以TMV为模式建立基于宏基因组学的植物病毒检测方法  

Metagenomics-based Plant Virus Detection Method with TMV Model

作　　者：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 

机构地区：[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

出　　处：《云南农业大学学报（自然科学版）》2017年第2期197-203,共7页Journal of Yunnan Agricultural University:Natural Science

基　　金：国家自然科学基金项目(31470018)

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。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 lev- el display Tobamovirus accounted for 99. 913%. The experiments proved that the method of dsRNA ex- traction 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 adapta- tion in agricultural cultivation and non farming areas.

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 

分 类 号：Q933[生物学—微生物学] S432.41[农业科学—植物病理学]