毛白杨PtoIAA9基因对木材次生发育的调控机制研究

Wood is widely used in pulp and paper manufacturing and recently has been considered as a promising source for industrial bio-based products and advanced biofuels. Secondary phloem and xylem tissues are produced through the activity of vascular cambium, which determines timber production. Identification and functional analysis of genes regulating wood secondary development are the preconditions for molecular breeding in tree species. . Auxin is considered as one of the key regulators in vascular tissue morphogenesis. Aux/IAA proteins are transcriptional repressors that control auxin signaling by interacting with auxin response factors (ARFs). Despite Aux/IAA have been characterized with regulation on vascular cambium activity and the differentiation of xylem cells in Arabidopsis, it remains to explore about their effects on wood development. . To validate Aux/IAA functions in woody plant and reveal the regulation mechanism in wood formation, Populus was used as a model. In this project, we selected the PtoIAA9 as the most promising candidate Aux/IAA genes. A series of experiments are designed and processed to analyze cell wall contents and the expression levels of marker genes, in both PtoIAA9 overexpression and CRISPR-Cas9 transgenic lines. Then the interacted proteins (ARFs) will be identified by yeast-two hybrid system, BiFC and Pull-down. We will also reveal the target genes with transcriptome sequencing, Effector-Reporter, EMSA, ChIP, etc. At the same time, the selected ARF and target gene will be overexpressed in the PtoIAA9 transgenic background plants to restore the phenotype. Finally, with both biochemical and genetic evidences, we will clarify the role and the regulation mechanism of Aux/IAA in wood formation, and the relationship among Aux/IAA and other transcription factors, which provides a genetic strategy for traits improvement in woods.

木本植物的次生维管组织决定着木材的产量，如何有效调控次生生长对于林木分子育种具有重要的意义。生长素被认为是决定次生木质部形成最重要的激素信号，但木本植物中生长素调控次生发育的研究还鲜有报道。我们在前期工作中发现毛白杨PtoIAA9在次生维管组织特异表达，但其具体功能尚不清楚。为此，本项目拟获得PtoIAA9的超表达和敲除转基因植株，测定细胞壁组成和相关基因表达变化，解析PtoIAA9在木材次生发育中的功能；通过酵母双杂交、BiFC、Pull-down等方法鉴定出PtoIAA9互作蛋白；进一步利用转录组测序预测下游靶基因，通过Effector-Reporter、EMSA、ChIP等鉴定下游靶基因；通过遗传互补实验，综合生化和遗传证据揭示PtoIAA9的调控机理，建立PtoIAA9与其它转录因子间的调控关系，阐述生长素信号调控木材发育的分子机制，为筛选和培育优质的林木种系奠定基础。

木本植物的次生维管组织决定着木材的产量，如何有效调控次生生长对于林木分子育种具有重要的意义。生长素被认为是决定次生木质部形成最重要的激素信号，但其调控木材次生发育的研究还鲜有报道。我们在前期工作中发现毛白杨PtoIAA9是一个典型的Aux/IAA蛋白，受生长素诱导，在次生维管组织形成层及发育的木质部特异高表达，但其具体功能尚不清楚。为此，本项目获得了IAA9蛋白稳定存在的PtoIAA9m超表达转基因植株，发现其显著抑制了次生发育。通过酵母双杂交、BiFC等方法鉴定出PtoIAA9互作蛋白PtoARF5，其PtoIAA9通过III,IV结构域与PtoARF5互作，PtoARF5能部分回复PtoIAA9m-OE的表型。进一步利用转录组测序、Effector-Reporter、ChIP-PCR等鉴定出下游靶基因HD-ZIP III型转录因子HB，通过遗传互补实验，综合生化和遗传证据揭示了Aux/IAA-ARF-HB模块调控木材次生发育的调控机理，阐述生长素信号调控木材发育的分子机制，为筛选和培育优质的林木种系奠定基础。