PtoWRKY40基因调控毛白杨次生生长的分子机理研究

The production of genetically engineered new varieties of forest trees for fast-growing and fine quality can effectively improve timber yield and quality of man-made forest. Therefore, the research of molecular mechanism of wood formation has been a focus in research field of forestry molecular biology. In our previous study, PtoWRKY40 of Populus tomentosa Carr. may be involved in the regulation of secondary wall formation through regulates the expression of secondary wall biosynthetic genes PtoCCoAOMT1 and Pto4CL3. The interaction between PtoWRKY40 and several proteins such as Elongation factor protein, Alpha-xylosidase family protein and Sucrose synthase was observed in the GST pull down assay and Co-immunoprecipitation (Co-IP) tests. In this research, mechanisms of interaction between PtoWRKY40 and PtoWRKY40-interacting protein was further identified by investigations using Yeast two-hybrid (Y2H) assay, etc. Mechanism of PtoWRKY40 regulate the expression of PtoCCoAOMT1 and Pto4CL3 was illuminated by Arabidopsis thaliana protoplast transient expression system, etc. Effect of PtoWRKY40 overexpression or downregulation on the xylem of transgenic poplar plants was analyzed by paraffin section. Through this project, it could elucidate the molecular mechanism of PtoWRKY40 in regulating secondary growth in Populus tomentosa, and provide molecular basis and technical approaches for the molecular genetic improvement of wood-property of forest trees.

利用转基因技术培育速生优质林木新品种，能有效提高人工林木材产量与质量，因此木材形成的分子机理研究已成为树木分子生物学研究的热点。项目组前期研究发现毛白杨PtoWRKY40基因能够调控次生壁合成相关基因PtoCCoAOMT1和Pto4CL3的表达，且与Elongation factor protein、Alpha-xylosidase family protein、Sucrose synthase等蛋白可能有相互作用。在此基础上，本项目拟通过酵母双杂交等实验进一步阐明PtoWRKY40与互作蛋白的互作机制，以及运用拟南芥原生质体瞬时表达系统等研究PtoWRKY40对PtoCCoAOMT1和Pto4CL3表达的调控作用，结合其过表达及抑制表达对转基因植株木质部的影响，最终解析PtoWRKY40调控毛白杨次生生长的作用及分子调控机制，为木材性状的分子改良提供理论基础和技术途径。

我国森林覆盖率低，木材供求矛盾突出，迫切需要营造速生优质人工林，加速木材品质性状遗传改良，木材形成机理的研究可以为培育速生优质林木提供理论指导。. 本项目创制了PtoWRKY40超表达或RNAi抑制表达后转基因银腺杨植株，分析了转基因杨树的木质部和韧皮部径向宽度、次生壁厚度变化、木质素含量、木材密度和总生物量，以及木材形成相关基因的表达变化。运用BiFC、GST pull down等实验研究PtoWRKY40与互作蛋白PtoSOD4的互作机制，通过酵母单杂交、烟草瞬时转化等技术，研究PtoWRKY40调控下游靶基因PtoCCoAOMT1和Pto4CL3表达的作用机制，以及在PtoSOD4的参与下，PtoWRKY40调控PtoCCoAOMT1和Pto4CL3表达的方式。对转基因杨树开展了干旱胁迫处理，研究了PtoSOD4与PtoWRKY40模块响应干旱胁迫的机制。.　 研究发现PtoWRKY40基因在毛白杨未成熟木质部和形成层中的表达量较高，PtoWRKY40超表达植株的高度、茎干粗度明显降低，木质部和韧皮部径向宽度变窄，次生壁变薄，木质素、木材密度和总生物量均降低。RNAi表达植株的高度、茎干粗度明显增加，木质部和韧皮部径向宽度变宽，次生壁加厚，木质素、木材密度和总生物量均增加。PtoWRKY40能够直接结合到木质素合成相关基因PtoCCoAOMT1和Pto4CL3的启动子上，负调控PtoCCoAOMT1和Pto4CL3的表达。研究进一步发现，逆境响应因子PtoSOD4蛋白能与PtoWRKY40发生相互作用，干旱处理下，PtoWRKY40过表达植株中PtoWRKY40与PtoSOD4的互作增强，并且在PtoSOD4的作用下，缓解了PtoWRKY40负调节PtoCCoAOMT1和Pto4CL3表达的能力，从而促进了PtoCCoAOMT1和Pto4CL3表达，增加木质素合成，增强了转基因杨树对干旱胁迫的适应性。. 该研究对毛白杨PtoWRKY40基因调控木质部发育和次生生长的作用机制开展了系统研究，发现了毛白杨PtoWRKY40介导的木质素生物合成新通路，揭示了PtoWRKY40通过与PtoSOD4互作，在干旱下促进木质素合成，从而提高杨树抗旱性的新机制，为阐明杨树次生壁形成的分子机制以及林木优质、抗逆分子设计育种提供了重要依据。