基于组学的小球藻耐受沼液胁迫分子机制研究

The untreated swine slurry is one of the important factors causing water pollution at present. Microalgae can transform nutrients such as nitrogen (N) and phosphorus (P) from biogas slurry into valuable algal biomass, which can not only purify wastewater, but also obtain high value-added products of microalgae. However, the presence of high ammonia nitrogen and high turbidity in the biogas slurry has an adverse effect on the growth of microalgae. Thereby, most microalgae cannot grow in high concentration biogas slurry, which seriously restricts the large-scale application of microalgae in the treatment of swine wastewater. To solve this problem, a Chlorella vulgaris has been isolated from the swine wastewater, which has strong tolerance to high concentration swine slurry. Whole-genome bisulfite sequencing (BS-seq) and transcriptome sequencing (RNA-seq) are used to analyze the differences in DNA methylation, transcriptional level and gene expression level in the whole genome range of the microalgae grown under normal BG11 medium and swine slurry stress environment. The project is aimed to reveal the DNA methylation pattern of Chlorella vulgaris and the regulation mechanism of gene expression related to stress response and to discover the genes of Chlorella vulgaris responding to stress and epigenetic modification. The regulation of epigenetic modification of Chlorella vulgaris on stress environment and the molecular mechanism of Chlorella vulgaris’s tolerance to high concentration swine slurry are analyzed, which provides the basis for molecular breeding of microalgae to tolerate extreme environment.

生猪养殖过程产生的沼液未加处理的排放是引起水资源污染的重要因素之一。微藻能将沼液中引起富营养化的氮磷等物质转化成有价值的细胞生物量，不仅能净化废水，还能获得高附加值产品。但沼液中存在的高氨氮、高浊度等对微藻生长不利，导致大多数微藻不能在高浓度沼液中生长，严重制约了微藻处理猪沼液的大规模应用。针对这一问题，本项目已从原始猪沼液中分离得到对高浓度猪沼液具有强耐受性的小球藻，拟采用全基因组甲基化测序(BS-seq)和转录组测序(RNA-seq)技术，分析BG11培养基正常环境和猪沼液胁迫环境下生长的微藻全基因组范围内DNA甲基化差异、转录水平和基因表达水平差异，揭示小球藻DNA甲基化模式以及对逆境响应相关基因表达的调控机制，挖掘小球藻对逆境胁迫应答基因和表观修饰应答基因。探明表观遗传修饰如何调控微藻对逆境的胁迫应答，解析该小球藻耐受高浓度猪沼液的分子机理，为选育耐受极端环境微藻分子育种提供基础。

生猪养殖过程产生的沼液富含氮磷等能够引起自然水体富营养化的物质，而微藻能将其转化成有价值的细胞生物量，在净化废水的同时获得高附加值产品。但沼液中存在的高氨氮、高浊度等因素导致大多数微藻不能在高浓度沼液中生长，严重制约了微藻处理猪沼液技术的大规模应用。本研究从实验室分离获得的一株耐受高浓度猪沼液的Chlorella vulgaris MBFJNU-1（简称C. vulgaris MBFJNU-1）普通小球藻出发，利用转录组测序(RNA-seq)和全基因组甲基化测序(BS-seq)技术分析猪沼液胁迫环境和游离氨（FA）环境下生长的C. vulgaris MBFJNU-1全基因组范围内随时间变化的基因表达水平差异和DNA甲基化差异，揭示了小球藻对逆境响应相关基因表达的调控机制。研究发现小球藻对猪沼液和游离氨的胁迫响应主要发生在48小时内，其抗胁迫策略主要为三点：1.通过半乳糖代谢、糖酵解加速精氨酸的合成来消耗氨氮；2.加快电子生产和传递效率，增加光合作用中ATP的合成量，以确保固碳需要的能量供应；3.通过调节还原型谷胱甘肽(GSH)的合成和利用，加速自由基和活性氧（ROS）的清除，同时调节甲基化供体甜菜碱的合成，以控制基因转录水平来缓解氧化应激效应。此外，68个参与响应和耐受胁迫的关键基因被发现。本项目为微藻净化猪沼液等高氨氮污水工艺的优化和选育耐受极端环境微藻分子育种奠定了基础。