核糖体失活蛋白（α-MC）亚细胞定位及对TMV的抑制作用

doi:10.3864/j.issn.0578-1752.2017.05.007

摘要/Abstract

摘要： 【目的】克隆获得苦瓜α-MC、商陆PAP，在烟草中异源表达观察两个蛋白在细胞中的定位情况。研究α-MC对烟草花叶病毒（Tobacco mosaic virus，TMV）的抑制作用及其引起的抗性防御反应。【方法】根据已报道的商陆抗病毒蛋白PAP基因全序列和苦瓜素基因全序列，设计并合成扩增PAP和α-MC基因全长引物，通过RT-PCR及基因克隆方法，从苦瓜和商陆春叶克隆得到苦瓜α-MC、商陆PAP；用WolfPSORT预测蛋白定位，将苦瓜α-MC、商陆PAP分别融合在GFP和DsRed2的N端，构建融合蛋白表达载体，采用GFP和DsRed2标记进行亚细胞定位，验证预测结果；通过农杆菌介导在本氏烟中瞬时表达α-MC，再接种烟草花叶病毒，利用酶联免疫吸附试验（ELISA）和实时荧光定量PCR(qRT-PCR)分别检测病毒在接种叶片的蛋白积累量和RNA表达量，分析瞬时表达α-MC的抗病毒效果。利用qRT-PCR分析植物防卫相关基因NPR1、PR1、PR2的表达，探究其抗病毒机理。【结果】克隆得到基因α-MC和PAP全长，分别为861和939 bp。Wolf PSORT预测显示α-MC和PAP主要定位于细胞质膜上。共聚焦荧光显微镜下观察发现，分别用GFP和DsRed2标记的α-MC和PAP均定位在本氏烟叶片表皮细胞质膜上，与Wolf PSORT预测的α-MC和PAP定位结果相一致。异源表达的PAP对植物细胞毒性作用强，导致表达部位细胞坏死，异源表达α-MC的植物细胞无明显毒性，表达部位细胞完整。在本氏烟中异源表达α-MC后，再接种TMV-GFP，在紫外灯下观察发现α-MC处理后的本氏烟在接种TMV-GFP 48 h后没有出现绿色荧光，而对照组出现荧光。72 h后处理组出现零星荧光，但对照组的绿色荧光开始扩散，连续观察，处理组几乎没有变化，接种TMV-GFP 6 d后，发现处理组的绿色荧光几乎没有扩大的趋势，而对照组的绿色荧光已扩散至心叶；ELISA检测表明，在接种TMV-GFP 6 d后的叶片中，对照组与健康植物的OD₄₉₂比值几乎已达到处理组的10倍以上；qRT-PCR检测TMV RNA的含量，结果显示对照组TMV RNA表达量是处理组的149倍左右，表明α-MC对TMV复制和移动均有明显抑制；qRT-PCR结果分析显示，NPR1在只注射TMV、单独表达α-MC以及表达α-MC后注射TMV的本氏烟中均被诱导表达，但后者的表达量是前两个处理的约2.5倍左右，在只接种α-MC和表达α-MC后注射TMV的本氏烟中均检测到PR1、PR2，但后者的表达量显著高出前者5—7倍，表明异源表达α-MC可诱导植物中防卫相关基因NPR1、PR1、PR2的表达，从而引起更强的防御反应。【结论】异源表达α-MC显著抑制TMV，能够激活植物防卫反应，且对植物细胞无明显毒性。研究结果为利用异源表达α-MC方法开发控制植物病毒新产品提供了参考依据。

