Expression of Pns10 Protein of Rice ragged stunt virus in Rice Protoplasts

doi:10.16819/j.1001-7216.2017.6148 232

Abstract

Abstract:

【Objective】Pns10 of Rice ragged stunt virus (RRSV) forms viroplasm-like inclusion bodies which are essential for virus infection to vector insects. However, the expression pattern and function of Pns10 in rice remain unknown.【Method】Polyclonal antibody against Pns10 protein was prepared by immunizing rabbits with a bacterially expressed Pns10; the cellular distribution of Pns10 protein in rice protoplasts was observed with an immunofluorescence microscopy; the accumulation of Pns10 RNA in rice protoplasts infected by RRSV was quantified with real-time quantitative RT-PCR and that of the Pns10 protein was investigated using Western blot.【Result】Pns10 ORF was cloned into the Gateway prokaryotic expression vector pDEST17 and its expression was induced by IPTG. Polyclonal antiserum against the Pns10 was obtained and it can react with the Pns10 protein from the naturally RRSV-infected rice by Western blot detecting. Pns10 formed viroplasm-like inclusion bodies in protoplasts. The Pns10 RNA was first detected at 8 h post RRSV inoculation. The accumulation of Pns10 RNA peaked at 24 h post virus inoculation but began to decline thereafter. The Pns10 protein was first detected at 24 h post virus inoculation. A high level of Pns10 protein was maintained until 60 h post virus inoculation.【Conclusion】Antiserum against RRSV Pns10 protein was obtained. Pns10 was expressed and viroplasm-like inclusion bodies formed in rice protoplasts. In addition, the expression of Pns10 RNA was earlier than that of the Pns10 protein.

Key words: Rice ragged stunt virus, rice protoplast, Pns10 antibody, Pns10 RNA, Pns10 protein

摘要：

【目的】水稻齿叶矮缩病毒(Rice ragged stunt virus, RRSV) Pns10蛋白在介体昆虫细胞内可形成类似病毒原质(viroplasm)的内含体,是RRSV侵染介体所必需。然而Pns10蛋白在水稻寄主中是否具有类似功能及其表达情况如何未见报道。【方法】利用大肠杆菌系统表达Pns10蛋白,免疫家兔制备多克隆抗体;通过水稻原生质体病毒侵染体系,利用免疫荧光技术分析Pns10蛋白在水稻原生质体内的分布情况,利用实时定量PCR技术和Western blot技术分别检测Pns10 RNA和Pns10蛋白在水稻原生质体内的积累情况。【结果】将Pns10基因克隆到Gateway系统原核表达载体pDEST17中,IPTG诱导表达成功后,制备融合蛋白抗血清。Western blot检测显示,该抗血清可检测感病水稻叶片中的Pns10蛋白。病毒侵染水稻原生质体后, Pns10蛋白可形成类似病毒原质的内含体;Pns10 RNA在病毒接种8 h后开始积累,24 h后达到最大值,随后开始下降;Pns10蛋白在24 h后开始表达,之后维持较高水平,60 h后略有下降。【结论】成功获得了Pns10抗血清;Pns10在水稻原生质体内成功表达,可形成类似病毒原质的内含体,并且Pns10 RNA的表达先于其蛋白的表达。

关键词: 水稻齿叶矮缩病毒, 水稻原生质体, Pns10抗体, Pns10 RNA, Pns10蛋白

CLC Number:

Jie ZHANG, Xiaomin CHEN, Jinhong WU, Chongqing ZHU, Xinlun DING, Zujian WU. Expression of Pns10 Protein of Rice ragged stunt virus in Rice Protoplasts[J]. Chinese Journal OF Rice Science, 2017, 31(3): 232-237.

张洁, 陈晓敏, 吴锦鸿, 朱重庆, 丁新伦, 吴祖建. 水稻齿叶矮缩病毒Pns10蛋白在水稻原生质体内的表达[J]. 中国水稻科学, 2017, 31(3): 232-237.

Figures/Tables 6

Fig. 1. PCR analysis of the bacterium harboring the pDEST17-Pns10 expression vector. M, DNA Marker Ⅲ; Lanes 1 to 3, Pns10 gene.

Fig. 2. SDS-PAGE analysis of the expression of Pns10 protein in Escherichia coli Rosetta strain. M, Protein Marker; 1, Pns10 protein induced by IPTG; 2, Pns10 protein without IPTG induction.

Fig. 3. Analysis of the antiserum against Pns10 by Western blot. M, Protein marker; 1, RRSV infected rice plants; 2, Uninfected rice plants.

Fig. 4. Analyzing the Pns10 expression in rice protoplasts 48 h post virus inoculation by confocal microscope.

Fig. 5. Analyzing the RNA expression of Pns10 gene in rice protoplasts at different time post virus inoculation by real-time quantitative RT-PCR.

Fig. 6. Analyzing the expression of Pns10 protein in rice protoplasts at different time post virus inoculation by Western blot.

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