基于流式细胞仪高通量分选的深海微生物单细胞培养  

Isolation of deep-sea microorganisms by flow cytometry–based high-throughput cell sorting and single cell cultivation

作　　者：阮楚晋 郑小伟 王丽[1] 王铱 朱雅新[1] 王剑[1] 贠娟莉 董志扬[1] 陆祖军[4] 黄英[1] 杜文斌[1,3] 黄力 戴欣[1,2] Chujin Ruan;Xiaowei Zheng;Li Wang;Yi Wang;Yaxin Zhu;Jian Wang;Juanli Yun;Zhiyang Dong;Zujun Lu;Ying Huang;Wenbin Du;Li Huang;Xin Dai(State Key Laboratory of Microbial Resources,Institute of Microbiology,Chinese Academy of Sciences,Beijing 100101,China;College of Life Sciences,University of Chinese Academy of Sciences,Beijing 100049,China;Savaid Medical School,University of Chinese Academy of Sciences,Beijing 100049,China;College of Life Sciences,Guangxi Normal University,Guilin 541006,Guangxi Province,China)

机构地区：[1]中国科学院微生物研究所,微生物资源前期开发国家重点实验室,北京100101 [2]中国科学院大学生命科学院,北京100049 [3]中国科学院大学存济医学院,北京100049 [4]广西师范大学生命科学学院,广西桂林541006

出　　处：《微生物学报》2021年第4期816-827,共12页Acta Microbiologica Sinica

基　　金：国家自然科学基金(91951105);中国大洋矿产资源研究开发协会十三五项目(DY135-B-02);“科学”号高端用户项目(KEXUE2019GZ05)。

摘　　要：【目的】建立适用于海洋微生物的流式细胞分选与高通量单细胞培养的方法,通过该方法从印度洋深海样品中分离微生物纯培养菌株。【方法】利用流式细胞仪单细胞分选功能,以前向角(FSC)和侧向角(SSC)散射光信号代替荧光信号作为分选逻辑,对深海水体和沉积物样品中微生物进行单细胞高通量分选和培养。【结果】确定了流式细胞分选的区域和条件,发现所建立方法适于分离海洋水体微生物,而不是沉积物微生物。从印度洋深海水体样品中获得61个潜在新菌株,分属于6个新属种,占分离菌株总数的26.29%,其16S rRNA基因序列与已培养的模式菌株相似性为89.79%–95.37%。【结论】本研究所建立的方法有助于提高发现海洋微生物新物种的效率,获得更多新的海洋微生物资源。[Objective]To test a single cell cultivation method involving flow cytometry-based high-throughput cell sorting and single cell cultivation in the isolation of microorganisms from deep-sea samples.[Methods]The flow cytometer sorted to microbial cells of interest based on the size and complexity of the particles using forward versus side scatter gating(SSC vs.FSC)without the need for fluorescence labeling.Then,the single cells were cultivated in 96-well plates and further transferred to agar plate for scale-up cultivation and taxonomic identification.The performance of this method to cultivate microorganisms from deep-sea water and sediment samples was evaluated.[Results]An optimal sort region was chosen to sort microbial cells from the deep-sea samples,following by high-throughput single-cell broth cultivation.A total of 61 potential novel microbial strains,which belong to 6 novel genus or species,were obtained from deep-sea samples from the Indian Ocean.The novel strains accounted for 26.29%of the total isolates and shared 89.79%–95.37%similarity at the 16S rRNA gene sequence level.[Conclusion]The FCM-based high-throughput cell sorting and single-cell cultivation method is more suitable for sorting and cultivating of sea-water microorganisms.This methed may help increase the efficiency of identifying novel species from deep seas.

关 键 词：微生物培养 单细胞 流式细胞仪 深海 

分 类 号：Q93-335[生物学—微生物学]