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。

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.