Tissue- and cell-specific transcriptomics of pyrrolizidine alkaloidbiosynthesis

吡咯里西啶生物碱（PAs）系统是研究植物次生代谢途径进化的一个模型。多胺对脊椎动物和昆虫有毒，被认为是针对食草动物的化学防御的一部分。对于第一个途径特异性酶，homospermidine合酶（HSS），我们能够证明，它起源于被子植物进化过程中至少五次独立的初级代谢基因的重复。这一观察结果表明，PA的完整途径也有几个独立的起源。在本项目中，我们打算进一步确定PA生物合成的三种酶，以研究它们的进化，并将这些数据与HSS进化的广泛知识进行比较。利用我们对HSS的组织和细胞特异性表达的了解，我们成功地生成了几种PA生产物种的各种组织的转录组数据库。通过比较来自不同组织的序列数据与生物信息学工具，我们能够命名的序列，编码酶假定参与PA生物合成。我们将使用该项目的第二阶段，以证明这些候选序列在PA生物合成中的功能作用，通过比较它们的生化特性与转录组数据库中也可获得的旁系同源序列。除了系统发育分析，这些数据应该允许PA生物合成的这些酶的进化的结论。

The system of pyrrolizidine alkaloids (PAs) is a model for our studies about the evolution of pathways in plant secondary metabolism. PAs are toxic to vertebrates and insects and are believed to be selected as part of the chemical defence against herbivores. For the first pathway-specific enzyme, homospermidine synthase (HSS), we were able to show that it originated by duplication of a gene involved in primary metabolism at least five times independently during angiosperm evolution. This observation suggests that also the complete pathway of PAs has several independent origins. In the present project, we intend to identify three further enzymes of PA biosynthesis to study their evolution and compare that data with the extensive knowledge about HSS evolution. Using our knowledge about the tissue- and cell-specific expression of HSS we successfully generated transcriptome databases of various tissues of several PA-producing species. By comparing the sequence data from different tissues with bioinformatic tools, we were able to denominate sequences that encode enzymes postulated to be involved in PA biosynthesis. We will use this second phase of the project to prove the functional role of these candidate sequences in PA biosynthesis by comparing their biochemical properties with paralogous sequences that are also available in the transcriptome databases. This data in addition to phylogenetic analyses should allow conclusions about the evolution of these enzymes of PA biosynthesis.