Elucidation of mechanisms required for convergent evolution of secondary metabolite pathways in plants

植物合成大量次生代谢物。可变性似乎对这些特殊代谢物与环境沟通的功能以及作为化学防御的武器库至关重要。次级代谢途径的建立是以共同的初级代谢为基础的。然后特定的酶参与产生次生代谢物，例如细胞色素P450单加氧酶、2-氧戊二酸依赖的双加氧酶、甲基转移酶和糖基转移酶。在基因组中，这些修饰酶的大基因家族构成了工具箱，从中招募到独特的酶。关于如何选择这个军火库的成员，如何塑造它以适应生物合成中不同步骤的要求，以及协调反应序列的原则，人们知之甚少。分离和比较趋同进化途径的全套生物合成基因将有助于深入了解这些机制。苯并恶嗪类化合物的生物合成非常适合于这样的分析。苯并恶嗪类化合物是与防御相关的次生代谢物，存在于被子植物的三个目：Poales、Ranunculales和Lamiales。这种复杂的生物合成是在生化和基因水平(Bx基因)上完全被阐明的。所有这8个生物合成基因都位于一个簇中，同时也发现了这样一个基因组组织，用于进一步防御相关的生物合成途径。在草本植物中，Bx基因是单系进化的。我们从毛茅科的东方锦鸡儿和灯盏花科的Lamium Galeobdolon中分离到了该途径的第一个基因Bx1。Bx1是色氨酸合成酶α亚基的同系物，在三个目中独立招募。最近，我们发现侧柏的UDP-葡萄糖基转移酶和β-葡萄糖苷酶是通过重复进化而进化的，这些基因家族的成员与单子叶植物Bx基因谱系不是同源的。目前还没有拉米亚基因的相关数据。我们建议分离所有缺失的东方华支睾吸虫和华支睾吸虫的苯并恶嗪特异基因。三个序列的生物合成基因的比较将揭示反应步骤序列的保存，可能存在的从给定的池中招募基因的限制，阐明招募的基因的修饰是否遵循共同的模式，以及基因簇的形成是否平行于生物合成途径的建立。候选基因将通过转录组学进行鉴定，并通过异源系统的功能特征进行验证。为了进一步获得分离苯并恶嗪类特异基因的独立证据，我们打算建立病毒诱导的基因沉默，最初是针对东方拟青虫。

Plants synthesise a multitude of secondary metabolites. Variability seems to be essential for the function of these Specialised metabolites in communication with the environment and as an arsenal for chemical defence. Establishment of secondary metabolic pathways is based on the common primary metabolism. Specific enzymes are then involved to generate the secondary metabolites, these are e.g. cytochrome P450 monooxygenases, 2-oxoglutarate dependent dioxygenases, methyltransferases, glycosyltransferases. In the genomes large gene families of these modifying enzymes constitute the Toolbox from which unique enzymes are recruited. Little is known about the principles to choose a member of this arsenal, to shape it for the requirements of a distinct step in biosynthesis and to coordinate the reaction sequence. Isolation and comparison of complete sets of biosynthetic genes for pathways that result from convergent evolution will allow getting insight into these mechanisms. Benzoxazinoid biosynthesis is well suited for such an analysis.Benzoxazinoids are defence-related secondary metabolites found in three orders of the angiosperms: Poales, Ranunculales and Lamiales. The complex biosynthesis is biochemically and on gene level (Bx-genes) completely elucidated in maize. All of the 8 biosynthetic genes are located in a cluster, such a genomic organisation has meanwhile been detected for further defence-related biosynthetic pathways. In the grasses the Bx-genes are monophyletic. We have isolated the first gene of the pathway, Bx1, from Consolida orientalis (Ranunculaceae) and Lamium galeobdolon (Lamiaceae). BX1 is a homolog of the alpha-subunit of tryptophan synthase and has been recruited in each of the three orders independently. Recently we could show for C. orientalis that the specific UDP-glucosyltransferase and beta-glucosidase of the pathway evolved by repeated evolution recruiting members of the respective gene families that are non-orthologous to the monocot Bx-gene lineages. No data for these genes are available for the Lamiales.We propose to isolate all missing benzoxazinois-specific genes of C. orientalis and L. galeobdolon. The comparison of the biosynthetic genes of the three orders will reveal preservation of the sequence of reaction steps, possibly existing restrictions for the recruitment of genes out of the given pool, elucidate whether modifications of recruited genes follow a common pattern and whether the formation of a gene cluster parallels for the establishment of the biosynthetic pathway. Candidate genes will be identified by Transcriptomics and verified by functional characterisation in heterologous systems. To get a further independent proof for the isolation of benzoxazinoid-specific genes we intend to establish Virus Induced Gene Silencing, initially for C. orientalis.