杨树木质素单体合成的分子调控机制研究

Lignin content and composition play pivotal roles in the properties and applications of wood. Ferulate 5-hydroxylase (F5H) has been proven a key enzyme in the regulation of S/G ratio in lignin, thus it is critical for the molecular breeding of trees for wood production to elucidate the regulatory mechanism of F5H. We have previously identified several factors that show influence on F5H expression in Populus, including PtrCSE1/2, the enzymes involved in lignin metabolism, PtrMYB10/128, MYB family transcription factors, and miR6443, a microRNA. However, it remains unclear how these various factors are involved in the regulation of F5H expression. Therefore, we plan to: 1) determine the metabolite composition, gene expression and lignin composition in PtrCSE1/2 knock-out poplar to clarify how F5H expression and lignin composition areinfluenced by metabolite cascade; 2) analyze the morphology and composition of thesecondary cell wall in PtrMYB10/128 over-expression and knock-out poplar to elucidate their biological functions; 3) screen target genes of PtrMYB10/128 by transcriptome sequencing and clarify the regulatory network for F5H transcription; 4) elucidate the regulatory mechanism of miR6443 against F5H by RACE and genetical analysis; 5) determine the influence of miR6443 on lignin composition in transgenic poplar. Finally, establish a coordinated regulatory mechanism on lignin monomer biosynthesis through metabolite cascade, transcription factor and epigenetic regulations on F5H expression in poplar, so as to set up theoretical foundations for the breeding of poplar with modified lignin composition.

杨树木质素组分的调控对于其生产应用具有重要意义，已证实F5H是调控木质素S/G比例的关键酶。因此解析F5H的调控机制对于林木育种十分重要。课题组前期在杨树中鉴定到木质素代谢相关酶PtrCSE1/2、PtrMYB10/128转录因子和miR6443小RNA影响F5H表达，但调控机制尚不清楚。为此本项目拟：检测PtrCSE1/2敲除杨树的代谢产物、基因表达和木质素组分，探索代谢流影响F5H表达和木质素组分机理；分析超表达和突变体杨树次生壁形态与组分，解析PtrMYB10/128的功能；转录组测序筛选PtrMYB10/128的靶基因，阐明F5H的转录调控网络；RACE和遗传分析解析miR6443对F5H的调控机制；在转基因杨树中探究miR6443对木质素组分的作用。最终阐明代谢流、转录因子和表观作用三个层次的F5H调控方式在杨树木质素单体合成中的协同机制，为杨树木质素组分改良育种奠定理论基础。

木质素的含量和组分决定了林木木材的材性和生产利用。围绕杨树次生壁合成中木质素单体组分调控这一关键科学问题，从转录因子、表观作用和代谢流三个层次开展研究，筛选鉴定到杨树中MYB128、miR6443等多个参与木质素单体合成的调控因子。表达谱分析发现miR6443在杨树的茎中特异地定位于发生木质素沉积的细胞中；通过降解组、RACE等技术证明了miR6443可特异性靶向和抑制F5H2的表达；利用超表达和内源抑制的转基因株系，解析了miR6443对杨树F5H2的表达的调控和对S型木质素单体合成的促进作用，揭示了miR6443在杨树木质素单体合成中的生物学功能和机制。同时，还发现MYB128是一个在杨树茎的木质部中特异表达的转录因子，具有促进木质部细胞次生壁形成、激活木质素、纤维素等生物合成和提高木质素中S型单体比例的功能。通过高通量测序等技术，证明MYB128既可以结合和调控NAC家族的次生壁开关基因，又可以直接激活木质素合成的关键酶基因表达，阐明了MYB128在多层次调控网络中参与次生壁和木质素调控的分子机制。此外，研究还发现利用遗传调控miR6443和MYB128水平，可显著提高从杨树木材中提取纤维素的效率，降低对酸、碱的依赖，促进纤维素分解为单糖和提升乙醇的产率。本项目研究阐明了MYB128转录因子和miR6443介导的表观调控在调节杨树F5H表达水平和控制杨树木质素单体合成中的功能和机制，为培育林木优良品种，实现对木材的高效、环保利用和生物能源生产提供了新思路，具有十分重要的生产应用价值。项目支持下发表SCI论文9篇，其中生物学和植物学一区论文6篇。