素B，青蒿酸含量。【结果】通过基因组PCR检测确认得到10个DBR2、9个ALDH、4个DBR2-ALDH转基因毛状根；成功制得青蒿素标品浓度-峰面积标准曲线【结论】成功建立转基因青蒿毛状根体系，青蒿素标品浓度-峰面积标准曲线重复性稳定性良好，为进一步利用代谢工程技术培育青蒿素高产转基因黄花蒿提供了良好基础。89837

毕业论文关键词：青蒿素；DBR2;ALDH；气相色谱；青蒿；毛状根

Abstract Artemisinin is a sesquiterpenoid extacted from the ground part of a traditional Chinese medicine, Artemisia annua L。 Artemisinin-based combination (ACTs) is a effective medicine for the treatment of malaria 。 In the present study, it was also found that the two key enzyme genes DBR2 and ALDH (aldehyde dehydrogenase) play a role in artemisinin biosynthesis。 DBR2 belongs to the family of enol reductase, which is used to catalyze the formation of dihydroartroic acid。ALDH can cata the conversion of dihydroartroic acid to dihydroartroic。Dihydroartemisinic acid translate into the intermediate product of perhydrol by singlet oxygen Ene reaction and the intermeditate product of Hock fault under mild conditions and triplet oxygen promotion。 Then dehydrate and form into Peroxide bridge and internal fat structure。 But the function of ALDH and DBR2 is only verified in vitro。 the function in Biology is unknown yet。 [Objective] establishment of ALDH,DBR2 and DBR2-P1301-ALDH transgenic hairy root in Artemisia annua L, promoting the accumulation of Artemisinin based on fast-growing hairy root。 Using GC to analysis standard of Artemisinin and draw standard curve then mensurate content of Artemisinin in artemisinin, besides, we can mensurate Arteannuin B,Dihydroartemisinic acidand Artemisinic acid。Confirm the function of ALDH and DBR2 in biology。 [Methods] Using SAAT to structure over express DBR2、ALDH hairy root in Artemisia annua。 Using PCR to identify if it succeed。 Useing GC to confirm the content of Arteannuin B,Dihydroartemisinic acid,Artemisinic acid and Artemisinin。 [Results] Get 9 ALDH,10DBR2 and 4 DBR2-ALDH transgenic hairy root,drawing the curve of standard Artemisinin [Conclusion] The results showed that the reproducibility of Artemisia selenium was higher than that of Artemisia annua L, which provided a good basis for further cultivating artemisinin high yield transgenic Artemisia annua L。 using metabolic engineering technology。