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

植物合成大量的次生代谢物。变异性似乎对这些特殊代谢物与环境沟通的功能至关重要，并作为化学防御的武器库。次级代谢途径的建立是基于共同的初级代谢。然后涉及特定的酶以产生次级代谢物，这些酶是例如细胞色素P450单加氧酶、2-酮戊二酸依赖性双加氧酶、甲基转移酶、糖基转移酶。在基因组中，这些修饰酶的大基因家族构成了独特酶被招募的基因组。我们对选择这一武器库中的一员、根据生物合成中不同步骤的要求塑造它以及协调反应顺序的原则知之甚少。分离和比较完整的生物合成基因的途径，导致从趋同进化将允许深入了解这些机制。苯并恶嗪类化合物是被子植物中与防御相关的次生代谢产物，存在于禾本科、毛茛目和唇形科三个目中。复杂的生物合成是生物化学和基因水平（Bx基因）完全阐明玉米。所有的8个生物合成基因都位于一个簇中，这样的基因组组织同时被检测到用于进一步的防御相关的生物合成途径。在禾本科植物中，Bx基因是单系的。我们已经从毛茛科的Consolida orientalis和唇形科的Lamium galeobdolon中分离到了该途径的第一个基因Bx 1。BX 1是色氨酸合酶α-亚基的同源物，并且已经独立地在三个订单中的每一个中募集。最近，我们可以展示C。orientalis的研究表明，该途径的特异性UDP-葡糖基转移酶和β-葡糖苷酶通过重复进化而进化，招募与单子叶植物Bx基因谱系非直系同源的相应基因家族的成员。我们建议分离所有缺失的苯并恶嗪类特异性基因。orientalis和L. galeobdolon。三个订单的生物合成基因的比较将揭示反应步骤的顺序的保存，可能存在的限制，为招聘的基因的给定池，阐明是否修改招聘的基因遵循一个共同的模式，以及是否形成一个基因簇平行的生物合成途径的建立。候选基因将通过转录组学鉴定，并通过异源系统中的功能表征进行验证。为了获得苯并恶嗪类特异性基因分离的进一步独立证据，我们打算建立病毒诱导的基因沉默，首先对C。东方

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.