杨树防御应答相关miRNA及其靶基因的鉴定与功能研究

Poplar is one of the important fast-growing timber and tree species which plays an important role in forestry sustainable development in China. The fast-growing planting of new varieties of high-yield poplar promotes the great development of poplar planting industries. But the poplar concentrated plantations and homogeneous forest structure is very likely to lead to forest disease outbreak and epidemic, so there is an urgent need to study the poplar defense mechanism and also can provide a theoretical basis for poplar disease-resistant breeding. miRNAs are now known to have greatly expanded roles in a variety of plant developmental processes and play an important role in the fine regulation of signal transduction, and the response to environmental stress and pathogen invasion. Based on Marssonina brunnea and poplar genome sequence resources, we take new-generation semiconductor sequencing technology as the starting point, to build and improve a platform to identificate and functional verificate poplar defense-related miRNAs and their target gene, to resolute the molecular mechanisms of interaction between poplar and sMarssonina brunnea, to excavate and validate of miRNAs and their target genes regulating defense mode, to analyze the regulation network of miRNAs in poplar disease-resistance, then to provide theoretical guidance in breeding lasting broad-spectrum resistance new varieties of poplar.

杨树是我国重要的速生工业用材树种和绿化造林树种之一，对我国无性系林业可持续发展发挥了重要作用。随着速生高产杨树新品种的不断推广，各地杨树种植产业迅猛发展，但是林分结构单一，可能引发森林病害的爆发和流行，因此迫切需要研究杨树防御应答机制，为杨树抗性育种提供理论基础。miRNA(microRNA)是一类时序性调控生长发育进程的小分子RNA，在植物防御应答的精细调节中发挥重要作用。本研究基于杨生褐盘二孢菌全基因组序列信息和杨树基因组信息，以新一代半导体测序技术(PGM)为平台，开展杨树防御应答相关miRNAs及其靶基因的鉴定和功能研究，解析杨树-杨生褐盘二孢菌（Marssonina brunnea）相互作用的分子机制，挖掘和验证相关miRNAs及其靶基因的防御调控模式，分析杨树黑斑病防御反应中miRNAs的调控网络，为选育具有持久广谱抗性的杨树新品种提供理论依据。

杨树(Populus)是我国的速生用材树种之一，但杨树人工林林分结构单一，导致遗传多样性降低，病原物易于侵染和传播，一旦爆发，将严重影响杨树的生长。杨生褐盘二孢菌（Marssonina brunnea）是杨树黑斑病的致病因子，受害杨树提早落叶，光合效率下降，严重影响树木生长量。因此，系统而深入地研究杨生褐盘二孢菌致病机制、杨树抗病机制以及二者间相互作用机制，具有非常重要的科学意义，同时也为杨树抗病育种和遗传改良提供理论基础。本研究结果如下：.1、构建了4个NL-895杨受杨生褐盘二孢菌诱导和非诱导的miRNA库，并通过深度测序，建立miRNA和EST表达谱，筛选出显著表达（上调或下调）的miRNAs，并挖掘杨树防御应答特有的miRNA。.2、使用stem-loop RT-qPCR(茎环引物反转录的实时定量PCR)检测法，定量检测miR156a、miR164a在不同侵染阶段时杨树miRNAs的表达情况；克隆杨树miR164a及其靶基因PeNAC1.1和PeNAC1.2，miRNA393家族的5个靶基因：PeTIR1，PeAFB2-1，PeAFB2-3，PeAFB3和PeAFB5；利用Gateway技术构建目的基因mRNA164a，PdNAC1.1，PdNAC1.2.和PeTIR1的植物过量表达载体，采用农杆菌介导法转化欧美杂交山杨T89以及山新杨，经抗生素筛选，以及获得了一批再生植株。.3、已发表SCI论文1篇，中文核心刊物论文2篇，培养研究生2名。. miRNA在植物抵抗细菌、真菌和病毒的过程中发挥作用，而植物防御系统的平衡取决于由植物miRNA直接导致的RNA干扰或miRNA特异靶基因的降解。同时miRNA在植物抗生物和非生物胁迫的途径中存在着交叉，这种复杂的miRNA调控网络使植物具有适应经常改变的环境的能力。