Gene profiling during symbiosis between the actinorhizal tree Alnus glutinosa and the actinobacteria Frankia alni ACN14. Alnus glutinosa

Alnus glutinosa belongs to a family of angiosperms called actinorhizal plants because they can develop nitrogen-fixing nodules in association with the soil bacteria Frankia. The aim of this transcriptomic study was to get a global view of the plant symbiotic genetic program and to identify new key plant genes that control nodulation during symbiosis in A. glutinosa. Symbiosis between A. glutinosa and Frankia was obtained after inoculation of young plant with a concentrated culture of the bacteria. Inoculation was performed in a medium depleted in nitrogen which favors the induction of nitrogen fixing symbiosis. For this study we considered two stages of symbiosis: - an early stage where inoculated roots were harvested 7 days after inoculation with the bacteria and compared to two controls (non-inoculated roots grown with or without nitrogen and harvested at the same time) - a late stage where nodules (nitrogen-fixing specific organs) were harvested 21 days after inoculation and compared to non-inoculated roots harvested on the day of inoculation (which is our reference time 0d). Three biological replicates were used for each condition. Overall design: Microarrays were designed by Imaxio (Clermont Ferrand, France ; http://www.imaxio.com/index.php) which has been accredited by Agilent Technologies (Palo Alto, CA, USA; http://www.home.agilent.com/agilent/home.jspx) as a certified service provider for microarray technologies. Based on 14327 annotated unigenes for A. glutinosa, 60mers probes were designed using eArray software (1 probe per unigene) and custom 8 x 15K Oligo Microarrays were manufactured by Agilent

欧洲桤木（Alnus glutinosa）隶属于被子植物中的放线菌根共生植物（actinorhizal plants）类群，因其可与土壤弗兰克氏菌（Frankia）共生形成固氮根瘤。本转录组学研究旨在全面解析该植物的共生遗传程序，并鉴定其在共生结瘤过程中的关键新型植物基因。
欧洲桤木与弗兰克氏菌的共生体系，可通过将浓缩菌液接种至幼株构建获得。接种实验在缺氮培养基中开展，该培养条件有利于固氮共生过程的诱导。
本研究设置两个共生阶段开展分析：
- 早期阶段：接种细菌7天后收获接种根系，并与两个对照组（同期收获的、分别在有氮和无氮条件下培养的未接种根系）进行比较；
- 晚期阶段：接种21天后收获根瘤（固氮特异性器官），并与接种当日收获的未接种根系（即参考时间点0d）进行比较。每个实验条件均设置3次生物学重复。
整体实验设计：基因微阵列由法国克莱蒙费朗的Imaxio公司（http://www.imaxio.com/index.php）定制开发，该公司已获安捷伦科技（Agilent Technologies，美国加利福尼亚州帕洛阿尔托，http://www.home.agilent.com/agilent/home.jspx）认证，为微阵列技术的合格服务提供商。研究基于欧洲桤木的14327条注释单基因簇（unigene），使用eArray软件设计60聚体探针（每个单基因簇对应1条探针），并由安捷伦制造了定制化8×15K寡核苷酸微阵列。