Yuan Jian, Li Meizhen, Lin Senjie
The State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen, Fujian, China; Marine Biodiversity and Global Change Research Center, Xiamen University, Xiamen, Fujian, China.
The State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen, Fujian, China; Marine Biodiversity and Global Change Research Center, Xiamen University, Xiamen, Fujian, China; The Department of Marine Sciences, University of Connecticut, Groton, Connecticut, United States of America.
PLoS One. 2015 Jul 28;10(7):e0133060. doi: 10.1371/journal.pone.0133060. eCollection 2015.
Marine phytoplankton are highly diverse with different species possessing different cell coverings, posing challenges for thoroughly breaking the cells in DNA extraction yet preserving DNA integrity. While quantitative molecular techniques have been increasingly used in phytoplankton research, an effective and simple method broadly applicable to different lineages and natural assemblages is still lacking. In this study, we developed a bead-beating protocol based on our previous experience and tested it against 9 species of phytoplankton representing different lineages and different cell covering rigidities. We found the bead-beating method enhanced the final yield of DNA (highest as 2 folds) in comparison with the non-bead-beating method, while also preserving the DNA integrity. When our method was applied to a field sample collected at a subtropical bay located in Xiamen, China, the resultant ITS clone library revealed a highly diverse assemblage of phytoplankton and other micro-eukaryotes, including Archaea, Amoebozoa, Chlorophyta, Ciliphora, Bacillariophyta, Dinophyta, Fungi, Metazoa, etc. The appearance of thecate dinoflagellates, thin-walled phytoplankton and "naked" unicellular organisms indicates that our method could obtain the intact DNA of organisms with different cell coverings. All the results demonstrate that our method is useful for DNA extraction of phytoplankton and environmental surveys of their diversity and abundance.
海洋浮游植物种类繁多，不同物种具有不同的细胞覆盖物，这给在DNA提取过程中彻底破碎细胞同时保持DNA完整性带来了挑战。虽然定量分子技术在浮游植物研究中越来越多地被使用，但仍缺乏一种广泛适用于不同谱系和自然组合的有效且简单的方法。在本研究中，我们根据以往经验开发了一种珠磨法，并针对代表不同谱系和不同细胞覆盖物硬度的9种浮游植物进行了测试。我们发现，与非珠磨法相比，珠磨法提高了DNA的最终产量（最高可达2倍），同时还保持了DNA的完整性。当我们的方法应用于在中国厦门的一个亚热带海湾采集的现场样本时，由此产生的ITS克隆文库揭示了浮游植物和其他微型真核生物的高度多样化组合，包括古菌、变形虫、绿藻、纤毛虫、硅藻、甲藻、真菌、后生动物等。具壳甲藻、薄壁浮游植物和“裸”单细胞生物的出现表明，我们的方法可以获得具有不同细胞覆盖物的生物体的完整DNA。所有结果表明，我们的方法对于浮游植物的DNA提取及其多样性和丰度的环境调查是有用的。
