水稻矮缩病毒Pns6和P8蛋白在病毒侵染水稻原生质体后的表达

doi:10.16819/j.1001-7216.2019.8109

摘要/Abstract

摘要：

【目的】为明确水稻矮缩病毒(Rice dwarf virus, RDV) Pns6和P8蛋白在病毒侵染水稻原生质体后的表达动态,【方法】利用PEG介导的病毒侵染原生质体体系,通过免疫荧光和电镜技术分析Pns6、P8蛋白以及病毒粒体在水稻原生质体中的定位;同时,通过Western blot和实时荧光定量PCR分析Pns6和P8蛋白及其RNA在水稻原生质体中的积累量。【结果】病毒接种水稻原生质体48 h后,Pns6蛋白在细胞质中可以形成类似于病毒原质(viroplasm)的点状内含体,P8蛋白也大量的表达。同时,病毒粒体在水稻原生质体中也形成内含体状的结构。病毒接种水稻原生质体12 h后,均可检测到Pns6和P8蛋白的表达,并且在36 h后达到最高值。病毒接种水稻原生质体后,Pns6 RNA在24 h时表达量达到最高,P8 RNA在36 h时表达量达到最高。【结论】RDV侵染水稻原生质体后,Pns6和P8蛋白均有表达,并且病毒也可能通过形成病毒原质来完成病毒在寄主细胞的复制和装配。

关键词: 水稻矮缩病毒, 水稻原生质体, Pns6蛋白, P8蛋白

Abstract:

【Objective】In order to clarify expression dynamics of Pns6 and P8 proteins of Rice dwarf virus after virus infecting rice protoplasts, 【Method】subcellular localization of Pns6 and P8 proteins as well as viral particles in rice protoplasts were conducted with a PEG mediated virus infecting protoplast system by immunofluorescence and electron microscope. Meanwhile, accumulation of Pns6 and P8 proteins and their RNAs in rice protoplasts were measured by Western blot and real-time fluorescence quantitative PCR. 【Result】Pns6 protein could form viroplasm-like punctate inclusion bodies in cytoplasm 48 h after virus inoculation, and P8 protein was also expressed in large quantities. Meanwhile, viral particles could form inclusion body-like structure in rice protoplasts. Expression of Pns6 and P8 proteins was both detected at 12 h after virus inoculation, and reached the maximum level after 36 h. At the same time, the expression level of Pns6 RNA peaked at 24 h and that of P8 RNA at 36 h after virus inoculation. 【Conclusion】Pns6 and P8 proteins were both expressed after virus infection, and Rice dwarf virus may also formed viroplasm-like inclusion bodies to complete virus replication and assembly in host cells.

Key words: Rice dwarf virus, rice protoplast, Pns6 protein, P8 protein

中图分类号:

张洁, 宛柏杰, 尚鹏祥, 丁新伦, 杜振国, 吴祖建. 水稻矮缩病毒Pns6和P8蛋白在病毒侵染水稻原生质体后的表达[J]. 中国水稻科学, 2019, 33(2): 118-123.

Jie ZHANG, Baijie WAN, Pengxiang SHANG, Xinlun DING, Zhenguo DU, Zujian WU. Expression of Pns6 and P8 Proteins of Rice Dwarf Virus After Virus Infecting Rice Protoplasts[J]. Chinese Journal OF Rice Science, 2019, 33(2): 118-123.

图/表 3

图1 电镜观察水稻矮缩病毒在水稻原生质体中的定位 A-阴性对照;B-激光共聚焦显微镜观察Pns6蛋白在水稻原生质体中的定位;C-激光共聚焦显微镜观察P8蛋白的定位;D-透射电镜观察水稻矮缩病毒的定位,箭头示病毒粒体。

Fig. 1. Subcellular localization of Rice dwarf virus in rice protoplasts under an electron microscope. A, Negative control; B, Subcellular localization of Pns6 with a laser scanning confocal microscopy; C, Subcellular localization of P8 with a laser scanning confocal microscopy; D, Subcellular localization of Rice dwarf virus by transmission electron micrograph observation. Arrows show viral particles in viroplasm-like inclusion bodies.

图2 Western blot检测水稻矮缩病毒(RDV)在侵染水稻原生质体后Pns6和P8蛋白的表达 A-蛋白的Western blot; B-相对蛋白表达量。

Fig. 2. Expression of Pns6 and P8 in rice protoplasts after virus inoculation by Western blot. A, Western blot analysis; B, Relative protein level.

图3 实时定量PCR检测水稻矮缩病毒在侵染水稻原生质体后Pns6和P8 RNA的表达量

Fig. 3. Expression of Pns6 and P8 RNA in rice protoplasts after virus inoculation by real-time quantitative RT-PCR.

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