水平基因转移在寄生植物菟丝子基因组中的全面鉴定及其进化意义和发生机制研究

Compared with natural mutation, HGT (horizontal gene transfer) introduces evolutionary novelties to recipient organisms and thus is critical to evolution. Due to the insufficient genome information in key species and technical limitations in HGT identification, HGT currently is well recognized in prokaryotes, unicellular eukaryotes and the organelles of eukaryotes, but has been poorly investigated in the nuclear genomes of higher plants, despite of the potential important evolutionary significance. Due to large-scale exchanges of biomolecules between host plants and parasitic plants, studies on HGT in parasitic plants have shed light on HGT in higher plants. In the holoparasitic plant dodder (Cuscuta australis), we generated 95-fold whole-genome shotgun sequence coverage (26.6 Gb) using a single-molecule, real-time (SMRT) sequencing platform and acquired 95% genome sequence of the estimated 281 Mb genome size, total 267 Mb from 250 contigs (contig N50=3.6 Mb). The release of genomes from taxonomically related species, especially Ipomoea nil from the same family Convolvulaceae, eliminates the last barrier of HGT identification in dodders. Following the efforts on the development and improvement of HGT screening methods based on the protein sequences, we firstly designed and performed whole-genome HGT scanning in the nuclear genome of C. australis using the window sliding method for the transferred DNA fragments and found thousands of HGT candidates, including gene regions and non-coding regions that are escaped from previously protein-based HGT screening methods. In this project, we plan to optimize the methods of large-scale HGT identification both at protein and DNA levels, carry out functional analysis and experimental verification on the important HGT originated genes or fragments, and then evaluate the impact of HGT on the evolution of higher plants. In addition, the reliable isolation of the boundary sequence and the flanking gene arrangement of the transferred DNA fragments would allow us to explore the underlying molecular mechanisms of HGT and test the previously proposed homologous recombination, non-homologous end joining, and transposon-mediated HGT hypotheses. This project will provide insights into the evolutionary significance and the molecular mechanisms of HGT in higher plants, besides developing new methods and pipelines of genome-wide HGT identification that can also be applied to other species. Furthermore, the prospective results of this project may provide ideas for the development of new transgenic methods.

水平基因转移（HGT）对物种进化具有重要意义。但受基础数据和研究方法的限制，目前对高等植物HGT的研究难以深入开展。寄生植物因和寄主间存在大量的生物分子交流，已成为研究植物HGT的重要模型。我们使用三代测序技术获得了寄生植物菟丝子的高质量全基因组序列，其近缘物种基因组数据也相继公布，这使全面鉴定菟丝子基因组中的HGT成为可能。我们前期在蛋白质水平上开发了HGT鉴定方法，并首次在DNA水平进行了全基因组HGT扫描，取得了较好的鉴定结果。本项目拟在蛋白质和DNA水平上进一步优化HGT鉴定方法，选取重要的HGT基因进行功能分析和实验验证，解析HGT对寄生植物进化和发育的影响。此外，通过对转移片段边界序列的精确界定，拟揭示菟丝子中HGT发生的分子机制，包括对同源重组、非同源末端连接或转座子介导等已有假说的检验。本研究在开发新的HGT鉴定方法基础上，将加深对HGT进化意义和发生机制的理解。

水平基因转移对高等植物进化的影响还尚未定论，尤其是与其他植物建立有“亲密连接”的寄生植物更是如此。本研究通过重点开发和优化DNA与蛋白水平的HGT筛选工具，并利用它鉴定寄生植物菟丝子中HGT发生规模和分子机制。结果在菟丝子中发现相当多的转座子介导的HGT事件，并且菟丝子极大可能作为“桥梁”介导了转座子在不同寄主之间的转移；菊科和豆科的基因在菟丝子基因中占比最多，这可能反映了其近期寄主的偏好性。这表明HGT不但影响寄生植物的适应性进化，还能反映物种进化历史。利用以上开发的HGT筛选工具，在小立碗藓基因组中发现了富含真菌基因的两个DNA大片段，其中编码的基因极可能参与小立碗藓与真菌之间的相互作用，具体与免疫应答、自我识别及程序性细胞死亡相关。推测这些HGT基因有助于苔藓防御真菌和其它微生物病原体，这可能是苔藓不含菌根真菌的原因之一。该研究为苔藓植物与真菌的相互作用研究提供了全新视角。此外，为了在组学水平提供功能基因研究的线索，本项目建立了植物的高通量单细胞核转录组技术体系，成功获得了玉米叶表皮条和发育叶片的细胞图谱，并获得了一系列调控气孔发育和运动的关键基因，为后续功能基因的深入研究提供了良好的工作基础。