珠江河口水体环境DNA提取方法的建立及优化

李红婷; 张帅; 邹柯姝; 陈作志; 陈晓雷; 蒋佩文; 曹漪婷; 李敏

doi:10.12131/20210304

珠江河口水体环境DNA提取方法的建立及优化

doi: 10.12131/20210304

李红婷^{1, 2, 3, 4,},
张帅^{1, 4},
邹柯姝⁵,
陈作志^{1, 2},
陈晓雷^{1, 4},
蒋佩文^{1, 4},
曹漪婷^{1, 6},
李敏^{1, 2, 3, ,}

1.
中国水产科学研究院南海水产研究所/农业农村部外海渔业可持续利用重点实验室/广东省渔业生态环境重点实验室，广东广州 510300
2.
南方海洋科学与工程广东省实验室 (广州)，广东广州 511458
3.
广东珠江口生态系统野外科学观测研究站，广东广州 510300
4.
上海海洋大学海洋科学学院，上海 201306
5.
华南农业大学海洋学院，广东广州 510642
6.
大连海洋大学水产与生命学院，辽宁大连 116023

基金项目: 国家科技基础调查专项 (2019FY101905)；广东省科技计划项目(2019B121201001)；南方海洋科学与工程广东省实验室 (广州) 人才团队引进重大专项 (GML2019ZD0605)；中国水产科学研究院南海水产研究所中央级公益性科研院所基本科研业务费专项资金 (2019TS13, 2021SD18)

详细信息

作者简介:
李红婷 (1997— )，女，硕士研究生，研究方向为海洋生物环境DNA。E-mail: 1604790319@qq.com

通讯作者:
李　敏 (1984—)，男，副研究员，博士，从事海洋生物多样性保护研究。E-mail: limin@scsfri.ac.cn

中图分类号: S 932
计量
- 文章访问数: 151
- HTML全文浏览量: 39
- PDF下载量: 23
- 被引次数: 0
出版历程
- 收稿日期: 2021-10-21
- 修回日期: 2021-11-22
- 录用日期: 2021-11-25
- 网络出版日期: 2021-12-09

Establishment and optimization of environmental DNA extraction method from water of Pearl River Estuary

LI Hongting^{1, 2, 3, 4
,},
ZHANG Shuai^{1, 4},
ZOU Keshu⁵,
CHEN Zuozhi^{1, 2},
CHEN Xiaolei^{1, 4},
JIANG Peiwen^{1, 4},
CAO Yiting^{1, 6},
LI Min^{1, 2, 3
, ,}

1.
South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences/Key Laboratory for Sustainable Utilization of Open-Sea Fishery, Ministry of Agriculture and Rural Affairs/Guangdong Provincial Key Laboratory of Fishery Ecology and Environment, Guangzhou 510300, China
2.
Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Guangzhou 511458, China
3.
Scientific Observation and Research Field Station of Pearl River Estuary Ecosystem, Guangdong Province, Guangzhou 510300, China
4.
College of Marine Sciences, Shanghai Ocean University, Shanghai 201306, China
5.
College of Marine Science, South China Agricultural University, Guangzhou 510642, China
6.
College of Fisheries and Life Science, Dalian Ocean University, Dalian 116023, China

摘要

摘要: 近年来，环境DNA (eDNA) 技术广泛应用于水生生物多样性研究。以珠江河口咸淡水生态系统为研究区域，采用滤膜法富集eDNA，选取直径47 mm、孔径0.45 μm的硝酸纤维膜、醋酸纤维膜、玻璃纤维膜和聚碳酸酯膜共4种材质的滤膜，采用4种滤膜保存方法，结合2种试剂盒提取eDNA，评估不同组合方案对eDNA提取效果的影响。结果显示，滤膜材质和保存方法对eDNA产量具有显著影响；其中，醋酸纤维膜获取的eDNA浓度最高。在DNA提取物质量相当的情况下，海洋动物组织基因组DNA提取试剂盒提取的eDNA浓度高于DNeasy Blood and Tissue kit试剂盒。在不同保存条件下，“液氮”保存法获取的eDNA浓度最高。在不具备冷冻条件时，可添加乙醇，常温保存滤膜。采样后立即过滤水样能够有效防止eDNA降解。文章建立了珠江口咸淡水生态系统水样eDNA的获取、保存和提取方案，可为相似水域的eDNA研究提供参考。
- 珠江河口 /
- eDNA获取 /
- eDNA保存 /
- eDNA提取
Abstract: Environmental DNA (eDNA) technology has been widely used in the study of aquatic biodiversity in recent years. In this study, different filter membrane, storage method of membrane and DNA extract kits were used to evaluate effectiveness of eDNA enrichment from the brackish water in the Pearl River Estuary. Four kinds of filter membranes with diameter of 47 mm and pore size of 0.45 μm (Nitrocellulose membrane, cellulose acetate membrane, glass fiber membrane and polycarbonate membrane) were selected. Four kinds of membrane preservation methods were adopted and two kinds of DNA extract kits were used. The results show that filter membrane materials and preservation methods had a significant impact on the yield of eDNA. The highest concentration of eDNA was obtained by cellulose acetate membrane. Under the condition of the same quality of DNA yield, the concentration of eDNA extracted by Marine Animal Tissue Genomic DNA Kit was higher than that by DNeasy Blood and Tissue Kit. The concentration of eDNA obtained from ‘liquid nitrogen’ storage method was the highest among the four storage conditions. However, the concentration of eDNA was acceptable for alcohol preserved method at room temperature when cryopreservation was not available. Filtering water samples immediately after sampling can prevent eDNA degradation effectively. This study established a systematic protocol for eDNA extraction of water samples from the brackish ecosystem in the Pearl River Estuary, providing references for eDNA research in similar waters.
- Pearl River Estuary /
- eDNA capture /
- eDNA preservation /
- eDNA extraction

HTML全文

图 1 4种滤膜与2种试剂盒的实验设计方案

Figure 1. Experimental design of four kinds of filtration membranes and two kinds of kits

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图 2 滤膜与保存方法的不同组合

Figure 2. Different combinations of membrane and preservation methods

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图 3 滤膜与试剂盒组合提取的总eDNA电泳图

Figure 3. Electrophoresis diagram of total eDNA extracted by combination of membrane and kit

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图 4 滤膜与试剂盒组合获得的eDNA浓度比较

Figure 4. Comparison of eDNA concentration obtained by combination of membrane and extraction kit

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图 5 滤膜与试剂盒组合获得的eDNA纯度比较

Figure 5. Purity comparison of eDNA obtained by combination of membrane and extraction kit

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图 6 不同保存方法下提取的eDNA浓度比较

Figure 6. Comparison of eDNA concentrations extracted by different preservation methods

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图 7 不同保存方法下提取的eDNA纯度比较

Figure 7. Purity comparison of eDNA extracted by different preservation methods

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图 8 不同滤膜过滤1 L水样所需时间比较

Figure 8. Comparison of time used for filtration of 1 L water sample by different membranes

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