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云南农业大学学报(自然科学) 2017, Vol. 32 Issue (2) :197-203    DOI: 10.16211/j.issn.1004-390X(n).2017.02.002
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以TMV为模式建立基于宏基因组学的植物病毒检测方法
刘悦1, 孔宝华1, 王连春2, 王崇德1, 吴德喜1, 谢琼1, 甘晓帆1
1. 云南农业大学 植物保护学院, 省部共建云南生物资源保护与利用国家重点实验室, 云南 昆明 650201;
2. 玉溪师范学院 资源环境学院, 云南 玉溪 653100
Metagenomics-based Plant Virus Detection Method with TMV Model
LIU Yue1, KONG Baohua1, WANG Lianchun2, WANG Chongde1, WU Dexi1, XIE Qiong1, GAN Xiaofan1
1. State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, College of Plant Protection, Yunnan Agricultural University, Kunming 650201, China;
2. College of Resources and Environment, Yuxi Normal University, Yuxi 653100, China

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摘要 用感染烟草花叶病毒(TMV)的烟株做阳性对照,提取dsRNA,采用随机引物扩增、高通量测序方法进行了序列分析。PCR产物宏基因组测序结果显示:TMV的检出频率为99.84%;属水平分类和丰度统计结果显示:Tobamovirus占99.913%,成功建立了以植物病毒dsRNA提取与高通量测序相结合的植物病毒探测方法。按照病毒类似颗粒提取的方法对感染TMV的烟株提取病毒类似颗粒,获得TMV病毒粒子,从中提取RNA,应用TMV特异引物,进行RT-PCR,结果显示:扩增产物序列与TMA序列同源性为99%。结果表明:用该病毒类似颗粒抽提方法能成功提取TMV病毒粒子及RNA,满足宏基因组分析要求。应用本研究构建的dsRNA及病毒类似颗粒的方法,提取20个表现植物病毒病症状的植物样本dsRNA与病毒类似颗粒的DNA,核酸检测质量较好,成功检测到10多种植物病毒。所构建的方法为宏基因组技术探测农业栽培区及非耕作区植物病毒种群与生态适应奠定了基础。
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刘悦
孔宝华
王连春
王崇德
吴德喜
谢琼
甘晓帆
关键词宏基因组学   植物病毒   TMV   双链RNA   病毒类似颗粒   检测   方法     
Abstract: The tobacco leaves infected with Tobacco mosaic virus (TMV) was positive control, and the dsRNAs were extracted and the sequences analysis were performed by random primers amplification and high throughput sequencing. The PCR products of metagenomic sequencing results showed that the detection rate of TMV was 99.84%, and statistical results of classification and abundance of genus level display Tobamovirus accounted for 99.913%. The experiments proved that the method of dsRNA extraction method of metagenomic sequencing to plant virus were established successfully. According to the method of extracting virus like particles from tobacco infected by to TMV, then extracted RNA from the virus like particles, RT-PCR was conducted using TMV specific primers, and the results showed that the TMV sequence homology was 99%. The results proved that the method of nucleic acid of virus like particles extraction was established successfully and met the requirements of metagenomic analysis. The dsRNA and virus DNA of the like particles of 40 viral samples were constructed by this study. The nucleic acid of the all the samples had good quality. 20 kinds of plant viruses were detected from those samples. This study lays a foundation for the detection of plant virus populations and ecological adaptation in agricultural cultivation and non farming areas.
Keywordsmetagenomic   plant viruses   TMV   dsRNA   virus-like particles   detection   method     
Received 2016-08-10;
Fund:国家自然科学基金项目(31470018)。
Corresponding Authors: 孔宝华(1962-),女,云南昆明人,博士,教授,主要从事植物病毒研究。E-mail:baohuakong@126.com     Email: baohuakong@126.com
About author: 刘悦(1990-),女,黑龙江七台河人,在读硕士研究生,主要从事植物病毒研究。E-mail:liuyue0504@126.com
引用本文:   
刘悦, 孔宝华, 王连春, 王崇德, 吴德喜, 谢琼, 甘晓帆.以TMV为模式建立基于宏基因组学的植物病毒检测方法[J]  云南农业大学学报(自然科学), 2017,V32(2): 197-203
LIU Yue, KONG Baohua, WANG Lianchun, WANG Chongde, WU Dexi, XIE Qiong, GAN Xiaofan.Metagenomics-based Plant Virus Detection Method with TMV Model[J]  Journal of Yunnan Agricultural University, 2017,V32(2): 197-203
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