Molecular Biology and Biosynthesis of Lolines by Grass Endophytes

草内生菌的分子生物学和黑麦草生物合成

• 批准号: 0213217
• 负责人: Christopher Schardl
• 金额: $ 38.5万
• 依托单位: University of Kentucky Research Foundation
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2006-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0213217&HistoricalAwards=false
• 关键词: Molecular; Biology; Biosynthesis; Lolines; Grass

Neotyphodium and Epichloe species, known as fungal endophytes of grasses, are systemic, maternally transmitted symbionts that protect their host plants from herbivory, and can enhance drought tolerance, disease resistance, nutrient acquisition, biomass production, and competitiveness. The endophytes produce bioprotective alkaloids of four distinct chemical classes. This project concerns one such class, the saturated 1-aminopyrrolizidine alkaloids (loline and related compounds), which are highly insecticidal, complex heterocyclic molecules, and can reach levels of 2% total biomass in plant-endophyte symbioses. It is conceivable that these abundant alkaloids are also involved in drought tolerance or any of several other host fitness enhancements, but genetic and molecular genetic investigations are needed to address their true ecological and physiological roles. Thus, the goals of this project are to elucidate the genetics and biosynthetic pathways of the loline alkaloids, and to develop background information and tools to study the roles of these alkaloids in plant fitness. Two gene clusters (lol) have been identified that are associated with loline alkaloid production by the endophyte species, Neotyphodium uncinatum. In addition metabolic precursors of loline alkaloids have been determined. The first objective in this project is to complete DNA sequence determinations for the two lol gene clusters. The next objective is to utilize molecular genetic techniques to seek confirmation that the lol cluster genes are involved in loline alkaloid biosynthesis. The third objective is to test possible biosynthetic pathways for lolines by synthesizing putative intermediates labeled with stable isotopes, feeding these to loline alkaloid-producing cultures, and employing mass spectrometry and nuclear magnetic resonance to detect and identify the labeled atoms incorporated in the lolines. The information and mutants developed in this project will be a resource for future studies detailing the natural roles of lolines in grass-endophyte symbioses.Symbioses of grasses with fungal endophytes are protective mutualisms,whereby the endophytes ward off a variety plant pests and parasites. Among the fungal products involved in protecting host plants are the loline alkaloids, which are highly insecticidal, abundant in the symbiotic plant, and chemically complex. The pathway of loline production is unknown, and elucidating this pathway will provide new insights into enzyme mechanisms and evolution of symbiotic microorganisms. This project will employ a combination of modern techniques in molecular biology and chemistry to identify the steps in the loline production. Fungal mutants that are produced in the study will be a resource for future research to determine the specific roles and activities of lolines in endophyte-grass symbioses. The research topic is relevant to the ecological interactions of plants, fungi and herbivores, and also to the potential use of natural products for environmentally friendly biological protection of plants.

Neotyphodium 和 Epichloe 物种被称为草类真菌内生菌，是系统性、母系传播的共生体，可以保护宿主植物免受草食性侵害，并可以增强耐旱性、抗病性、养分获取、生物量生产和竞争力。内生菌产生四种不同化学类别的生物保护生物碱。该项目涉及这样一类，即饱和 1-氨基吡咯里西啶生物碱（黑麦草碱和相关化合物），它们是高度杀虫的复杂杂环分子，在植物内生菌共生体中可达到总生物量 2% 的水平。可以想象，这些丰富的生物碱也与耐旱性或其他几种宿主适应性增强有关，但需要进行遗传和分子遗传学研究来解决它们真正的生态和生理作用。因此，该项目的目标是阐明黑麦草生物碱的遗传学和生物合成途径，并开发背景信息和工具来研究这些生物碱在植物适应性中的作用。已鉴定出两个与内生菌 Neotyphodium uncinatum 产生黑麦草碱生物碱相关的基因簇（笑）。此外，还测定了黑麦草生物碱的代谢前体。该项目的首要目标是完成两个 lol 基因簇的 DNA 序列测定。下一个目标是利用分子遗传学技术来确认 lol 簇基因参与黑麦草生物碱生物合成。第三个目标是测试黑麦草碱可能的生物合成途径，方法是合成用稳定同位素标记的假定中间体，将这些中间体喂入黑麦草碱生物碱生产培养物中，并采用质谱和核磁共振来检测和识别黑麦草碱中掺入的标记原子。该项目中开发的信息和突变体将成为未来研究的资源，详细说明黑麦草在草-内生菌共生中的自然作用。草与内生真菌的共生是保护性互利共生，内生菌借此抵御多种植物害虫和寄生虫。涉及保护宿主植物的真菌产品包括黑麦草碱生物碱，其具有高度杀虫性，在共生植物中含量丰富，并且化学成分复杂。黑麦草生产的途径尚不清楚，阐明该途径将为共生微生物的酶机制和进化提供新的见解。该项目将结合分子生物学和化学的现代技术来确定黑麦草生产的步骤。研究中产生的真菌突变体将成为未来研究的资源，以确定黑麦草在内生菌-草共生中的具体作用和活性。该研究课题涉及植物、真菌和食草动物的生态相互作用，以及天然产物对植物进行环境友好型生物保护的潜在用途。