Artemisinin Biosynthesis: Control of Transcription

说明(申请人提供)：青蒿素(AN)及其衍生物已被证明是有效的抗疟疾治疗药物，也被证明具有广谱抗微生物和抗癌治疗的有效活性。AN是一种复杂的化合物，最好还是由植物青蒿素产生。然而，人们对生物合成是如何调控的知之甚少，尽管人们对其生产有以下了解：更多的AN或其前体青蒿酸(AA)是在植物从营养生长转移到开花时产生的，或者是在葡萄糖而不是蔗糖的基础上生长的(我们最近的数据)。由于这些条件对产量影响很大，我们建议使用它们来研究生物合成基因的转录调控。我们的假设：1.青蒿素的生物合成在早期(FDP前)和晚期(FDP后)的多个步骤中受到调节。2.发育阶段和碳源对该途径中多个基因的转录起调控作用。3.青蒿素和甾醇合成的协同调控发生在转录水平。技术目标：我们工作的长期目标是对青蒿素中萜类化合物产生的调控有一个基本的了解，以便最终不仅可以为青蒿素，也可以为其他重要的治疗植物生产的萜类药物开发萜类化合物的代谢调控。这项工作需要在2.5年内完成3个目标：1.比较二磷酸法尼基(FDP)前和FDP后的萜类生物合成基因的相对表达，导致AN与AA、DHAA和AN的水平，当它们从营养生长转移到诱导开花的条件时。2.比较在葡萄糖、果糖和蔗糖中生长的青木耳幼苗中导致AN的FDP前和FDP后的萜类生物合成基因的相对表达与AA、DHAA和AN的水平。3.在这些研究中包括对SQS表达的分析，以确定表达的协调控制。不幸的是，人们仍然很少知道像AN这样的重要植物产品是如何在植物中产生的，而且在低成本的田间种植的植物中高产量地生产萜类化合物仍然是具有挑战性的。这一信息很重要，因为几乎没有全面的努力来充分了解这种非常重要的治疗性倍半萜的调节，以及可能具有潜在非常有益的医学应用的其他植物来源的萜类。可用于治疗疟疾的最有效的疗法是青蒿素，它是在“甜安妮”植物中生产的，但含量太低，无法提供足够的剂量来每年治疗全世界发生的数百万病例。这项拟议的研究是帮助我们了解如何控制青蒿素的生物合成，以最终在低成本田间种植的植物中增加这种药物和其他化学相似药物的产量的第一步。

DESCRIPTION (provided by applicant): Artemisinin (AN), and its derivatives are proven effective antimalarial therapeutics that also been shown to have effective activity as broad spectrum antimicrobials, and anticancer therapeutics. AN is a complex compound and still best produced by the plant Artemisia annua. Little is known, however, about how AN biosynthesis is regulated, although the following is known about its production: more AN or its precursor, artemisinic acid (AA), is made when plants shift from vegetative growth to flowering, or grown on glucose instead of sucrose (our recent data). Since these conditions strongly affect AN production, we propose to use them to study the transcriptional regulation of AN biosynthetic genes. OUR HYPOTHESES: 1. Artemisinin biosynthesis is regulated at multiple steps in the pathway, both early (pre- FDP) and late (post-FDP). 2. Developmental stage and carbon source regulate the transcription of several genes in this pathway. 3. Coordinate control of artemisinin and sterol production occurs at the level of transcription. TECHNICAL OBJECTIVES: The long-term goal of our work is to develop a fundamental understanding of the regulatory control of terpene production in Artemisia annua so that metabolic manipulation of terpenoid biosynthesis can eventually be developed not only for artemisinin but other therapeutically important plant produced terpenoid drugs. This work has 3 objectives to be accomplished over 2.5 years: 1. To compare the relative expression of the terpenoid biosynthetic genes pre farnesyl diphosphate (FDP), and post FDP leading to AN with levels of both AA, DHAA, and AN production in A. annua seedlings as they are shifted from vegetative growth to conditions that induce flowering. 2. To compare the relative expression of the terpenoid biosynthetic genes pre farnesyl diphosphate (FDP), and post FDP leading to AN with levels of both AA, DHAA, and AN production in A. annua seedlings grown in glucose, fructose, and sucrose. 3. Include analysis of SQS expression in these studies to determine coordinate control of expression. Unfortunately little is still known about how important plant products like AN are produced in the plant and high production of terpenoids in low cost field-grown plants still remains challenging. This information is important because there has been little comprehensive effort to fully understand the regulation of not only this very important therapeutic sesquiterpene, but also other plant-derived terpenes that could have potentially very beneficial medicinal applications. The most potent therapeutic available for curing malaria, artemisinin, is produced in the plant, "Sweet Annie", but at levels too low to be able to provide adequate doses to annually treat the millions of cases that occur worldwide. The proposed study is a first step in helping us understand how to control artemisinin's biosynthesis to ultimately increase the production of this drug, and others that are chemically similar, in low cost field-grown plants.