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蝴蝶兰中建兰花叶病毒和齿兰环斑病毒复合侵染的鉴定及其vsiRNAs特征分析

兰汉红

兰汉红. 蝴蝶兰中建兰花叶病毒和齿兰环斑病毒复合侵染的鉴定及其vsiRNAs特征分析 [J]. 福建农业学报,2024,39(9):1−8
引用本文: 兰汉红. 蝴蝶兰中建兰花叶病毒和齿兰环斑病毒复合侵染的鉴定及其vsiRNAs特征分析 [J]. 福建农业学报,2024,39(9):1−8
LAN H H. Identification, Characterization, and vsiRNAs of Cymbidium Mosaic/Odontoglossum Ringspot Viruses Co-infection in Phalaenopsis equestris [J]. Fujian Journal of Agricultural Sciences,2024,39(9):1−8
Citation: LAN H H. Identification, Characterization, and vsiRNAs of Cymbidium Mosaic/Odontoglossum Ringspot Viruses Co-infection in Phalaenopsis equestris [J]. Fujian Journal of Agricultural Sciences,2024,39(9):1−8

蝴蝶兰中建兰花叶病毒和齿兰环斑病毒复合侵染的鉴定及其vsiRNAs特征分析

基金项目: 国家自然科学基金项目(31601613);福建省自然科学基金项目(2018J01465);漳州市自然科学基金(ZZ2017J03)
详细信息
    作者简介:

    兰汉红(1982 — ),男,博士,副教授,主要从事分子病毒学研究,E-mail:lanhanh@163.com

  • 中图分类号: S682

Identification, Characterization, and vsiRNAs of Cymbidium Mosaic/Odontoglossum Ringspot Viruses Co-infection in Phalaenopsis equestris

  • 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等特征。  结论  CymMV和ORSV两种病毒在蝴蝶兰植株中的存在复合侵染;CymMV和ORSV viRNAs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,病毒复合侵染鉴定及其病毒来源vsiRNAs特征的分析加深了病毒与蝴蝶兰之间互作的理解,对于开发蝴蝶兰病毒性病害的防治措施具有重要的理论意义。
  • 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: 21 nt vsiRNAs 5'或3'端的碱基overhang;D: Total vsiRNAs和21 nt vsiRNAs 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 (blue line) and viruses-free (red line) 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 (red line) and ORSV (black line) genome in P. equestris plants; D: relative frequency of 5′-terminal nucleotide of vsiRNAs from CymMV and ORSV genome in P. equestris plants.

    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.

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  • 收稿日期:  2024-04-17
  • 修回日期:  2024-05-23
  • 网络出版日期:  2024-11-11

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