转录因子TcLBD15对红豆杉韧皮部分化、发育的调控机理研究

The taxol of separation and extraction is mainly from yew bark which is composed of the phloem tissue, The lateral organ boundaries domain (LBD) proteins (also known as ASYMMETRIC LEAVES2-Like proteins) are a family of plant-specific transcription factors, and play a potential regulational role in plant lateral organs pattern formation. Based on the analysis of tissues regeneration after bark girdling in Taxus chinensis, our experiments showed that the mRNA expressions of some TcLBDs were most highly expressed in the phloem with cambium, simultaneously had increased significantly during the phloem formation stages, indicating a potential role of TcLBDs in the phloem differentiation and development. In this research, TcLBD15 with high expression level during phloem regeneration was obtained by the analysis of the analysis of the tissues expression patterns and the regeneration system of secondary vascular system. These transgenic plants generated with the promoter, overexpressed and low expression of TcLBD15 were analyzed through the molecular and cytological level, and determination of paclitaxel content, to elucidate the biology functions of TcLBD15 in regulating the phloem differentiation and development. The target genes were identified via RNA-seq and ChIP-seq. Therefore, this research will reveal a signaling pathway mediated by TcLBD15 and the mechanisms underlying this pathway, which will benefit the genetic breeding of Taxus chinensis.

紫杉醇分离提取主要以红豆杉树皮为原料，韧皮部是树皮的最重要部分。LBD在植物侧生器官建成中起重要的调控作用。基于红豆杉剥皮后组织再生分析，TcLBDs在形成层形成时期、韧皮部再生时期表达非常活跃，表明TcLBDs在韧皮部分化、发育过程中具有潜在的作用，但调控机制尚不明确。本研究通过组织表达特性和红豆杉次生维管组织再生系统分析，获得了韧皮部再生时期表达量较高的基因TcLBD15。创制TcLBD15启动子驱动的转基因材料，TcLBD15过表达和TcLBD15-RNAi转基因材料，对其进行分子鉴定、细胞学分析和紫杉醇含量的测定，明确TcLBD15在红豆杉韧皮部分化、发育的生物学功能。采用转录组和ChIP-seq等方法，解析TcLBD15调控的下游靶基因。这些研究将有助于揭示TcLBD15所介导的信号通路调控韧皮部分化、发育的机制，为红豆杉定向遗传培育提供理论依据和技术手段。

紫杉醇传统的分离提取法以红豆杉树皮（韧皮部为主）为材料。韧皮部分化、发育是复杂的动态过程，对其机制的解析尚不清楚。LBD基因被报道参与植物愈伤组织、根、茎、叶及花的发育方面调控，但其在木本植物（裸子植物）生长发育尤其是韧皮部分化、发育中的作用尚未明确。本项目利用红豆杉转录组数据共鉴定36个LBD转录因子，基于红豆杉次生维管再生系统，TcLBD15在形成层形成期（DAG16至DAG30）和韧皮部再生时期（DAG44以后）表达水平活跃，同时TcLBD15在韧皮部（含形成层)中高表达。表型观察和组织切片分析表明TcLBD15过表达拟南芥，主茎粗而且茎秆韧皮部细胞层数增多，细胞体积增大。TcLBD15过表达杨树植株的高度增加，茎部直径增粗并且次生韧皮部的厚度也增加了，第16节间已有完整的次生韧皮纤维环。进一步通过差异基因表达和转录组等分析，植物维管组织韧皮部的发育是有APL/LBD、NAC86/LBD基因之间的反馈调节环来完成。TcLBD15促进细胞扩展基因EXPA5、EXPA8与EXPA15与纤维素合成基因CesA4、CesA7和CesA6基因表达，抑制次生壁相关的基因NAC的表达，调控细胞扩展和细胞壁加固两个过程，进而最终影响韧皮部分化、发育。本研究揭示TcLBD15对维管组织尤其是韧皮部分化、发育调控的作用，为培育韧皮部增多（树皮增厚）的转基因植株提供理论依据和候选基因。依托本项目的实施，发表学术论文3篇，获批专利2项，培养了5名本科生。