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.

新肾脏和epichloe物种，被称为草的真菌内生植物，是系统性的，母亲传播的共生体，可保护其宿主植物免受草食性的侵害，并可以增强耐受性，耐旱性，营养，营养，养分，生物量产生和竞争力。内生产生四种不同化学类别的生物保护生物碱。该项目涉及这样一个类别的一个类别，即饱和的1-氨基吡咯烷生物碱（Loline和相关化合物），它们是高度杀虫剂，复杂的杂环分子，可以在植物 - 内植物中的植物 - 内植物中达到2％总生物量的水平。可以想象，这些丰富的生物碱也参与了干旱耐受性或其他几种宿主健身性的增强中的任何一种，但是需要进行遗传和分子遗传研究来解决它们的真正生态和生理作用。因此，该项目的目标是阐明洛琳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健身中的作用。已经确定了两个基因簇（LOL），这些基因簇与内生菌的新生型neothodium uncinatum相关。另外，已经确定了萝卜生物碱的代谢前体。该项目的第一个目标是完成两个LOL基因簇的DNA序列确定。下一个目标是利用分子遗传技术寻求确认LOL簇基因与Loline生物碱生物合成有关的确认。第三个目标是通过合成标记为具有稳定同位素的假定中间体，将其喂食，将其馈送到洛琳生物碱产生的培养物，并采用质谱和核磁共振共鸣，以检测和识别包含在Lolines中的标签原子，并使用质谱和核磁共振结构来测试洛琳的可能生物合成途径。该项目中开发的信息和突变体将是一种未来研究的资源，详细介绍了洛琳在基层共生中的自然作用。具有真菌内生植物的草的生物是保护性互助，其中内生植物可以抵消各种植物的生病和寄生虫。在保护寄主植物的真菌产品中，有洛琳生物碱，它们在共生植物中高度杀虫剂，并且化学复杂。洛林生产的途径尚不清楚，阐明这种途径将为酶机理和共生微生物的进化提供新的见解。该项目将采用分子生物学和化学中现代技术的结合，以确定洛林生产的步骤。研究中生产的真菌突变体将是未来研究的资源，以确定洛琳在内生菌 - 草菌共生中的特定作用和活动。该研究主题与植物，真菌和食草动物的生态相互作用有关，也与天然产品在环境友好的植物保护中的潜在使用有关。