杨树Aux/IAA转录因子对木材发育的调控机制研究

Wood is the most abundant biomass on earth and has major economic importance for humans. To improve wood quality with molecular breeding techniques, it is critical to understand the molecular and biochemical mechanisms controlling wood formation. Wood, also termed secondary xylem, derives from a secondary meristem, the vascular cambium. Wood formation is a complex biological process, including cell division, cell expansion, secondary cell wall thickening and programmed cell death (PCD). In the past decade, significant progress has been made to uncover the molecular players in various developmental stages of wood formation. Hormonal signalling, peptide–receptor–transcription factor signaling pathway, and many transcription factors have been shown involved in wood formation...The plant hormone auxin has also been demonstrated to play a key role in regulating wood formation through its effects on vascular cambium activity and the differentiation of xylem cells. Auxin/Indole-3-Acetic Acid (Aux/IAA) is one of the well-known auxin response mediators which have been mainly characterized in model plants such as Arabidopsis. In contrast, they remain largely uncharacterized in tree species; especially their role on cambium activity and xylogenesis is still unclear. Our previous work showed that overexpressing Eucalyptus IAA4, 9A and 20 in Arabidopsis could impact cambium activity, fiber cells differentiation and secondary cell wall composition. To validate their functions in woody plant and reveal the regulation mechanism in wood formation, we will use Populus as a model. In this project, first we need select the most promising candidate Aux/IAA genes to construct overexpression and RNAi transgenic Populus. Then we will analyze their impacts on cambium activity and xylem differentiation through histological analysis. At the same time, their regulation mechanism will be revealed by biochemical and molecular ways, like transcriptome sequencing, Pull-down, etc. Finally, we will figure out the role of Aux/IAA in wood formation.

木材形成是一个受到严格调控的复杂生物学过程，为实现通过分子育种技术定向改良木材品质，首先需深入了解木材形成过程的分子机制。已经证明，植物激素如生长素在木材形成层活动及维管组织分化中起着极其重要的作用。Aux/IAA是植物生长素响应的主要转录调控蛋白，课题组在前期工作中发现，桉树IAA4、IAA9A和IAA20蛋白可以抑制拟南芥形成层活动及木纤维细胞发育，但其分子机制尚不清楚。由于桉树转基因技术还不成熟，本项目拟在杨树基因组中筛选参与木材形成的Aux/IAA基因，构建超表达及RNAi抑制载体转化杨树，通过组织切片、显微分析等方法观察转基因植株形成层及次生木质部发育受到的影响，同时检测木质部各组分含量的变化，并利用转录组测序、Pull-down等技术分析鉴定其调控的下游靶标基因，搞清Aux/IAA转录因子在杨树木材形成过程中的调控机制，揭示生长素信号调控木材形成的分子机理。

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。在本项目中我们筛选出Aux/IAA3.2作为杨树参与木质部形成的候选基因。通过转基因及生化分析，初步弄清了生长素对木材发育的调控机制。主要结果如下：.超表达PtoIAA3.2m的转基因杨树的株高、茎直径等都有明显下降。茎横切片甲苯胺蓝染色结果显示，在过表达株系中，木质部发育受到明显抑制，木质部细胞层数减少，相对面积下降。酵母双杂交和双分子荧光互补实验证明PtoIAA3.2m 和 PtoARF5.1互作。将去掉III / IV 结构域的 ARF5.1Δ过表达载体转入 PtoIAA3.2m-OE 转基因植株中，发现PtoARF5.1Δ/PtoIAA3.2m-OE会完全回复PtoIAA3.2m-OE的表型。荧光定量 PCR 结果显示，在 PtoIAA9m-OE 转基因株系中，细胞壁合酶基因表达量受到明显下调，而在PtoARF5.1Δ/PtoIAA3.2m-OE中其表达回复到WT水平。.综上所述， PtoIAA3.2通过和 PtoARF5.1 相互作用形成蛋白复合体，并调控下游细胞壁合酶基因的表达，从而调控木质部的发育。以上结果将为解释生长素信号调控木材发育提供理论依据，为木材资源的优化奠定分子基础。