Characterization of vsiRNAs derived from Cymbidium Mosaic/Odontoglossum Ringspot Viruses in co-infected Phalaenopsis equestris
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摘要:
目的 探明蝴蝶兰和病毒（建兰花叶病毒、齿兰环斑病毒）之间的相互作用，进而为制定蝴蝶兰病毒性病害的有效防控措施提供理论基础。 方法 首先通过RT-PCR特异扩增建兰花叶病毒（cymbidium mosaic virus, CymMV）和齿兰环斑病毒（odontoglossum ringspot virus, ORSV）外壳蛋白基因片段，通过电镜负染和切片技术明确CymMV和 ORSV在蝴蝶兰细胞中的存在；然后通过小RNA深度测序技术鉴定分析病毒来源的小干扰RNA（vsiRNAs）的丰度、长度、碱基偏好性和正负义来源等特征。 结果 RT-PCR能够特异地扩增到病毒外壳蛋白基因片段，电镜负染和超薄切片结果中都能够观察到长约300 nm棒状的CymMV病毒粒子和长约500 nm线性的ORSV病毒粒子的存在；小RNA深度测序分别获得7 563 892和6 133 689个读数的CymMV和 ORSV来源的vsiRNAs，vsiRNAs在丰度、长度、碱基偏好性和正负义来源等方面具有一定的普遍性和特异性。 结论 CymMV和ORSV两种病毒在蝴蝶兰植株中存在复合侵染；CymMV和ORSV vsiRNAs的丰度、长度、悬挂、碱基偏好性、正负义链分布、Hotspot和Coldspot等特征具有普遍性和特异性。 Abstract:Objective Interactions between Phalaenopsis equestris and cymbidium mosaic virus (CymMV) and odontoglossum ringspot virus (ORSV) were studied to aid the effort in developing effective preventive and control means against the diseases caused by the pathogens. Method The coat protein (CP) genes of CymMV and ORSV were amplified using RT-PCR. Under an electron microscope, viral morphology and size of CymMV and ORSV particles in P. equestris cells were examined. Abundance, length, base preference and origin of virus-derived vsiRNAs were analyzed applying the small RNA deep sequencing technology. Result The amplifications of CP genes of CymMV and ORSV were specifically obtain by RT-PCR. The electron microscopy revealed the lengths of the rod-like CymMV to be approximate 300 nm, while the linear ORSV, 500 nm. The small RNA deep sequencing yielded 7 563 892 CymMV-derived and 6 133 689 ORSV-derived vsiRNAs exhibiting the universality and specificity in abundance, length, base preference and sense strand distribution. Conclusion Co-infections of CymMV and ORSV on P. equestris were clearly demonstrated in this study. The vsiRNAs of CymMV and ORSV displayed characteristic patterns in abundance, length, base preferences and sense strand distribution. -
Key words:
- Phalaenopsis equestris /
- virus /
- vsiRNAs /
- interaction
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图 1 CymMV和ORSV在蝴蝶兰叶片的共侵染的鉴定
A：病毒侵染的蝴蝶兰样品；B：CymMV和ORSV外壳蛋白基因的RT-PCR扩增产物的琼脂糖凝胶电泳， M为DNA marker MD102; 1和2分别为 CymMV和ORSV外壳蛋白基因产物； C：CymMV和ORSV病毒电镜观察的负染图；D：CymMV和ORSV病毒电镜观察的超薄切片图。
Figure 1. Identification of CymMV/ORSV co-infections in P. equestris
A: P. equestris plant infected by viruses; B: agarose gel electrophoresis of RT-PCR products of CP genes of CymMV and ORSV; M: DNA marker MD102; 1 and 2: RT-PCR products of CP genes of CymMV and ORSV, respectively; C: electron microscopic negative staining images of CymMV and ORSV; D: electron microscopic images on thin sections of CymMV and ORSV.
图 2 蝴蝶兰细胞中CymMV和ORSV来源的vsiRNAs特征
A：健康蝴蝶兰和病毒侵染蝴蝶兰中siRNAs长度分布；B： 病毒侵染蝴蝶兰中vsiRNAs长度分布； C:病毒侵染的蝴蝶兰中5'端距离为1-21 nt vsiRNAs读数；D： Total vsiRNAs（左）和21 nt vsiRNAs（右） 5'端第一个碱基偏好性。
Figure 2. Characterization of CymMV- and ORSV-derived vsiRNAs in P. equestris
A: Size distribution of total small RNAs in virus-infected and viruses-free P. equestris plants; B: size distribution of vsiRNAs matching CymMV and ORSV genomes in virus-infected P. equestris plants; C: reads of 21-nt vsiRNAs with 1-21 nt distance between 5′ ends from CymMV and ORSV genome in P. equestris plants; D: relative frequency of 5′-terminal nucleotide of total vsiRNAs (left) and 21 nt vsiRNAs (right).
图 3 CymMV和ORSV vsiRNAs的生成前体分析
A：CymMV和ORSV vsiRNAs的正负义链分布；B：vsiRNAs在CymMV基因组上高低频切割位点；C：vsiRNAs在ORSV基因组上高低频切割位点； D：CymMV正链基因组的二级结构预测；E：ORSV正链基因组的二级结构预测。方框I为图3B和图3D中高频切割位点和基因组茎环结构的对应关系。方框II为图3C和图3E中高频切割位点和基因组茎环结构的对应关系。
Figure 3. Biogenesis precursors of CymMV and ORSV vsiRNAs
A: Polarity distribution of vsiRNAs matching CymMV and ORSV genomes from co-infected P. equestris plants; B and C: vsiRNAs hotspots and cold spots distribution along CymMV and ORSV genomes, respectively; D and E: secondary structures of CymMV and ORSV RNAs predicted with RNAfold server. Box I shows the corresponding relationship between the high frequency cutting sites in Figures 3B and the stem ring structure in Figure 3D. Box II shows the corresponding relationship between the high frequency cutting sites in Figures 3C and the stem ring structure in Figure 3E.
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