杨树转录因子WRKY3调控木质素合成的分子机制研究

Poplar (Populus trichocarpa) is one of the most important tree species for papermaking. A novel and important strategy has been developed by reducing lignin content or altering its components to satisfy the yield and quality of wood pulp while cutting pollution.WRKY proteins are plant-spcific transcriptional factors involved in regulation of ligin biosynthesis. On the basis of previous substantial investigation, we used over-expression and RNAi-mediated suppression of WRKY3 to generate transgenic hybrid poplar and analysis them with respect to the content of lignin and cellulose in the stems as well as used the high-throughput transcriptome sequencing technology to detect the target genes regualated by WRKY3 and the signaling pathway related in transcription and expression. Based on the relationship between the expression level of WRKY3 and micro-distribution of lignin and cellulose in poplar stalk, we shall further reveal the regulating mechanisms of WRKY3 expression related to lignin biosynthesis to stalk lodging at the molecular level. So far, the functional analysis and application of this vital gene have not been reported. This research will provide the novel insights in the function of WRKY transcription factors on lignin biosynthesis and enrich the theory about the molecular mechanisms of regulating lignin synthesis. The results will benefit wood breeding aimed at good quality plant.

杨树是重要的造纸资源树种，降低木质素含量或改变其组分是提高纸浆得率和质量及保护环境的重要新途径。WRKY是一类植物特异的转录因子，参与木质部形成的转录调控。本项目在前期大量研究工作的基础上，将获得的WRKY3基因在杨树中过量表达和RNAi抑制表达，分析过量表达与RNAi转基因株茎杆木质素等化学组成的变化，同时采用高通量转录组测序技术，在全基因组水平上分析确定WRKY3所调控的靶标基因及其转录表达所涉及的信号通路，探明转基因株WRKY3表达水平对靶基因表达活性的影响及其与茎杆木质素含量、组分和微区分布变化的关系，明晰WRKY3基因在杨树木质素合成中的作用。该基因在调控杨树木质素合成中的功能分析和应用研究国内外未见报道，其研究结果对揭示WRKY转录因子调控木质素合成的分子机制，创新和丰富木质素调控的理论，培育优质杨树新品种具有重要的理论意义和应用价值。

WRKY转录因子在植物木质素合成调控过程中起到重要作用。本课题依据基因芯片GSE13990中的数据，筛选了一个在木质部高水平表达的候选基因PtWRKY3。从南林95杨中克隆到了该基因，并通过过量表达技术对PtWRKY3 在转基因植物木质素合成过程中的调控功能进行了验证。组织表达模式分析表明PtWRKY3在木质部中的表达水平最高。该蛋白定位在细胞核上且在酵母中不具有转录激活特性。PtWRKY3能够特异性结合W盒序列，并与W盒结合抑制下游基因表达。植物组织切片间苯三酚染色发现PtWRKY3转基因株系次生壁较野生型也有减少的趋势。测定Klason木质素及综纤维素含量的结果显示：转基因植株Klason木质素含量明显低于野生型，而综纤维素含量则显著高于野生型。通过对杨树中多个木质素合成相关基因的表达变化分析发现，这些基因在转基因植株中的表达水平显著低于野生型植株。综合上述结果，本研究为探讨PtWRKY3调控植物木质素合成的分子机制奠定了坚实的工作基础。项目资助发表SCI论文13篇、EI收录论文1篇；申请发明专利3项，培养博士研究生2名，硕士生8名，7名同学都已取得博士/硕士学位。