miRNA与生长素信号互作对杨树不定根发生的调控研究

Deep roots give rise to flourishing leaves, the two complement each other. However, up to now, genetic mechanisms underlying adventitious rooting for forest trees remain elusive. Poplar is accepted as a model system for molecular tree biology, and its rooting traits play an important role in the genetic improvement of poplars. Based on the interactions between miRNA and its target genes as a starting point, with the auxin signaling network as the focus, this project will study the molecular mechanism of poplar adventitious rooting. By establishing and improving the experimental system of poplar adventitious rooting, this study will investigate its morphogenesis and reveal auxin signal sensitivity of specific tissues or cell in root primordia. Meanwhile, miR160、miR167、miR390、miR393 and their target genes (ARF and TIR1/AFB) will be detected and validated by degradome sequencing technology, and the interactions between the above miRNAs and their target genes will be tested by PCR mutagenesis strategy and using transient expression assay in poplar protoplasts. Finally, we will systematically analyze their functions in adventitious rooting by expression vector construction and poplar genetic transformation experiments to explore the molecular regulatory networks of poplar adventitious rooting. This will provide a theoretical basis for the improvement of poplar rooting traits.

根深叶茂，两者相辅相成。林木不定根发生的遗传调控机理迄今尚无明确的结论。杨树是林木分子生物学研究的模式树种，生根性状在杨树遗传改良中占有重要地位。本项目以miRNA及其靶基因互作为切入点，围绕生长素信号转导途径，开展杨树不定根发生的分子调控研究。首先，建立和完善杨树不定根发生的实验系统，阐明不定根形态建成，揭示根原基形成中特定组织或细胞对生长素信号的发根感受性；同时，利用降解组测序技术，检测和验证miR160、miR167、miR390、miR393及其ARF和TIR1/AFB家族靶基因，结合靶基因剪切位点同义突变，在杨树原生质体中定性和定量地分析它们之间的相互作用；最后，通过表达载体构建和杨树遗传转化，对转基因杨树进行系统地分子检测和表型分析，结合生根试验和不同激素诱导处理，解析上述miRNA与生长素信号互作对杨树不定根发生的调控作用，为改良杨树生根性状和种质创新提供理论依据。

根深才能叶茂，根系是植物繁茂和逆境适应的基础。杨树是林木分子生物学研究的模式树种，本研究围绕miRNA与生长素信号互作模式，主要开展了如下工作：（1）构建了DR5::GUS转基因杨树株系，并基于组织切片和GUS染色追踪了不定根形态建成过程中特定组织对内源生长素的动态富集模式；（2）结合微扦插生根相关小RNA、降解组及转录组数据，综合分析完成了杨树不定根发生特异miRNA及其靶基因筛选和鉴定工作，共鉴定出89个已知miRNA的359个靶基因、6个novel miRNA的14个靶基因，即373对miRNA与mRNA表达调控组合，这些靶基因多与生长素信号转导相关；（3）基于降解组测序与RACE实验，探究了miR390-TAS3-ARFs和miR393-AFBs模块在杨树生长发育过程中的表达调控模式；（4）采用5'-RLM-RACE、多位点同义突变及原生质体瞬时表达等技术手段验证了miR160、miR167及其生长素信号转导途径相关靶基因的互作关系，并检测了它们在不同激素处理下的转录表达变化趋势；（5）通过各类表达载体构建、遗传转化及转基因杨树株系扦插生根试验，系统地揭示了miR160/miR167-ARFs模块对杨树不定根发育的调控功能；（6）对全转录组与降解组数据进行了联合分析，探讨了参与不定根生长发育调控的lncRNA/circRNA-miRNA- mRNA网络。综上所述，本研究运用了小RNA组、转录组和降解组等技术，并佐以系统地实验验证，所得到的上述研究结果，不仅有助于丰富植物器官发生与形态建成的理论基础，也将为改良林木生根性状及其种质创新提供科学依据。此外，在项目的资助下，已发表学术论文13篇，其中SCI收录9篇；2项发明专利获得授权；并培养了5名研究生。