毛白杨PtoWRKY40在木材形成过程中的作用解析

The production of fast-growing genetically engineered trees with an excellent quality can increase the yield and improve the quality of wood materials. Therefore, the research of molecular mechanism of wood formation has been a focus in research field of forestry molecular biology. In our previous study, cDNA microarray and microRNA microarray were used to analyze gene expression profiles and microRNA expression profiles during regeneration of secondary vascular system in Populus tomentosa Carr., and PtoWRKY40 showed transcript-level differences at the different regeneration stages.Thus, PtoWRKY40 was suggested in association with wood formation. Full-length cDNAs of PtoWRKY40 was isolated and nine WRKYs have been found in the genome of Populus trichocarpa Torr. & Gray. It indicated that the expression of PtoNAC and PtoC3H involved in wood formation were regulated by PtoWRKY40. In this research, the expression pattern of PtoWRKY40 in different tissues was detected by the quantitative real-time PCR analysis. The thickness of secondary cell wall and expression levels of wood formation associated genes in PtoWRKY40 overexpressing or RNAi(RNA interference) transgenic Populus alba L.× Populus glandulosa Dode cv.‘84k’ plants were analyzed by paraffin section and RNA-Seq, and the yeast two-hybrid experiments and transcriptional activation assay were carried out to identify the regulational network and molecular mechanism of PtoWRKY40 gene participating in P. tomentosa wood formation. Through this project, it could lay the foundation for the molecular genetic improvement of wood-property of forest trees.

利用基因工程技术培育速生优质林木，能提高木材产量与质量，因而林木木材形成的分子机理研究目前已成为树木分子生物学研究的热点。项目组前期通过毛白杨次生维管系统再生过程的cDNA基因芯片和microRNA芯片研究发现，毛白杨PtoWRKY40基因在不同时期的表达量存在差异性，可能与木材形成有关。分离该基因发现毛果杨WRKY基因家族有9个成员，并且PtoWRKY40可能主要调控材性相关基因PtoNAC、PtoC3H的表达。在此基础上，本项目拟运用qRT-PCR技术分析PtoWRKY40在毛白杨不同组织的表达特性，利用石蜡切片技术和转录组测序技术分析PtoWRKY40超表达或RNAi抑制表达后转基因银腺杨植株的次生壁厚度变化和木材形成相关基因的表达变化，以及酵母双杂交和转录激活实验研究PtoWRKY40 参与木材形成的调控网络，分析其参与毛白杨木材形成的分子调控机制，为木材性状的分子改良提供基础。

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。本项目运用qRT-PCR技术分析PtoWRKY40在毛白杨不同组织的表达特性，利用石蜡切片和qRT-PCR技术分析PtoWRKY40超表达或RNAi抑制表达后转基因银腺杨植株的次生壁厚度变化和木材形成相关基因的表达变化。通过对毛白杨形成层组织的GST pull down和CoIP筛库实验，研究PtoWRKY40 与互作蛋白间的互作机制，以及通过EMSA、酵母单杂交、烟草瞬时转化技术和ChIP-PCR等技术，研究PtoWRKY40调控下游靶基因表达的作用机制。. 研究发现PtoWRKY40基因在未成熟木质部和形成层中的表达量较高，PtoWRKY40蛋白定位在细胞核。获得了PtoWRKY40超表达和RNAi抑制表达的银腺杨植株，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带变宽，次生壁加厚，木质素、木材密度、总生物量和固碳量均增加。相反，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带变窄，次生壁变薄，木质素、木材密度、总生物量和固碳量均降低，这些研究初步阐述了PtoWRKY40基因参与毛白杨木材形成的生物学功能。. 进一步研究发现，PtoWRKY40蛋白可能与Tubulin、Elongation factor protein、Alpha-xylosidase family protein、Sucrose synthase、Dynamin-related protein、F-box/FBD/LRR-repeat protein和Zinc finger family protein等18个与木材形成相关的蛋白发生相互作用。另外，PtoWRKY40能够结合到PtoCCoAOMT1和Pto4CL3的启动子上，调控靶基因PtoCCoAOMT1和Pto4CL3的表达，从而初步解析了PtoWRKY40基因参与毛白杨木材形成的分子调控网络。