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.

新芽孢属和表观属植物被称为禾本科真菌内生菌，是一种系统的母传共生体，可以保护寄主植物免受草食性的侵害，并可以提高抗旱性、抗病能力、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性生物碱。该项目涉及一类饱和的1-氨基吡咯里西丁生物碱(洛林及其相关化合物)，它们是高度杀虫的复杂杂环分子，在植物-内生真菌共生体中的总生物量可达到2%的水平。可以想象，这些丰富的生物碱也与抗旱性或其他几种寄主适应性增强有关，但需要进行遗传和分子遗传学研究，以解决它们真正的生态和生理作用。因此，本项目的目标是阐明洛林生物碱的遗传和生物合成途径，并开发背景信息和工具来研究这些生物碱在植物适合性中的作用。有两个基因簇(LOL)已经被鉴定出来，它们与内生植物钩吻新伞(Neotyphdium Uncinatus)产生洛林生物碱有关。此外，还测定了洛林生物碱的代谢前体。本项目的第一个目标是完成两个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寻求确认LOL簇基因参与了洛林生物碱的生物合成。第三个目标是测试可能的生物合成途径，方法是合成标记了稳定同位素的假定中间体，将这些中间体喂入产生洛林生物碱的培养物中，并使用质谱学和核磁共振来检测和鉴定包含在洛林中的标记原子。在这个项目中开发的信息和突变将成为未来详细研究莲碱在草-内生菌共生中的自然作用的资源。草与真菌内生菌的共生体是保护性的互惠关系，内生菌通过这种共生体抵御各种植物害虫和寄生虫。在参与保护寄主植物的真菌产品中有洛林生物碱，它具有高度的杀虫活性，在共生植物中含量丰富，化学成分复杂。洛林产生的途径尚不清楚，阐明这一途径将为了解共生微生物的酶机制和进化提供新的见解。该项目将采用分子生物学和化学的现代技术相结合的方法来确定洛林生产的步骤。研究中产生的真菌突变体将成为未来研究的资源，以确定莲碱在内生植物-草共生中的具体作用和活性。该研究主题涉及植物、真菌和食草动物之间的生态相互作用，以及天然产品在植物环境友好型生物保护方面的潜在用途。