宏基因组学技术分析山羊瘤胃病毒的多样性

doi:10.11843/j.issn.0366-6964.2023.07.024

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (7): 2932-2941.doi: 10.11843/j.issn.0366-6964.2023.07.024

宏基因组学技术分析山羊瘤胃病毒的多样性

吴祎程^1,2, 冉涛^3,4*, 周传社^1,2*, 谭支良^1,2

1. 中国科学院亚热带农业生态研究所亚热带农业生态过程重点实验室畜禽养殖污染控制与资源化技术国家工程实验室湖南省动物营养生理与代谢过程重点实验室农业部中南动物营养与饲料科学观测实验站, 长沙 410125;
2. 中国科学院大学, 北京 100049;
3. 兰州大学草地农业科技学院, 兰州 730020;
4. 兰州大学草种创新与草地农业生态系统全国重点实验室, 兰州 730020

收稿日期:2022-08-30 出版日期:2023-07-23 发布日期:2023-07-21
通讯作者: 周传社,主要从事反刍家畜蛋白质营养调控与农牧复合生态系统研究,E-mail:zcs@isa.ac.cn;冉涛,主要从事传统动物营养学、分子营养学及胃肠道微生物组学研究,E-mail:rant@lzu.edu.cn
作者简介:吴祎程(1997-),女,湖北武汉人,硕士生,主要从事瘤胃环境病毒提取及功能研究,E-mail:wuyicheng19@mails.ucas.ac.cn,Tel:0731-84615204
基金资助:
国家自然科学基金（U20A2057）；兰州大学"双一流"建设人才项目（561120213）

Evaluation of the Viral Community Composition in Goat Rumen Fluid, Based on Metagenomic Analysis

WU Yicheng^1,2, RAN Tao^3,4*, ZHOU Chuanshe^1,2*, TAN Zhiliang^1,2

1. South Central Experimental Station of Animal Nutrition and Feed Science in the Ministry of Agriculture, Hunan Provincial Key Laboratory of Animal Nutritional Physiology and Metabolic Process, National Engineering Laboratory for Pollution Control and Waste Utilization in Livestock and Poultry Production, Key Laboratory for Agro-Ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, China;
2. University of Chinese Academy of Sciences, Beijing 100049, China;
3. College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730020, China;
4. State Key Laboratory of Herbage Improvement and Grassland Agro-ecosystems, Lanzhou University, Lanzhou 730020, China

Received:2022-08-30 Online:2023-07-23 Published:2023-07-21

摘要/Abstract

摘要： 反刍家畜瘤胃内微生物丰富多样，病毒（主要是噬菌体）亦是其重要组成部分。瘤胃内所有的病毒可统称为瘤胃病毒组（ruminal virome），可以利用宏基因组学（metagenomics）技术进行研究。解析山羊瘤胃病毒组的组分及功能有望更全面地了解其在瘤胃微生态区系中的地位和作用。为使测序结果能覆盖瘤胃中绝大部分病毒群落，不仅在样品的预处理阶段要尽量避免病毒样颗粒的损失，而且要保证瘤胃病毒组总DNA的提取质量和产量均满足宏基因组测序的要求。本研究比较了制备瘤胃液病毒组分的4种流程：（P1）离心+稀释、（P2）离心+稀释+过滤、（P3）离心+稀释+过滤+聚乙二醇沉淀、（P4）离心+稀释+过滤+聚乙二醇沉淀+氯仿处理。提取核酸后采用Illumina Novaseq 6000平台对瘤胃病毒基因组DNA进行测序，并对病毒群体结构进行分析比较，以筛选出瘤胃病毒组样品制备的最优流程。结果表明，聚乙二醇沉淀有助于病毒核酸提取，而氯仿处理过度影响病毒组分产量；山羊瘤胃内病毒群落主要是尾状病毒目的长尾病毒科、肌尾病毒科和短尾病毒科噬菌体。研究结果为深入分析噬菌体在山羊瘤胃微生态区系中的作用奠定了基础。

关键词: 瘤胃病毒, 病毒宏基因组学, 高通量测序技术, DNA提取, 多样性

Abstract: Viruses, mainly bacteriophage, are significant components of the ruminal microbiota in ruminants. All the ruminal viruses are called ruminal virome, and could be studied by metagenomics. Understanding the components and functions of goat ruminal viruses is expected to provide a more comprehensive understanding of rumen microecology. Accurate profiling of complex rumen virome requires DNA extraction methods that provide adequate quality and quantity, as well as sufficient coverage of the original community. In this study, four procedures to extract viruses-like particles (VLPs) from rumen fluid were compared:(P1) centrifugation + dilution, (P2) centrifugation + dilution + filtration, (P3) centrifugation + dilution + filtration + polyethylene glycol (PEG) precipitation, (P4) centrifugation + dilution + filtration + PEG precipitation + chloroform treatment. The nucleic acid in four procedures was extracted and DNA concentration, purity and integrity were compared. Then genomic DNA sequencing was performed using Illumina Novaseq 6000 and virus population structure was analyzed. The results showed that there were positive effects of filtration and PEG precipitation on viral nucleic acid quality. The chloroform treatment, severely reducing virus-like particles (VLPs) yield, should be used with caution when performing high-throughput sequencing. The viral communities in the rumen were dominated by bacteriophages belonging to the viral order Caudovirales (i.e., Myoviridae, Podoviridae, and Siphoviridae). The results provide the basis for future investigations regarding the ecological importance of viruses in rumen microbial ecosystems.

Key words: rumen virus, viral metagenomics, high-throughput sequencing technology, DNA extraction, diversity

中图分类号:

S852.65

吴祎程, 冉涛, 周传社, 谭支良. 宏基因组学技术分析山羊瘤胃病毒的多样性[J]. 畜牧兽医学报, 2023, 54(7): 2932-2941.

WU Yicheng, RAN Tao, ZHOU Chuanshe, TAN Zhiliang. Evaluation of the Viral Community Composition in Goat Rumen Fluid, Based on Metagenomic Analysis[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 2932-2941.

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宏基因组学技术分析山羊瘤胃病毒的多样性

Evaluation of the Viral Community Composition in Goat Rumen Fluid, Based on Metagenomic Analysis

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