中文摘要
青蒿素是药用植物青蒿产生的一种倍半萜内酯，是目前最有效的抗疟药物，但在植物中含量较低。本项目对青蒿素的生物合成及其调控进行了研究。构建了青蒿腺体毛EST文库，测序得到1300多个独立基因（uni-gene）。通过基因芯片分析，获得了596个差异表达克隆。分离鉴定了青蒿1个新的倍半萜合酶-没药醇合酶，1个α, β-双键还原酶，以及2个棉花P450还原酶。对青蒿素生物合成途径中的关键酶（紫穗槐二烯合酶，ADS）进行定点突变，使其催化效率提高近2倍，可用于青蒿素代谢基因工程。研究了光信号和植物激素对倍半萜生物合成的影响，发现茉莉酸酯信号因子MYC2起重要的的调控作用。对青蒿AP2类转录因子ZAP1和ZAP2展开研究，初步表明它们能够调控青蒿素生物合成途径中几个酶基因的转录。通过过量表达ADS，ZAP1和ZAP2，获得了若干青蒿素含量增加的转基因青蒿。由于青蒿素在腺毛中积累，项目还研究了miR156及其靶基因SPL对植物表皮毛分布和发育的影响，已获得相关转基因青蒿。已发表标注SCI论文4篇，申请国内和国际发明专利各1项。完成项目规定的研究任务。
英文摘要
Artemisinin, a sesquiterpene lactone isolated from Artemisia annua, is an effective drug for treatment of malaria，its accumulation in plant, however, is relatively low. In this research, we investigated the biosynthesis of artemisinin and its regulation. EST libraries were constructed for A. annua glandular hairs, from which more than 1,300 uni-genes were obtained. Furthermore, microarray analyses revealed 596 differentially expressed clones. We also identified a new sesquiterpene synthase (bisabolol synthase) and an α, β-double bond reductase from A. annua, in addition to two cotton P450 reductase. Amorpha-4,11-diene synthase (ADS) is the key enzyme of artemisinin biosynthesis. Though site-directed mutagenesis, the ADS activity was nearly doubled, and the mutant enzyme has application potential in synthetic biology for engineering artemisinin production. We studied that the regulation of sesquiterpene biosynthesis by light and phytohormones, and found that JA signaling factor MYC2 plays a key regulatory role. Two AP2 transcription factors, ZAP1 and ZAP2, were also found to affect the expression level of several key enzyme genes in the artemisinin pathway. We have generated transgenic Artemisia lines over-expressing ADS, ZAP1 and ZAP2, respectively, and some lines showed higher levels of artemisinin accumulation
结题摘要
青蒿素是药用植物青蒿产生的一种倍半萜内酯，是目前最有效的抗疟药物，但在植物中含量较低。本项目对青蒿素的生物合成及其调控进行了研究。构建了青蒿腺体毛EST文库，测序得到1300多个独立基因（uni-gene）。通过基因芯片分析，获得了596个差异表达克隆。分离鉴定了青蒿1个新的倍半萜合酶-没药醇合酶，1个α, β-双键还原酶，以及2个棉花P450还原酶。对青蒿素生物合成途径中的关键酶（紫穗槐二烯合酶，ADS）进行定点突变，使其催化效率提高近2倍，可用于青蒿素代谢基因工程。研究了光信号和植物激素对倍半萜生物合成的影响，发现茉莉酸酯信号因子MYC2起重要的的调控作用。对青蒿AP2类转录因子ZAP1和ZAP2展开研究，初步表明它们能够调控青蒿素生物合成途径中几个酶基因的转录。通过过量表达ADS，ZAP1和ZAP2，获得了若干青蒿素含量增加的转基因青蒿。由于青蒿素在腺毛中积累，项目还研究了miR156及其靶基因SPL对植物表皮毛分布和发育的影响，已获得相关转基因青蒿。已发表标注SCI论文4篇，申请国内和国际发明专利各1项。完成项目规定的研究任务。