比较转录组重构紫草目系统发育模型以及探索紫草素与不饱和脂肪酸代谢的分子进化史

Up to now, the external evolutional status and internal phylogeny of Boraginales remain unclear. Additionally, the metabolism of two kinds of characteristic natural products, e.g. naphthaquinone compounds (shikonin and its derivatives) and unsaturated fatty acids biosynthesized by some Boraginales plants, might have specific evolutionary trend in the phylogeny. Recently, many novel results have been achieved in the research fields of molecular evolution and phylogeny by comparative transcriptome analysis. This project will gather and cultivate about 50 species plants belonging to Boraginales, then construct transcriptome database of root, leaf, and seeds of those 50 species plants, respectively. Based on transcriptome database, we will reconstruct Boraginales’ phylogeny in order to clearly define the external evolutional status and internal phylogeny of Boraginales. Furthermore, on the basis of the reconstructed model of Boraginales’ phylogeny, we will screen those replicated genes and those genes under positive selection during the evolution of subfamily Boraginoideae / Trib. Lithospermeae, and explore the molecular evolution base of the metabolism of Shikonin and unsaturated fatty acids. Finally, the project will discover the expression profiles of related genes involved in the metabolism of Shikonin and unsaturated fatty acids between species via analyzing the expression of homologue genes crossing species. The completion of this project will optimize the present system of phylogeny and classification of Boraginales, and will clearly define the molecular evolution story of the metabolism of Shikonin and unsaturated fatty acids in Boraginales.

至今，紫草目的外部进化地位与内部系统发生依然不甚明确。此外，紫草目植物类群两大类特征天然产物即紫草素萘醌类与不饱和脂肪酸类的代谢，在系统发育中可能具有独特的进化趋势。近年来，比较转录组分析在分子进化与系统发育研究领域中取得了许多新颖的成果。本项目拟收集紫草目约50种代表性的物种并种植，构建各物种的根、叶、种子转录组数据库；采用比较转录组分析法重构紫草目的系统发育模型，用于明确紫草目外部的进化地位与内部的分类界限。然后，基于所构建的紫草目系统发育模型，筛选紫草亚科/紫草族进化支中的正选择基因与复制基因，用于探索紫草素与不饱和脂肪酸代谢的分子进化基础。最后，本项目还将通过跨物种的同源基因表达分析，探索紫草目植物类群中紫草素与不饱和脂肪酸代谢相关基因的物种间表达水平。由此可见，本项目的完成有望完善紫草目现行的系统发育与分类体系，并能进一步明确紫草素与不饱和脂肪酸代谢在紫草目中的分子进化历史。

本项目主要围绕紫草目外部进化地位与内部系统发生展开了较为深入而系统的研究。项目通过野外采集紫草目31个、室内栽培2个代表物种的根、叶及部分种子样品，提取RNA做转录组高通量测序；其间，从12个物种的根样品提取出了紫草素萘醌类化合物并作了高效液相色谱（HPLC）表征，另从公开数据库SRA（sequence read archive）下载了紫草目51个物种相应组织的转录组原始数据并严格过滤，经Trinity从头组装，构建了82个物种无参转录组及unigenes集，同时构建了室内栽培2个代表物种（硬紫草和车前叶蓝蓟）的有参转录组及unigenes集，然后，利用OrthoFinder软件平台，通过比较转录组的方法，重构了紫草目内部的系统发生树，明确了紫草目内部的分类界限。进一步，基于已发表的唇形支10个物种及其外部2个物种的基因组数据，结合本项目野外采集的紫草目10个代表物种以及从SRA下载的4个物种转录组数据，利用OrthoFinder软件平台，通过比较转录组与比较基因组的方法，重构了紫草目与外部近缘分类群的系统发生树，补充、改善了紫草目的外部进化地位。另外，基于所构建的紫草目系统发生模型，筛选紫草科/紫草族进化支中的一些正选择基因(例如，香叶基对苯二酚3〃-羟化酶、12-氧磷二烯酸还原酶)与复制基因，探索了紫草素代谢的分子进化基础。此外，本项目还通过跨物种的同源基因表达分析，探索了紫草目植物类群中紫草素萘醌类化合物代谢相关基因在野外采集的30个物种间的表达谱。项目通过比较转录组获得的结果，完善了紫草目现行的系统发生与分类体系。