Construction of stable infectious full-length and eGFP-tagged cDNA clones of Mirabilis crinkle mosaic virus via In-Fusion cloning
Construction of stable infectious full-length and eGFP-tagged cDNA clones of Mirabilis crinkle mosaic virus via In-Fusion cloning
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通过 In-Fusion 克隆构建稳定的感染性全长和 eGFP 标记的紫茉莉皱纹花叶病毒 cDNA 克隆
DOI：
10.1016/j.virusres.2020.198039
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发表时间：
2020-09-01
期刊：
影响因子：
5
通讯作者：
Wang, Jianguang
中科院分区：
文献类型：
--
作者：
Li, Xiaoqin;Li, Yu;Wang, Jianguang
2019年，紫茉莉皱叶花叶病毒（MiCMV）被初步归类为马铃薯Y病毒科马铃薯Y病毒属新成员。然而，根据外壳蛋白（CP）区域的序列相似性，认为它是basella rugose mosaic virus。在这项研究中，感染性的MiCMV的cDNA克隆的控制下的35 S启动子构建与In-Fusion克隆方法。用pMiCMV和pMiCMV-NIb/eGFP接种的紫茉莉和本氏烟草植物的系统感染的叶片在5dpi时具有花叶症状。通过免疫印迹分析、电子显微镜、RT-PCR和接种N.本塞姆亚那幼苗与后代病毒体。在烟草叶类似地接种后未观察到系统感染，仅在接种的叶中检测到eGFP荧光。有趣的是，pMiCMV和pMiCMV-NIb/eGFP诱导的症状与野生型MiCMV在落葵植物中引起的症状不相似。此外，还对B.用病毒特异性引物（MicpF和MicpR）对红花植物进行PCR扩增，结果表明扩增出了与MiCMV CP编码区对应的非靶片段。这是第一次报告的建设一个生物活性，全长cDNA拷贝的MiCMV RNA基因组。
Mirabilis crinkle mosaic virus (MiCMV) was tentatively classified as a new member of the genus Potyvirus in the family Potyviridae in 2019. However, it was considered to be basella rugose mosaic virus based on the sequence similarity of the coat protein (CP) region. In this study, infectious MiCMV cDNA clones under the control of the 35S promoter were constructed with an In-Fusion cloning method. Systemically infected leaves of Mirabilis jalapa and Nicotiana benthamiana plants inoculated with pMiCMV and pMiCMV-NIb/eGFP had mosaic symptoms by 5 dpi. Infections were confirmed by a western blot analysis, electron microscopy, RT-PCR, and the inoculation of N. benthamiana seedlings with progeny virions. Systemic infections were not observed after Nicotiana glutinosa leaves were similarly inoculated, with eGFP fluorescence detected only in the inoculated leaves. Interestingly, the symptoms induced by pMiCMV and pMiCMV-NIb/eGFP were not similar to those caused by the wild-type MiCMV in Basella rubra plants. Moreover, RT-PCR analyses of B. rubra plants with virus-specific primers (MicpF and MicpR) indicated that a non-target fragment corresponding to the MiCMV CP coding region was amplified. This is the first report of the construction of a biologically active, full-length cDNA copy of the MiCMV RNA genome.