浮萍休眠期储存物质淀粉合成的转录调控网络研究

Exploration of the promising alternative biofuel for fossil fuels is imperative due to energy deficiency and water environmental contamination. Aquatic biomass from duckweeds is attracting a lot of interest for the production of biofuels. They grow very fast in municipal wastewater rich of nitrogen and phosphate. They could develop abundant biomass and starch in a very short time. Interestingly, duckweeds have a unique life cycle with seasonal changes from vegetative growth to dormant stage, accumulating up to 60% starch of dry weight within two weeks. The starch synthesis involves many genes and regulators, which requires to be built up through transcriptional regulatory networks. As we all know, the studies of genomics and transcriptome would be critical to understand the network and mechanism of starch accumulation. The reference genome of duckweeds was published in 2014, while the transcriptome, especially the temporal and spatial variation of gene expression in starch accumulation of dormancy is missing. We will take advantage of the 2nd- (RNA-Seq) and 3rd-(ISO-Seq) generation sequencing platform to qualify the full-length cDNA sequence and to quantify gene expression in time series during the development of dormancy. We will infer the interactions among regulator genes and target genes to construct transcriptional regulatory networks. Our purpose is to find transcription factors expressed at specifically time and/or genes with significantly change causing essential phenotypical changes. The findings will be beneficial to genetically engineer duckweeds as a source for bioethanol production and a phytoremediation of wastewater.

能源短缺和水资源污染亟需开发可替代的绿色能源。水生植物浮萍能高效吸收污水中的氮磷并促进快速生长，而且积累的淀粉和其他生物质极易被降解成酒精，这些特性使浮萍备受关注，而研究淀粉累积机制成为利用浮萍的首要科学问题。浮萍从春夏的营养体过渡到秋冬的休眠芽时，淀粉在两周内累积到占干重的~60%，这为开发利用淀粉能源提供了极佳的模型。淀粉合成途径非常复杂，依赖于多基因的时空特异性表达和协同调控。浮萍基因组序列已经完成，但是转录组结构和表达信息的缺乏阻碍了淀粉机理的研究。因此，我们利用二代（RNA-seq ）和三代(ISO-seq)高通量的转录组测序，构建浮萍休眠芽形成过程中的全长cDNA文库并定量基因表达谱，解析淀粉迅速累积的转录调控网络, 挖掘淀粉合成时关键的转录因子和功能基因，即在时间和空间上特异性控制淀粉合成的开关，从而为开发利用浮萍作为清洁的生物能源酒精提供分子基础。

浮萍是世界上生长最快的开花单子叶高等植物。由于浮萍是水生植物，基本上不需要木质素来维持它向上生长，多余的能量被用于合成蛋白质和淀粉，其含量占总生物质的40%~70%。而研究浮萍淀粉的累积机理离不开该物种的参考基因组和基因注释。随着测序技术和生物信息学的快速发展，我们利用三代PacBio单分子长读长的测序优势，拼装出的序列连续性比上一版基因组提升了44倍，填补了上一版95.4%的序列丢失区域，鉴定到了更多的完整LTR序列和嵌套LTR区间。长读长直接获取的492,435条高质量的全长cDNA帮助精准注释了18,708个蛋白编码基因，提升的基因组序列和注释为浮萍的生物学研究提供了更优质的参考基因组。此外我们还利用同一套数据成功拼装注释了紫萍的叶绿体基因组，加上上述的全长转录本让我们深入解读了浮萍叶绿体基因结构（内含子），转录水平的RNA编辑和操纵子现象，为推动物种进化研究和叶绿体基因工程的开展奠定理论基础。在浮萍基因组和基因注释工作的基础上，我们追踪了浮萍在休眠过程中淀粉含量和基因表达量的动态变化。利用基因表达量之间的相关性，我们构建了一个完整精确的转录调控网络，找出了关键的转录因子和功能基因并进行验证，为更好地利用和挖掘浮萍生物能源的潜能奠定了基础。