杨树光敏色素互作因子PIFs调控次生壁合成的机理研究

次生细胞壁合成受到遗传和环境因素的调控。研究表明，光作为最重要的环境因子之一，光强和光质改变（红光与远红光比率）能够影响次生壁的生物合成，但其分子机制还不清楚。本项目前期研究发现，毛白杨光敏色素互作因子PtoPIF3.1和PtoPIF3.2可能参与次生细胞壁合成，为搞清其功能，拟创制PtoPIF3.1/3.2超表达和CRISPR突变体材料，结合表型分析、免疫组化染色和细胞壁组分测量等，阐明PtoPIFs在次生壁合成过程中的生物学功能。进一步利用转录组测序分析预测PtoPIFs下游基因调控通路，通过酵母单杂交试验初步筛选出直接靶基因，再通过凝胶阻滞迁移、ChIP-qPCR、Effector-Reporter和遗传回补等试验，验证PtoPIF3.1/3.2因子与候选靶基因的调控关系，从而阐明光信号通路调控次生壁合成的分子机理。预期研究成果可为培育和改良林木新品种提供新思路和奠定理论基础。

The biosynthesis of secondary cell wall (SCW) is regulated by genetic and environmental factors. It has been shown that light is one of the most important environmental factors, and changes in light quantity and quality (the ratio of red light to far red light) can affect SCW biosynthesis, yet the underlying molecular mechanisms remain unclear. Our preliminary studies suggest that Populus tomentosa PHYTOCHROME-INTERACTING FACTOR 3.1 (PtoPIF3.1) and PtoPIF3.2 may be involved in regulating SCW biosynthesis. To test this hypothesis, this project plan to generate populus PtoPIF3.1/3.2 overexpression and CRISPR mutant plants, and conduct phenotype analyses, immunohistochemical staining and cell wall component measurement to elucidate the biological function of PtoPIF3.1/3.2 in SCW biosynthesis. Next, we will perform transcriptome sequencing analyses to predict downstream gene regulatory pathways of PtoPIF3.1/3.2, and to identify direct target genes using yeast-one-hybrid assays. Further, electrophoretic mobility shift assays (EMSA), chromatin immunoprecipitation (ChIP)-qPCR, Effector-Reporter assays and genetic complementation experiments will be performed to verify the regulatory relationship between PtoPIF3.1/3.2 and putative target genes, consequently elucidating the molecular mechanisms for light-mediated regulation of SCW formation. Anticipated research results may provide novel insights and theoretical basis for the cultivation and improvement of new tree varieties.