毛白杨miR476a靶向调控PPR231基因促进不定根发生的分子机制研究

Adventitious root formation is a limited factor in vegetative propagation and industrialized seedling culture techniques of tree breeding. How to improve rooting ability of wood species is important for successful propagation of woody plants from cuttings. In a previous work, we have isolated a root-specific expression gene miR476a from Populus tomentosa Carr. Overexpression of miR476a resulted in a increase in the number of lateral and adventitious roots of transgenic plants. However, the mechanism of adventitious root formation regulated by miR476a is still unknown. In this project, the predicted target genes PPRs will firstly be confirmed by RACE method. To investigate their functions, the PPR genes, in which target sites specific for miR476a are mutant, will be introduced into transgenic plants overexpressing miR476a. Sucullular localization will be used to determine if PPR proteins are localized in mitochondia. Meanwhile, the constructs containing the 35S:PPR genes will be transformed into poplar plants, respectively. And also poplar ppr mutants will be generated by the CRISPR/CAS9 method. We will use Northern bloting to compare the transcripts of different genes in mitochondrial NAD genes. Using NBT、DAB and DCFH-DA staining, accumulation of reactive oxygen species (ROS) in transgenic plants overexpressing miR476a will be detected. The levels of phytohormones such as Auxin, GA, BR, SA, SL, ABA, will be determined by HPLC and GC-MS, respectively. Forthermore, transcriptome sequencing and qRT-PCR analysis are used to examine difference in expression level of genes involoved in hormone transduction. Our study will figure out the molecular mechanism of adventitious roots in poplar and provide a sight for improvement of rooting efficiency in vegetative propagation in poplar.

扦插是林木生产中主要的繁育方式之一，因此搞清树木不定根发生的分子机理对于林木育种具有十分重要的意义。课题组前期研究发现，超表达毛白杨miR476a降低了其预测靶基因PPR231的表达，促进了不定根的发生，但其机制尚不清楚。为此，本项目拟：①在过表达miR476a材料中验证PPR231功能；②采用Confocal对PPR231进行亚细胞定位；③转录组分析超表达及CRISPR/CAS9敲出PPR231转基因杨树中差异表达基因；④qPCR和蛋白纯化明确PPR231对线粒体NAD基因的调控作用；⑤组织化学分析不定根中活性氧ROS的积累，弄清其在不定根发育的作用；⑥检测不定根中生长素水平，qPCR分析生长素信号通路基因的表达谱，搞清不定根发生过程中ROS与生长素之间的调控关系。最终阐明miR476a通过调控PPR231的表达促进毛白杨不定根发生的分子机制，为提高杨树扦插生根效率奠定理论基础。

扦插是目前林木无性繁殖的主要方式，生根则是影响扦插成活的首要因素。在扦插损伤刺激下，插条伤口处的一部分细胞向根细胞发生命运转变，启动不定根再生。生长素已被发现在促进不定根发生过程中发挥关键作用，然而从扦插刺激到生长素启动干细胞命运转变的信号传递机理并不清楚。本项目在前期克隆到一个影响杨树不定根发育的小RNA基因miR476a的基础上，全面解析了该基因调控杨树不定根形成的表达模式和遗传功能，鉴定了其直接靶标基因RFL26/PPR231，揭示了miR476a-RFL26模块通过影响线粒体功能稳态并级联下游的生长素信号，从而调控不定根原基发生的分子机制。本项目取得的重要结果和关键数据包括：（1）通过构建过表达和表达抑制株系并开展生根表型分析，证实miR476a是促进杨树不定根原基发生的关键基因；（2）开展启动子报告株系分析，发现miR476a的表达水平在不定根原基建立阶段显著受诱导；（3）结合降解组数据、基因表达检测、荧光切割实验和遗传回补分析，鉴定了miR476a的直接靶标是线粒体定位的PPR蛋白编码基因RFL26；（4）通过线粒体功能指标检测，发现miR476a-RFL26模块调控了生根过程中线粒体功能的动态变化；（5）利用线粒体抑制剂外源处理，阐明了miR476a介导的不定根发育依赖于线粒体功能稳态；（6）发现miR476a介导的线粒体功能增强，诱导了PIN基因表达，从而促进了生长素向插条伤口处的累积，启动根原细胞命运建立并形成不定根原基。基于以上结果，我们提出了杨树miR476a-RFL26分子模块通过动态调节线粒体功能稳态并级联生长素信号促进扦插后不定根发生的工作模型，揭示了miR476a通过调节线粒体功能启动向根细胞命运转变的作用机制，建立了从扦插刺激到杨树不定根再生的信号传递的基本框架，形成了线粒体级联生长素信号从而调控林木细胞分化潜能的新认识。