Tissue- and cell-specific transcriptomics of pyrrolizidine alkaloidbiosynthesis

吡咯利西啶生物碱系统是研究植物次生代谢途径进化的一个模型。PAs对脊椎动物和昆虫是有毒的，被认为是作为对抗食草动物的化学防御的一部分。对于第一个途径特异性酶，同亚精胺合成酶（HSS），我们能够证明它起源于在被子植物进化过程中至少5次独立参与初级代谢的基因的复制。这一观察结果还表明，PAs的完整途径有几个独立的起源。在本项目中，我们打算进一步鉴定三种PA生物合成酶来研究它们的进化，并将这些数据与关于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，我们成功地建立了几种产生pa的物种的各种组织的转录组数据库。通过使用生物信息学工具比较来自不同组织的序列数据，我们能够区分编码酶的序列，这些酶被认为参与了PA的生物合成。我们将利用该项目的第二阶段来证明这些候选序列在PA生物合成中的功能作用，通过将它们的生化特性与转录组数据库中可用的类似序列进行比较。这些数据加上系统发育分析，应该可以得出这些PA生物合成酶的进化结论。

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.