紫茎泽兰肉桂酸四羟化酶基因及其在酚类代谢途径的调控机理

Eupatorium adenophorum,as a kind of noxious invasive alien weed, has seriously threatened the ecological system of forest and farmland in our country. Allelotaphy is one of important factors in aspect of efficient invasion, and research had showed that HHO is one of important allelochemicals in E.adenophorum.The previous research indicated that some genes in phenolic metabolic pathway of E.adenophorum, such as phenylalanine ammonia-lyase(PAL) gene, chalcone synthase gene, flavonoid 3'-hydroxylase gene and so on, were closely related to HHO. In this project, cinnamate-4-hydroxylase gene was cloned, we will further analyse the expression profile of this gene. We are also plan to overexpress or scilence the gene in E.adenophorum. Comparing with the wild type plant,quantitative RT-PCR will be used to analyze the expression difference of gene when the plant is induced by HHO in trangenic plant. We have obtained some transgenic plant of phenylalanine ammonia-lyase gene, chalcone synthase gene and flavonoid 3'-hydroxylase gene. Based on this work, we will study the effect of a gene to other genes when this gene is overexpressed or scilenced in the plant, we also need to separate and purify the product of cinnamate-4-hydroxylase and investigate the difference of the product in the yield. Ultimately, we will detect the relationship of phenolic metabolic pathway among these four key genes, and discuss the relationship between HHO and these four genes. Reveal the function of phenolic metabolic pathway in the generating process of allelochemical, will provide the theory basis in studying the mechanism of allelopathy in E. adenophorum.

紫茎泽兰是一种入侵我国的恶性杂草。化感作用是紫茎泽兰种群竞争扩张的一个重要成因，羟基泽兰酮（HHO）是紫茎泽兰的重要化感物质，我们已有的研究表明紫茎泽兰次生代谢途径中酚类代谢的PAL/CHS/F3'H等几个关键酶基因均和羟基泽兰酮高度相关。本项目从肉桂酸四羟化酶（C4H）基因入手，克隆C4H基因并进行表达谱分析；对C4H基因进行过量表达和RNAi干涉的遗传转化，获得转C4H基因的紫茎泽兰植株；对野生型和转基因植株的C4H基因的表达和受HHO诱导下差异分析，对野生型和转基因植株的HHO分离纯化，分析差异。在已有的转PAL/CHS/F3'H基因植株的基础上研究过量表达和RNAi一个基因对其他基因表达的影响，分离纯化酶产物，比较分析不同条件下4个关键节点的基因在酚类代谢途径的关系。探讨C4H基因与HHO的关系，研究该代谢通路在化感物质产生过程中的作用。为揭示紫茎泽兰化感的作用机理提供理论基础。

本项目从紫茎泽兰中克隆了一个长度为1518bp的肉桂酸四羟化酶编码基因并做了遗传背景分析；构建了植物过表达载体及干涉载体并转化烟草及棉花，分析发现：随着CA4H表达水平的降低，包括PAL、CHS、F3’H等在内的几个苯丙烷代谢途径关键基因的表达水平也随之下调，说明这些基因的表达在植物体内受到协同调控，相应的肉桂酸四羟化酶活性以及类黄酮的积累也随之减少，而上述这些关键基因以及类黄酮含量并未因CA4H的过量表达而提高，造成这一结果可能是由于CA4H转录本的过度积累导致该酶底物（肉桂酸）的大量积累，从而触发了反馈调节机制，为了避免肉桂酸过度积累，阻遏蛋白释放并抑制PAL的表达，从而使下游基因的表达及类黄酮化合物的产生受到封阻。对转基因烟草及棉花在抗逆性方面的研究进一步拓展了EaC4H基因潜在的生产应用价值；发现了紫茎泽兰化感物质对拟南芥种子萌发存在抑制作用。2.5mM的HHO粗提物对拟南芥种子胚根的萌发及子叶的展开均产生抑制，1%的蔗糖增强了它对于胚根萌发的抑制却降低了对子叶伸展的抑制，而无机盐的存在则封阻了HHO的抑制作用。进一步分析对比了ABA的抑制作用发现：HHO对于种子的抑制不依赖于ABA途径，1%的蔗糖强化了HHO对于种子萌发的抑制作用，而削弱了ABA对于种子萌发的抑制，相似的状况在Ler型拟南芥中也是如此，且对于ABA抑制种子萌发的封阻更显著。采用Rapid Pressurised Hot Water Extraction（PHWE）技术从紫茎泽兰中快速萃取主效化感物质，进一步优化了高效液相色谱及核磁共振技术，从紫茎泽兰中鉴定出一个全新的化合物，暂命名为DH-DTD，三种化合物DTD:DH-DTD:HHO的比例约为0.2:0.3:1.0。对三者在两种拟南芥生态型种子萌发的抑制分析发现：DH-DTD的抑制作用要优于DTD及HHO。上述研究将进一步推进紫茎泽兰入侵分子机制的揭示工作。