植物miRNA调节丛枝菌根共生的分子机制及演化模式

Arbuscular mycorrhiza (AM) is an ancient symbiosis that originated over 400 million years ago and widespread in over 80% of land plant species from all major land-plant lineages, including hornworts, liverworts, lycopods, ferns, and angiosperms. The formation of AM with arbuscular mycorrhizal fungi (AMF) would assist plants in the assimilation of water and nutrients from the soil, which may have played a key role in facilitating plant colonization of the terrestrial environment. Establishment of an efficient symbiosis relies on a set of highly conserved 'symbiotic genes' characterized mainly in legumes. These symbiotic genes are required for the perception of AMF signals, root colonization, arbuscule development and to control the level of root colonization. Loss of symbiotic genes often results in defective in formation of AM symbiosis. Currently, how plants have evolved to tune the expression of symbiotic genes so that they are not over-colonized by AMF but maintain an efficient symbiosis relationship are still poorly understood. Recently, the identification of several miRNAs involving in AM symbiosis opened a new era for studying the regulation of AM symbiosis at transcription level. To obtain further insights into the role of miRNAs in AM symbiosis, this project planned to investigate the molecular mechanism and evolutionary pattern of AM symbiosis-regulating miRNAs from the following aspects. Firstly, a global survey of AM-responsive miRNAs in representative species from two deep-diverged angiosperm lineages of dicot (tomato) and monocot (rice) will be performed by high-throughput sequencing and bioinformatics analysis, through which the conserved and species-specific AM-responsive miRNAs could be distinguished and the shaping forces could be traced. Secondly, the interaction of a set of conserved AM-responsive miRNAs and their predicted target genes will be tested and their exact functions in AM symbiosis will be determined. Finally, the origin and evolution of these conserved AM symbiosis-regulating miRNAs and target genes will be explored by searching their homologs from key land plant lineages. The progress made by this project would enhance our understanding on how miRNAs were recruited by plants to fine tune an anciently originated symbiotic relationship and provide new insights into the molecular mechanism and evolutionary pattern of AM symbiosis.

超过80%的陆地植物都能与丛枝真菌形成共生——丛枝菌根（AM），这种共生关系的建立依赖于一套起源古老并在不同植物类群中保守的植物共生基因。但是，植物如何调控共生基因表达，以维持和谐的共生关系？以及这种调控作用是如何演化的？目前尚不清楚。本项目拟在前期发现植物miRNA对AM共生调节的基础上，以被子植物不同演化分支的两个代表性物种（双子叶植物番茄和单子叶植物水稻）为切入点，结合高通量测序和生物信息学分析，比较两种植物共生响应miRNA的谱系组成，发现物种间保守的调节AM共生的植物miRNA，鉴定其靶基因，并通过转基因、基因敲除等技术对这些miRNA进行功能验证，探讨其调节AM共生的分子机制。最后，通过调查番茄和水稻间保守的共生响应miRNA在陆地植物关键演化节点的存在状况和调控特性，追溯植物miRNA对AM共生调节作用的起源及演化，从表达调控的全新角度揭示植物AM共生建立的分子和演化机制。

植物丛枝菌根（arbuscular mycorrhiza, AM）共生是一类起源古老、分布广泛的植物-微生物共生关系，超过80%的陆地植物都能与丛枝真菌形成AM共生。本项目以被子植物两大分化类群（双子叶植物和单子叶植物）中的两个代表性物种（番茄和水稻）为切入点，通过鉴定这两个物种在AM共生和非共生状态下表达存在显著差异的miRNA（简称AM共生响应miRNA），发现在番茄和水稻中分别有7个和53个AM共生响应miRNA家族；进一步整合公共数据库中发表的其它单、双子叶植物AM共生前后的sRNA测序数据，发现在烟草和玉米中各有5个miRNA家族的成员在AM共生时表达发生显著性改变；对上述四个物种中AM共生响应miRNA进行比较分析，发现仅有一个miRNA家族——miRNA399在不同被子AM共生过程中发生一致性的表达下调，而其它的共生响应miRNA多为物种特异性共生响应miRNA，暗示被子植物miRNA对AM共生的调控作用既存在保守性，又存在物种特异性的演化特征；在此基础之上，我们通过对番茄AM共生过程中的多维度RNA组学测序和分析，构建了一个以miRNA为核心的调控AM共生的ceRNA网络；为了深入揭示在多个被子植物中保守的AM共生响应miRNA——miR399在AM共生中的调控作用及其分子机制，我们构建了miR399过表达的番茄转基因植株，通过实验研究发现miR399过表达对植物AM共生具有负调控作用，表明miR399是AM共生的负调控因子，并且可能通过作用于其靶基因PHO2及下游基因PHT1发挥对AM共生的调控作用；比较基因组学分析发现，miR399及其靶基因PHO2的出现均可以追溯到种子植物的共同祖先，而miR399识别靶位点仅能在被子植物的PHO2基因上检测到，暗示miR399通过靶向PHO2对AM共生的调控作用可能在被子植物共同祖先中演化形成。综合而言，本项目揭示了番茄和水稻等被子植物AM共生响应miRNA的谱系组成，发现并深入研究了唯一一个在不同被子植物中保守的AM共生响应miRNA——miRNA399对AM共生的调控作用及其分子机制，追溯了miR399-PHO2调控模块在陆地植物不同类群中的存在情况和演化模式，揭示了miRNA对AM共生的调控作用的保守性与多变性共存的演化特征，为从RNA层面研究AM共生的调控作用拓展了新的研究思路。