基于单细胞拉曼分选耦合精准宏基因组的海洋原位固碳微生物解析

Marine microbes play important roles in the global carbon cycle. Therefore, mining of indigenous CO2-fixing microbes in the ocean is of great scientific significance. However, one key bottleneck that hinders the advance of the field has been the paucity of methods that recognize and sort those carbon-fixing cells in situ. Current research tools, such as metagenomics and single-cell genomics, are widely used, yet their setbacks are many, for example, low resolution of taxa recognition, inability to cover low abundance species, lack of cellular phenotype/function information, inability to connect genomes to their ecological functions. Therefore, here we propose a new strategy to identify and dissect the indigenous marine CO2-fixing microorganisms, in which Raman-based functional cell sorting was coupled to downstream precise mini-metagenomic analysis. Firstly, microbes in a marine microbial consortium that are actively fixing CO2 are identified based on Single-cell Raman Spectra (SCRS), by feeding the marine consortium with 13C-labeled carbon source substrates. Secondly, the cells with targeted functions as represented by the target SCRS were sorted into a number of subgroups. Single-cell genomic amplification will be then applied to the sorted groups, followed by high-throughput sequencing, to produce a mini-metagenome for the precisely functionalized cells. Finally, a model for the taxonomical diversity and functional mechanism of marine CO2 fixation will be constructed based on the single-cell phenotypes and the mini-metagenome data. In summary, the endeavors should provide a new tool for dissecting the in situ functioning mechanism of microbiota.

海洋微生物是全球碳循环的主要载体之一，因此海洋原位固碳微生物的发掘具有重要意义，然而阻碍其深入推进的瓶颈之一是细胞原位固碳功能识别的方法学。目前，以宏基因组学和单细胞基因组学技术为代表的研究方法存在诸多缺陷，如物种解析度低；难于覆盖低丰度物种；细胞表型/功能信息缺失；细胞基因型与其生态功能无法直接联系等。对此，本项目提出一种基于微生物单细胞拉曼分选与精准宏基因组学方法，用于海洋固碳微生物的解析。首先，对海洋微生物在13C碳源底物培养条件下进行单细胞水平固碳功能的拉曼表型识别和细胞筛选；其次，将目标细胞分选为不同的亚群，利用单细胞基因组扩增策略进行微小规模细胞群体的基因组扩增，应用高通量测序手段获得功能明确的精准宏基因组数据；最终，整合单细胞表型与功能性细胞群体基因组，建立原位固碳物种多样性及机制模型。本项目将为微生物组原位功能机制研究提供一种新工具。

海洋原位固碳微生物的资源挖掘具有重要意义，其瓶颈之一是细胞原位固碳功能识别方法的匮乏。本项目基于微生物单细胞拉曼分选与单细胞精度的定向精准宏基因组学方法，对海洋固碳微生物进行原位解析。通过对海洋微生物在13C碳源底物培养条件下进行单细胞水平固碳功能的拉曼表型识别和细胞筛选，随后将目标细胞利用单细胞基因组扩增策略进行定向精准宏基因组扩增。最终，建立单细胞精度的海洋微生物原位固碳机制模型。这一系列方法学的开发和应用将有力拓展拉曼介导的定向精准宏基因组技术在海洋诸多领域的应用，为海洋微生物组原位功能机制研究提供一个新工具。