基于ssRNA-seq的水杨酸诱导浮萍开花的关键基因挖掘及分子调控机制研究

Duckweed is a simple aquatic floating plant and has a great potential in sewage treatment and bioenergy production. However, in the natural conditions, duckweed reproduces mainly by asexual reproduction and the flowering in duckweed is rare, which greatly limits its germplasm collection and conservation, germplasm innovation, and heterosis usage. Our previous studies have demonstrated that salicylic acid (SA) can high-efficiently induce the flowering of duckweed, and have determined all the crucial parameters, including the optimal SA concentration and culture conditions, and the key time-points during the process of SA-induced flowering in duckweed. We also obtained the hybrid seeds through artificial-assisted hybridization. However, the molecular mechanism of flowering by SA-induction in duckweed remains unclear. In this project, strand-specific RNA-seq (ssRNA-seq) technology is employed to explore the differentially expressed genes involved in duckweed flowering by SA-induction at the transcriptional level. Subsequently, their expression patterns and the related gene regulation networks are revealed through gene co-expression analysis. At the meantime, the functional enrichment analysis of modules and the association analysis between the modules and physiological phenotypes are conducted, based on which a few key hub genes from SA and/or flowering related modules are selected to perform functional verification, in order to elucidate the molecular regulation mechanism of SA-induced duckweed flowering at the transcriptional level. The findings will not only contribute to the preservation and utilization of duckweed germplasm, but also extend the knowledge of flowering in other plants / crops.

浮萍是一种简单的水生漂浮植物，在污水净化、生物质能源利用等方面具有巨大的应用潜力。然而浮萍在自然条件下以无性繁殖为主，很难开花，极大地限制了其种质资源收集与保存、种质创新、和杂种优势的利用。本项目前期研究表明水杨酸（SA）可以高效诱导浮萍开花，并掌握了诱导浮萍开花最适的SA浓度和培养条件以及SA诱导浮萍开花的关键时间节点，通过杂交，获得了杂交种。但目前有关SA诱导浮萍开花的分子调控机制尚不清楚。本项目在前期基础上，以链特异性RNA-seq技术在转录组水平挖掘SA诱导浮萍开花的关键基因，通过基因共表达分析揭示它们的表达模式及相关基因调控网络，同时进行模块功能富集分析和模块与生理表型的关联分析，并在此基础上选择与SA和开花相关模块中的关键节点基因进行功能验证，以期从转录水平阐明SA诱导浮萍开花的分子调控机制。研究结果不仅有助于浮萍种质资源的保存和利用，而且还可以拓展其他植物/作物中开花的知识。

浮萍是一种简单的水生漂浮植物，在污水净化、生物质能源利用等方面具有巨大的应用潜力。然而浮萍在自然条件下以无性繁殖为主，很难开花，极大地限制了其种质资源收集与保存、种质创新、和杂种优势的利用。水杨酸（SA）可以高效诱导浮萍开花。本项目在已有SA诱导浮萍开花规律研究的基础上，选取关键时间节点构建ssRNA-seq文库，通过转录组比较挖掘SA诱导浮萍开花关键基因。转录组从头组装共获得了175,267个转录本，平均长度为568bp，N50长度为742bp。约70.8%的基因长度为100-500bp，而只有3.5%的基因长度大于2000bp。转录组比较分析鉴定了1,992个差异表达基因。在开花诱导阶段，浮萍生长受到SA抑制、光合作用和细胞壁基因表达下调；同时碳水化合物和氧化还原基因显著上调，为开花诱导提供足够的能量。在开花起始阶段，光反应和脂质代谢基因下调，淀粉降解和钙信号基因上调，在一定程度上反映了开花起始阶段对能量的持续需求。在开花阶段，碳水化合物和氧化还原代谢基因显著下调；同时细胞壁和木质素合成基因上调以支持花的发育。对拟南芥FLOR-ID数据库开展BLASTP分析，共鉴定13个与浮萍开花相关的差异基因，其中6个基因的表达在开花诱导阶段显著上调，因此它们被选作查询基因构建共表达网络。在这个共表达网络中，LgTEM1、LgSVP和LgFT1是最关键的开花枢纽基因。此外，该共表达网络还包括了转录因子、激素、氧化还原、淀粉代谢和钙信号相关基因，揭示了SA诱导浮萍开花的一个复杂的基因调控网络。最后，通过qRT-PCR验证了这些基因的表达模式，并提出了SA诱导浮萍开花的转录调控模型。这些结果初步阐明了SA诱导浮萍开花的分子机制，为进一步开展浮萍杂交育种、拓宽浮萍遗传资源、和培育浮萍新种质奠定了基础，同时也有助于拓展其他植物中开花相关理论知识。