关键词: 核糖体失活蛋白；&alpha, -MC；烟草花叶病毒；异源表达；亚细胞定位；防卫相关基因

Abstract: 【Objective】The objective of this study is to obtain the alpha-momorcharin (α-MC) and pokeweed antiviral protein (PAP) by cloning, observe the cellular localization of which in Nicotiana benthamiana by heterologous expression, and to evaluate the effects of α-MC on inhibiting Tobacco mosaic virus (TMV) and the resistance defense response of N. benthamiana.【Method】Based on the published sequences of α-MC and PAP, primer pairs for cloning PAP and α-MC were designed. RT-PCR and gene cloning were used to obtain the target genes α-MC and PAP from the leaves of Momordica charantia and Phytolacca acinosa, respectively. First, subcellular localization of the α-MC and PAP was predicted through Wolf PSORT, then the fusion protein vectors for verifying the subcellular localization were constructed by fusing the α-MC and PAP to the N-terminal of the GFP and DsRed2, respectively. The α-MC was transiently expressed in the N. benthamiana leaves by agro-infiltration, and the α-MC expressed leaves were inoculated with TMV-GFP. The accumulation of the virions and viral RNA were detected by indirect ELISA and real-time quantitative PCR (qRT-PCR). In order to understand the antiviral mechanism of the α-MC, the expression of plant defense-related genes including non-expressor of pathogenesis-related genes (NPR1, PR1, PR2) were evaluated by qRT-PCR. 【Result】 The length of genes α-MC and PAP obtained by RT-PCR were 861 and 939 bp, respectively. The subcellular localization showed that the coding proteins of α-MC and PAP were predicted by Wolf PSORT to distribute on the plasma membrane. Under the confocal laser scanning microscope, the fusion proteins α-MC-GFP and PAP-DsRed2 also distributed on the plasma membrane of the leaf epidermis cell of N. benthamiana, which is consistent with the predicted results. It was observed that the heterologously expressed PAP produced strong toxic effects on tobacco leaf cells, which led to necrosis of the cells, while the heterologously expressed α-MC showed no obvious toxic effect on tobacco leaf cells, which were kept intact. Additionally, following the heterologous expression of α-MC in N. benthamiana, TMV-GFP was inoculated. After 48 hours, there was no green fluorescence observed in the α-MC expressed leaves under UV light, but the green fluorescence could be observed in the control group. After 72 hours, sporadic green fluorescence was observed in the treatment group and the fluorescence in the control group started to spread. After 6 days, the green fluorescence spread to the spear leaf of the control group, while no obvious change was found in that of the treatment group. ELISA assay results showed that after 6 days of TMV-GFP inoculation, the value of OD₄₉₂ in the control group was over 10 times more than the value of samples under treatment. qRT-PCR data showed that the expression level of TMV in control group was 149 times than that of the level in group after treatment. Those data indicate that α-MC has a significant impact on both replication and movement of TMV. The expression levels of several defense-related genes in N. benthamiana leaves expressing α-MC with or without TMV were tested through qRT-PCR. Data showed that NPR1 was induced in both cases while the expression level was 2.5 times in plant with TMV injection than the one without injection. As to PR1 and PR2, the expression of these two genes was 5-7 times higher in plants with TMV injection. Combining all those data together, it was suggested that the resistance effect of heterologously expressed α-MC on plant viruses could induce the expression of responsive defense-related genes including NPR1, PR1 and PR2, resulting in much stronger plant defense response. 【Conclusion】 The heterologously expressed α-MC significantly inhibited TMV, activated the plant defense response, enhanced the defense response of N. benthamiana, and produced few toxicity to the plant cells. Therefore, the results will provide a reference for the development of new products for the control of plant viruses based on heterologous expression of α-MC.

Key words: ribosome-inactivating proteins, α-momorcharin, Tobacco mosaic virus (TMV), heterologous expression, subcellular localization, defense-related genes

魏周玲，彭浩然，潘琪，张永至，蒲运丹，吴根土，青玲，孙现超. 核糖体失活蛋白（α-MC）亚细胞定位及对TMV的抑制作用[J]. 中国农业科学, 2017, 50(5): 840-848.

WEI ZhouLing, PENG HaoRan, PAN Qi, ZHANG YongZhi, PU YunDan, WU GenTu, QING Ling, SUN XianChao. Subcellular Localization of the Ribosome-Inactivating Protein α-MC and Its Antiviral Effect on TMV[J]. Scientia Agricultura Sinica, 2017, 50(5): 840-848.

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