利用深度测序技术鉴定疑似桑花叶卷叶病感病桑树叶片中的相关病毒

Identification of Mulberry Mosaic Leaf-roll Associated Virus in Diseased Mulberry Leaf Sample by Deep Sequencing Technology

植物通过小干扰RNA(siRNA)介导的RNA干扰机制来防御外来病毒感染,通过对感病植物材料小RNA(sRNA)的深度测序,获得这一过程中产生的与病毒序列高度一致的siRNA序列信息,可以快速地鉴定侵染植物的病毒组成。以采集自浙江省桐乡市桑园疑似感染桑花叶卷叶病(原称桑花叶型萎缩病)的桑树叶片为材料提取总RNA,构建sRNA文库并进行深度测序,对测序得到的总sRNA进行组装后,比对鉴定出桑花叶卷叶病相关病毒(mulberry mosaic leaf roll-associated virus,MMLRa V),并分别得到占MMLRa V 2个组分全基因组14.19%和21.86%的核酸序列。进一步以感病桑叶组织的总RNA为模板,通过RT-PCR扩增获得部分MMLRa V基因组序列,经Sanger测序确定采集的感病桑树叶片中只存在MMLRa V。通过生物信息学方法分析表明,MMLRa V来源siRNA在基因组中的长度分布、5'端碱基偏好性及热点区分布等具有明显特点。研究结果显示,利用siRNA深度测序技术鉴定桑树病毒是非常高效的手段。

Plants defense against viruses through small interfering RNA（ siRNA）-mediated RNA interference mechanism.siRNA sequence information,which was highly consistent with viral sequence,can be obtained by small RNA（ sRNA）deep sequencing of plant samples. It enables quick identification of viruses in plant. In the present study,suspected mulberry mosaic roll leaf disease（ originally named mulberry mosaic dwarf disease）-infected mulberry leaf samples collected from Tongxiang City of Zhejiang Province were used as material to extract total RNA for sRNA library construction and deep sequencing. After assembly of total sRNA,mulberry mosaic leaf roll-associated virus（ MMLRaV） was identified through alignment,and 14. 19% and 21. 86% of full length genome nucleotide sequence of the two components of the previously reported MMLRaV were obtained.Partial genome sequence of MMLRaV was obtained by RT-PCR using total RNA of diseased mulberry leaf as template,and Sanger sequencing confirmed that only MMLRaV existed in the diseased mulberry leaf samples. Bioinformatics analysis showed that the length distribution,5＇-terminal nucleotide preference,and hotspot distribution of MMLRaV-derived sRNA has their own characteristics. Taken together,these data suggest that siRNA deep sequencing technology is an efficient tool for virus identification of mulberry.