油菜素内酯受体PtBRI1调控杨树木质形成的分子机理研究

Our previous study on the function of PtCYP85A5 gene demonstrated that Brassinosteroids (BRs) could play an important role in poplar wood formation. However, the exact molecular mechanisms of them are still unknown. Recently, we found that overexpression of the BR receptor gene PtBRI1 in poplar promoted wood formation and induced the expressions of PtWNDs and other NAC and Myb transcriptional factors and cell wall-biosynthesis genes involved in poplar wood formation. Therefore, we speculate that BR may control wood formation by regulating these wood-associated genes. To dissect the mechanism, PtBRI1 will be overexpressed, silenced and knocked out to change BR signaling in transgenic poplar. And the biological functions of PtBRI1 in wood formation will be examined at gene, protein, tissue and cellular levels. Then, RNA-seq will be performed to screen the down-stream genes of PtBRI1. Whether BR signaling is activated by PtBRI1 through BZR1/BES1 (two important transcriptional factors in BR signaling) will also be determined. Finally, whether the regulation of wood formation by BR is achieved via the regulation of wood-associated genes by BZR1/BES1 and other key transcription factors will be assessed by Chip-seq, qRT-PCR, double fluorescence experiment, yeast one hybrid and EMSA. The completion of this project will not only clarify the biology function of PtBRI1 and the molecular mechanism of BR in wood formation, but also provide research bases for the genetic improvement of poplar trees.

申请者前期研究发现油菜素内酯（BR）在杨树木质发育中起着重要作用，过表达油菜素内酯受体PtBRI1可以促进杨树木质部的发育，并上调PtWNDs及其它木质发育相关基因的表达。为阐明PtBRI1调控木质发育的分子机理，本研究拟通过过表达、沉默和敲除PtBRI1改变BR信号，并从基因、蛋白、细胞、组织等方面阐明其在木质发育中的生物学功能；通过RNA-seq挖掘PtBRI1下游关键转录因子，并确认BZR1/BES1转录因子是否参与激活BR信号；进一步通过ChIP-seq鉴定BZR1/BES1和其他关键转录因子的DNA结合序列，分析靶标基因，再用qRT-PCR及双荧光实验、酵母单杂及EMSA实验对转录调控和结合进行验证，综合分析揭示由PtBRI1介导的BR信号通路在杨树木质形成过程中的调控网络。研究结果在阐明PtBRI1的生物学功能和调控杨树木质发育分子机理的同时，还可以为杨树的遗传改良奠定基础。

杨树是重要的生物能源和造林树种，其木质部是主要的工业原料，也是重要的可再生生物能源。因此，改良杨树的林木产量及木质部的生产速度具有巨大的经济价值和生态效益。油菜素内酯（BRs）是一种重要的植物类固醇激素，参与了植物细胞分裂分化、细胞程序性死亡和抗逆等过程，并在杨树木质部发育中起重要作用，但其在此过程中的分子调控机制尚不清楚。项目前期，我们发现过表达BRs受体基因PtBRI1可以促进杨树木质部发育，并上调木质部发育相关基因的表达。为了探究相关的分子机制，我们从在毛果杨中克隆了与拟南芥AtBRI1序列高度同源的PtBRI1.2基因，亚细胞定位结果显示PtBRI1.2作为受体定位于细胞膜上。通过荧光定量PCR发现该基因在杨树顶芽、形成层和木质部等组织中的均有表达，且受到24-表芸苔素内酯（BL）的诱导。在拟南芥突变体中异源表达PtBRI1.2，可将拟南芥突变体矮化及结实率低等表型恢复至野生型水平。在杨树中过量表达PtBRI1.2可显著提高转基因株系的生长速度、株高和生物量等。通过转基因株系的田间试验进一步证实了过表达PtBRI1.2可导致杨树转基因植株的株高和茎粗生长速度加快。此外，通过免疫组织化学、蛋白质印迹和Dual-Luc等技术进一步研究发现PtBRI1.2可以增强BR信号途径中的关键转录因子PtBZR1的活性，并促进木质部中PtWNDs及纤维素和木质素合成相关基因的表达。本研究不仅揭示了BRs及其受体PtBRI1.2在杨树木质部发育中的作用机理，加深了我们对BRs在木质形成中调控机制的了解，而且为杨树等经济树种的遗传改良提供了宝贵的基因资源和理论基础。