侵染守宫木的曼陀罗黄脉病毒的分离鉴定及其基因组序列分析

Isolation, identification, and genomic sequence analysis of datura yellow vein virus infecting Sauropus androgynus

摘要:

守宫木具有较高的药用和食用价值，其生长过程受到多种病毒病害的危害，严重影响了守宫木的产量和质量。目前有关我国守宫木病毒病害研究的较少。目的 分离鉴定我国守宫木病毒病原。方法 分析本实验室前期守宫木叶片小RNA测序(small RNA sequencing, sRNA-seq)的数据；利用逆转录聚合酶链式反应(reverse transcription-polymerase chain reaction, RT-PCR)对10种不同品种的守宫木叶片样品进行检测；以阳性样品S4叶片的总RNA为模板，结合RT-PCR、cDNA末端快速扩增(rapid amplification of cDNA ends, RACE)和Sanger测序技术获得曼陀罗黄脉病毒(datura yellow vein virus, DYVV)守宫木分离物(DYVV-sa)全基因组序列。结果 基于sRNA-seq的数据分析发现守宫木中存在DYVV，不同品种样本经RT-PCR检测显示S4和S12表现为DYVV阳性。以S4品种为模板，克隆获得DYVV-sa基因组全长序列，其大小为13 185 nt，可编码6个开放阅读框(open reading frames, ORFs)，与分离自黑叶苏珊的DYVV序列相似性高达95.8%-98.1%，同时系统发育分析显示DYVV-sa与DYVV亲缘关系最密切，因此确定DYVV-sa为DYVV的分离株。对DYVV-sa病毒来源的小干扰RNA (virus-derived small interfering RNA, vsiRNA)进行分析发现，DYVV-sa-vsiRNA大小以21 nt和22 nt为主，其中21 nt更为丰富；DYVV-sa-vsiRNA的5′端第一个核苷酸优先偏好U和C；来源于负链的vsiRNAs所占比例高于正链，并在整个病毒基因组中均有分布，局部区域形成热点。结论 本研究发现DYVV可侵染守宫木植株并克隆获得DYVV-sa分离株基因组全长序列，研究结果拓展了DYVV的天然寄主范围，为DYVV的多样性研究和进化分析提供了基础，同时为守宫木病毒病害的防治提供了理论依据。

Abstract:

Sauropus androgynus has high medicinal and edible values. However, its growth is threatened by various viral diseases, which severely affect both the yield and quality of S. androgynus. Since research is limited regarding the viral diseases affecting S. androgynus in China.Objective To isotation, identifying the viral pathogens of S. androgynus in China.Methods The small RNA sequencing (sRNA-seq) data of S. androgynus leaves from our previous study were analyzed. RT-PCR was employed to detect the datura yellow vein virus (DYVV) in leaf samples of 10 different varieties of S. androgynus. With the total RNA of positive S4 leaves as a template, reverse transcription-polymerase chain reaction (RT-PCR), rapid amplification of cDNA ends (RACE), and Sanger sequencing were employed to determine the full-length genome sequence of the DYVV isolate from S. androgynus (named DYVV-sa).Results The analysis of the sRNA-seq data revealed the presence of DYVV in S. androgynus. RT-PCR detection of different varieties showed that only S4 and S12 tested positive for DYVV, and the full-length sequence of DYVV-sa was cloned based on S4. The genome of DYVV-sa was 13 185 nt in length and contained six open reading frames (ORFs). The DYVV-sa showed the identity as high as 95.8%-98.1% with the DYVV sequences isolated from Thunbergia alata. Moreover, the phylogenetic tree also demonstrated that DYVV-sa shared the closest genetic relationship with DYVV, clearly indicating that DYVV-sa was an isolate of DYVV. In addition, the majority of DYVV-sa virus-derived small interfering RNA (vsiRNA) were 21 nt and 22 nt, and those of 21 nt were more abundant. The first nucleotide at the 5′ termini of vsiRNAs derived from DYVV-sa preferred U and C. The proportion of vsiRNAs derived from the negative strand was higher than that from the positive strand. The distribution of vsiRNAs along the viral genome was generally even, with some hot spots formed in local regions.Conclusion This study found that DYVV can infect S. androgynus and successfully obtains the full-length genomic sequence of the DYVV-sa isolate. These findings expand the known natural host range of DYVV, provide crucial theoretical foundations for research on its genetic diversity and phylogenetic relationship, and offer clues for the prevention and control of viral diseases attacking S. androgynus.