落叶松不定根发育关键miRNA及其靶基因的多组学联合精准鉴定与分子功能研究

miRNAs mediate adventitious root development (ARD) by regulating the expression of their target genes. In the study of regulation mechanism for the "miRNA-target genes" involved in ARD, we find that the identification analysis of miRNA and its target genes is inefficient and poor in accuracy. Therefore, this project takes the hard-to-root tree species larch as the research material. On the basis of the completion of its genome sequencing and gene annotation, we will sequence smRNAome, degradome, transcriptome of the cuttings from two clones with significant rooting differences at the critical time node of ARD. The two clones generated from the same family. Using the existing miRNA and target gene information, we will develop method of multi-omics joint analysis of “genome – smRNAome – degradome - transcriptome” data by simulation and calculation. The key miRNAs and their target genes that regulate ARD will be deciphered by this method, and their molecular function will be parsed and verified using larch transgenic experiment etc. This study will reveal the molecular regulation mechanism of "miRNA-target genes" on ARD in larch. Thus, this project can help to solve the problem of hard rooting of larch by molecular biology technology, and provide theoretical basis and technical support for resource conservation and asexual expansion of excellent germplasm.

miRNA可通过调节其靶基因的表达介导不定根的发育。在研究“miRNA-靶基因”调控不定根发育机制时发现，对miRNA及其靶基因的鉴定存在效率低、准确性差等问题。因此，本项目以难生根树种落叶松为研究对象，在已完成其基因组测序及基因注释的基础上，利用前期筛得的同家系生根差异较大的两份无性系材料，对其在不定根发育关键时间节点的插穗进行smRNA组、降解组和转录组等多组学测序，利用已有miRNA及靶基因信息进行数据模拟及分析，研发一套“基因组-smRNA组-降解组-转录组”多组学联合精准鉴定调控不定根发育的关键miRNA及其靶基因的分析方法，确定出调控该过程的关键miRNA和靶基因，并通过落叶松转基因等实验对其功能进行解析和验证，揭示“miRNA-靶基因”调控落叶松不定根发育的分子机制。该研究有助于利用分子手段解决落叶松扦插难生根问题，为优良种质的资源保存和无性扩繁提供理论依据和技术支撑。

miRNA可通过调节其靶基因的表达介导不定根的发育。在研究“miRNA-靶基因”调控不定根发育机制时发现，对miRNA及其靶基因的鉴定存在效率低、准确性差等问题。本项目以难生根树种落叶松为研究对象，在已完成其基因组测序的基础上，运用Perl语言编程，构建了一种基于“基因组-smRNA组-降解组-转录组”多组学联合分析的植物高可信度miRNA及其靶基因鉴定方法；整合落叶松组学数据，应用该方法在落叶松全基因组水平完成了miRNA及其靶基因的精准注释，发现串联复制事件是落叶松保守MIRNA扩张的主要驱动力；鉴定出落叶松不定根发育相关的4个miRNA（lka-miR482k-5p、lka-miR167b-5p、lka-miR482d-5p和lka-miRN511-5p）及4个靶基因（含1个LkBBM和3个LkARF基因），并利用荧光定量、亚细胞定位、遗传转化等试验验证了上述靶基因在落叶松不定根发育过程中的功能。本项目的实施，为落叶松及其他针叶树种miRNA的研究提供了重要参考方法和数据资源，同时初步揭示了落叶松不定根发育的“miRNA-靶基因”分子调控机制，对落叶松遗传改良和扦插扩繁具有重要的理论意义和应用价值。