杨树ARF2因子调控木质素合成的分子机制研究

Lignin is one of important components in the plant secondary wall. Regulating the lignin content and ingredient of poplars through gene engineering to get new transgenic poplars plays key roles in environment protection and economy. Today, auxin plays important roles in lignin biosynthesis while the underlying mechanism remains elusive. Through studies in Arabidopsis, Auxin Responsive Factor 2 (AtARF2) is involved in the auxin-mediated vascular development. The poplar is the tree model plant and PtrARF2，the AtARF2 homolog，has proved to be preferentially and highly expressed in xylem cells and pholem cells in our previous analysis. ..In this project, we firstly clone the full-length of PtrARF2 gene, and analyze its response to auxin by real-time PCR. Using by the confocal laser scanning micoscope, the subcelluar location of PtrARF2 is identified. Through reverse genetics analysis in poplar, the specific role of PtrARF2 is revealed in lignin biothsynthesis depended on over-expressing or knocking out PtrARF2. Further research of differently expressed genes by RNA-sequencing uncovers the regulation network of PtrARF2 in the lignification. ..Moreover, the downstream PtrMYB or PtrLAC gene directed by PtrARF2 will be identified by the auxin responsive element (AuxRE) in their promoters and confirmed by in vivo transient effector-reporter analysis and the electrophoretic mobility shift assay (EMSA) in vitro. All together, the data provides insights into the molecular regulation of PtrARF2 in lignin biosynthesis.

木质素是植物次生壁中重要的组成成分，如何有效地调控木质素的合成对于林木资源的开放利用具有重要意义。研究表明，生长素在木材发育过程中起着重要作用，但其如何调控木质素合成的分子机制仍不清楚。在拟南芥中研究表明，AtARF2因子参与了维管束发育，但其具体作用知之甚少。课题组前期在杨树中发现，PtrARF2基因在次生木质部和韧皮部中特异表达。为搞清PtrARF2因子的功能，本项目首先克隆PtrARF2基因，分析其对生长素的响应情况；利用激光共聚焦方法确定其亚细胞定位；通过转基因超量表达和定点敲除技术，搞清PtrARF2因子在木质素合成中的具体作用；结合RNA测序，建立其下游分子调控网络；通过生长素响应元件（AuxRE）分析，利用瞬时共转化和EMSA实验，鉴定其靶向的下游PtrMYB或PtrLAC基因，最终阐明PtrARF2因子调控木质素合成的分子机制。

生长素应答因子(ARFs)是调节植物生长素应答基因在各种生物过程中表达的重要调控因子。在杨树基因组中，共鉴定出39个ARF成员，但其具体功能尚不清楚。在本研究中，6个PtrARF2成员从果杨中分离出来；表达谱分析显示，PtrARF2.1主要在叶片组织中表达，且外源性生长素处理显著抑制其表达水平。酵母单杂交和亚细胞定位分析表明，PtrARF2.1不具有转录激活活性且在细胞核中特异性表达。通过反向遗传学方法研究PtrARF2.1在杨树中被RNA干扰(RNAi)的敲除导致矮秆植株，叶片形状发生改变，叶片尺寸变小；而过表达PtrARF2.1导致植株高度减少，与野生型相比叶片表型相似。组织学染色显示PtrARF2.1-RNAi株系叶脉和叶柄中木质素异位沉积。PtrARF2.1-RNAi株系叶中RNA-测序(RNA-Seq)分析显示，有74个差异表达基因(DEGs)属于NAC、MYB、ERF和bHLH等12个转录因子家族，24个DEGs与木质素生物合成途径相关。通过PtrARF2.1与PtrMYB92蛋白相互作用分析揭示，PtrARF2.1是通过间接影响PtrMYB92进而调控木质素合成途径中关键酶基因的表达。综上所述，数据表明PtrARF2.1在杨树叶片发育过程中起着重要的调控作用，影响杨树木质素的生物合成。