Use of Interfering RNAs to Identify Gene Function in Medicago Truncatula

粮食和饲草类豆科作物的种植面积约为1.8亿公顷，约占地球可耕地面积的15%，占世界主要作物产量的27%。豆科植物的特点是它们与根瘤菌和菌根真菌形成共生关系的能力。在根瘤菌群落中，细菌固定大气中的氮，提供生物圈中生物可利用氮的主要来源。真菌共生促进了磷的获取。氮和磷是植物生长所需的主要矿质养分。尽管豆科植物在农业和生态方面具有重要意义，但人们对参与细菌或真菌共生发展的基因知之甚少。豆科研究界已经开发了许多关于模型豆科植物紫花苜蓿的资源。其中包括大量est（表达序列标签），从中已鉴定出许多在共生过程中在根中活跃的基因。有效的转化方案已经制定，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，以沉默基因表达和识别参与植物-微生物共生的基因。序列数据库分析和基因表达研究将用于识别可能参与共生的基因，其中1500个基因将通过RNAi进行沉默。将对转基因根进行沉默处理，并研究其对共生关系的影响。研究结果将通过一个公开的项目网站报告给研究界。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提供，不受研究界的限制。本研究项目整合了分子生物学、微生物学、植物生物学和生物信息学等多个科学领域。这项研究还采用了最新和最强大的基因组技术之一——RNAi，它被用于从基础生物学研究到人类治疗学开发的各种应用中。研究小组将向选定的本科生介绍研究的许多方面，使他们接触到现代生物学研究的兴奋。此外，Gustavus Adolphus学院（一所本科院校）的合作者将按照研究计划中描述的一般协议开发一门实验课程。通过研究项目开发的材料和协议是这项工作的优秀资源。这一努力的结果将是描述植物生物学、植物分子生物学或植物生理学的本科生在一个学期的课程中可以获得的一系列经验。生成的资源：根和共生表型和性状的公开访问数据库。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].