Use of Interfering RNAs to Identify Gene Function in Medicago Truncatula

全球约有1.8亿公顷（约占地球可耕地面积的15%）的土地种植着谷物和豆科牧草，占世界主要作物产量的27%。豆科植物的特点是能与根瘤菌和菌根真菌形成共生关系。在根瘤菌群中，细菌固定大气氮，提供生物圈中生物可利用氮的主要来源。真菌共生导致改善的磷获取。氮和磷是植物生长所需的主要矿质营养元素。尽管豆科植物在农业和生态学上具有重要意义，但人们对参与细菌或真菌共生体发育的基因知之甚少。豆科植物研究界围绕模式豆科植物蒺藜苜蓿（Medicago truncatula）开发了许多资源。这些包括大量的EST（表达序列标签），其中许多在共生过程中在根中活跃的基因已经被鉴定出来。已经制定了有效的转化方案，基因组测序项目正在进行中。这些资源和技术将与RNA干扰（RNAi）技术相结合，以沉默基因表达并识别参与植物-微生物共生的基因。对序列数据库和基因表达研究的分析将用于识别可能参与共生的基因，其中1,500个将通过RNAi进行沉默。沉默将在转基因根中进行，并将检查对共生的影响。研究结果将通过一个公开的项目网站向研究界报告。该基因靶向序列将在克莱姆森大学基因组学中心以一种易于使用的多功能载体的形式提供给研究界，该研究项目整合了包括分子生物学、微生物学、植物生物学和生物信息学在内的多种科学领域。该研究还采用了最新和最强大的基因组技术之一RNAi，该技术被用于从基础生物学研究到人类治疗学开发的各种应用。研究小组将向选定的本科生介绍研究的许多方面，这将使他们接触到现代生物研究的兴奋。此外，古斯塔夫·阿道夫学院（Gustavus Adolphus College）的合作者将开发一门实验室课程，该课程遵循研究提案中描述的一般协议。通过研究项目开发的材料和协议是这项工作的极好资源。这一努力的结果将是一系列的经验，在植物生物学，植物分子生物学，或植物生理学的本科生可以在一个学期course.Resources生成的描述：公开访问的根和共生表型和性状的数据库。有关基因组实验的信息将位于MtDB[http://medicago.org]、Concensus Legume数据库www.legumes.org和[http://decifr.ccgb.umn.edu]。

ABSTRACT Grain and forage legumes are grown on some 180 million Ha, or about 15% of the Earth's arable land and account for 27% of the world's primary crop production. Legumes are distinguished by their ability to form symbiotic associations with both rhizobial bacteria and mycorrhizal fungi. In the rhizobial association, the bacteria fix atmospheric nitrogen, providing the major source of biologically available nitrogen in the biosphere. The fungal symbiosis results in improved phosphorous acquisition. Nitrogen and phosphorus are the major mineral nutrients required for plant growth. Despite the agricultural and ecological importance of legumes, relatively little is known about the genes that are involved in the development of bacterial or fungal symbioses. The legume research community has developed many resources around the model legume Medicago truncatula. These include a large collection of ESTs (expressed sequence tags) from which many of the genes that are active in roots during symbiosis have been identified. Efficient transformation protocols have been developed and a genome sequencing project is ongoing. These resources and techniques will be combined with RNA interference (RNAi) technology to silence gene expression and identify genes involved in the plant-microbe symbioses. Analysis of sequence databases and gene expression studies will be used to identify genes that are possibly involved in symbiosis and 1,500 of these will be targeted for silencing via RNAi. Silencing will be carried out in transgenic roots and the effects on symbiosis will be examined. The results will be reported to the research community through a publicly available project web-site. The gene-targeting sequences will be made available at Clemson University Genomics Center without restriction to the research community in an easy to use, multifunctional vector.This research project integrates a variety of scientific fields including molecular biology, microbiology, plant biology and bioinformatics. The research also employs one of the latest and most powerful genomic technologies, RNAi, which is being used in a variety of applications from investigations into basic biology to the development of human therapeutics. The research team will introduce selected undergraduate students to many aspects the research which will expose them to the excitement of modern biological research. Additionally, collaborators at Gustavus Adolphus College, an undergraduate institution, will develop a laboratory course that follows the general protocols described in the research proposal. The materials and protocols that are developed through the research project are an excellent resource for this effort. The outcome of this effort will be the description of a series of experiences that undergraduate students in plant biology, plant molecular biology, or plant physiology can obtain in one semester course.Resources Generated: Publicly accessible database of root and symbiotic phenotypes and traits. Information on genomic experiments will be located at MtDB[http://medicago.org] , the Concensus Legume database www.legumes.org and[http://decifr.ccgb.umn.edu].