内生菌对黄花蒿腺毛发育及其青蒿素合成的诱导调控研究

The glandular trichomes are called as 'molecular farms' for producing pharmaceutically important terpenoids, phenylpropanoids and other phytochemicals to play a defensive function. Recently endophytic fungi, described as fungal intracellular symbionts of plants, are also found to be significant reservoirs and regulators of novel bioactive secondary metabolites. Therefore the exploration on the role of endophtic fungi on development and phytochemical biosynthesis in glandular trichomes, has important theoretical and practical significance to reveal the mutual relation between the endophytes and their host plants, to produce more medicinal value of phyto-constituents, and to enhance plant resistance to environmental stress. In this project, the isolated endophytes from Artemisia annua L. with potential elicitation on trichome-dependent artemisinin biosynthesis will be re-inoculated to aseptic plantlets of A. annua. The trichome differentiation, development and distribution in leaves and stems will be studied on treated plants. On the other hand, the secondary metabolites including phytohormones, oligosaccharide and cerebroside elictors from endophytes will be analyzed for the regulation roles on upstream branch of artemisinin biosynthesis in the plastid and cytoplasm of glandular trichomes. The structure-function relationship of those eliciting compounds corresponding to the stimulating artemisinin synthesis is going to be revealed. Furthermore, we will pay more attention on the signal cascades of small molecules such as reactive oxygen species (ROS) and nitric oxide (NO) to the key enzymes in the pathway of artemisinin synthesis and bioconversion of artemisinin derivates. The differential gene expression by cDNA-AFLP analysis and the characterization of some enzymes responding to the elicitation of endophytes will be obtained. This project will focus on the role of signal compound and secondary metabolites of endophytic fungi on glandular trichomes, the "micro-factory' for artemisinin. We believe our ongoing results will be helpful in the application of endophytes on agricultural and biotechnological regulation on artemisinin production. Our research will give hints or theoretical basis of endophyte application for other explorations on medicinal or ecological important terpernoids in plants.

具有生态防御功能的植物特化结构-腺毛被誉为活性物质合成的"微工场"，内生菌是与植物具有"亲密"关系的新型资源库和调节者。探讨内生菌对植物腺毛发育及其代谢物合成的调节，对了解内生菌与宿主的相互关系、活性物质合成与植物抗逆性都具有重要意义。以课题组筛选的能够促进青蒿素合成的黄花蒿内生真菌为对象，返接到黄花蒿无菌苗中，考察内生菌对茎叶腺毛结构、发育和生长的影响；探讨内生菌代谢物质（激素、寡糖和脂类）对腺毛中青蒿素合成的质体和细胞质路径的选择性诱导，比较内生菌代谢物的诱导构效；考察内生菌诱导的活性氧/氮信号在腺毛中的级联转导以及对青蒿素衍生物的生物转化与合成的作用；利用cDNA-AFLP差异表达分析，获得腺毛中对内生菌侵染有响应的青蒿素合成特异酶基因。本研究探讨内生菌代谢和信号物质在腺毛中的作用，建立内生菌在青蒿素生产中的调节方法，为内生菌在植物萜类药物生产和腺毛发育调控上的应用提供理论基础。

研究了黄花蒿内生菌对青蒿素合成“微工厂”-腺毛的分化、生长、代谢及基因调控的影响。分离出促进青蒿素产量的内生链霉菌Streptomyces sp. AC 1、蜡状芽孢杆菌 Bacillus cereus SZ1、草酸青霉菌Penicillium oxalicum B4。通过构建的内生菌-宿主愈伤组织、内生菌-发根、内生菌-无菌苗共培养体系，发现内生菌通过调控宿主植物内源生长素吲哚乙酸（IAA）、脱落酸(ABA)和赤霉酸(GA3)的合成，促进了分泌型腺毛(AaGST)密度和非分泌型腺毛(AaTNG)生长。内生青霉还可通过调整宿主三羧酸循环（TCA）中苹果酸、亮氨酸和缬氨酸的合成为菌丝生长提供营养，调整离子积累和渗透调节物质适合内生菌的共生，同时宿主通过合成桃叶珊瑚甙、青蒿酸、青蒿素B和青蒿素等萜类植保素，有效地控制内生菌在宿主细胞体内种群平衡。我们还首次发现内生菌激活了宿主戊糖磷酸途径的关键酶：葡萄糖-6-磷酸脱氢酶（G6PDH），产生的NADPH可供青蒿素合成反应的需要，同时G6PDH诱导的活性氧（ROS）和一氧化氮（NO）的产生，ROS不仅促成腺毛分泌细胞中青蒿酸向青蒿素的生物转化，形成6个新代谢中间体。还刺激了3-羟基-3-甲基戊二单酰辅酶A还原酶（HMGR）、紫穗槐二烯合酶（ADS）和紫穗槐二烯单加氧酶（CYP）等重要关键酶基因的表达。NO信号则诱导了胞质中HMGR基因的表达，促进青蒿素合成前体异戊二烯焦磷酸（IPP）的形成。我们首次克隆得到一个可能直接或间接地参与了腺毛的分化的转录因子基因AaWD40。通过转录组分析，从基因表达上证实了内源激素变化、ROS\NO信号迸发和防御反应的产生，我们通过构建RNAi-DXR（1-脱氧-D-木酮糖-5-磷酸还原异构酶）转基因黄花蒿与内生菌共培养，发现位于腺毛亚顶细胞叶绿体中的DXR可能是内生菌对青蒿素合成上游通路的调节点，而位于分泌性腺毛中青蒿素生物合成下游途径中紫穗槐二烯合酶(ADS)和细胞色素P450 单氧酶(CYP)基因是内生菌在青蒿素合成下游的调节点。本项目结果不仅为内生菌对青蒿素合成的调节机制提供新的线索，还为内生菌农业应用、抗生素生物降解和医药应用提供新方法和措施。