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浙江大学学报（农业与生命科学版）
浙江大学学报(农业与生命科学版)  2013, Vol. 39 Issue (1): 1-    DOI: 10.3785/j.issn.1008-9209.2012.05.101
生物科学与技术     
拟南芥AGO2的亚细胞定位分析
赵丽,  夏文强,  蔡新忠*
（浙江大学 农业与生物技术学院 生物技术研究所，杭州310058）
Identification of subcellular localization of Arabidopsis AGO2
ZHAO Li，  XIA Wenqiang，  CAI Xinzhong*
(Institute of Biotechnology, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058， China
)
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摘要： Argonaute (AGO)是一类高度保守的蛋白，对基因沉默和植物抗病性起重要调控作用。为进一步了解AGO作用机理，今采用生物信息学技术对拟南芥AGO蛋白各成员的细胞定位进行预测，并对AtAGO2进行了亚细胞定位分析。10个拟南芥AGO蛋白均不含信号肽序列，表明它们不是外泌蛋白。AtAGO1，AtAGO2，AtAGO3，AtAGO4和AtAGO9带有核定位信号，但携带的核定位信号肽序列各不相同，显示这些AGO蛋白可能定位于细胞核。通过克隆AtAGO2全长序列，构建获得与报告基因eGFP融合表达的细胞定位检测载体pCHF3eGFP∷AtAGO2，采用农杆菌介导的方法将其瞬时表达于本氏烟（Nicotiana benthamiana）叶片中，激光共聚焦显微镜观察结果表明，AtAGO2蛋白定位于细胞质和细胞核中。茉莉酸（jasmonic acid, JA）处理诱导AtAGO2蛋白在细胞质膜及其周围 “颗粒化”聚集，但抗病诱导剂苯并噻二唑（benzothiodiazole， BTH）处理不影响AtAGO2蛋白的亚细胞定位。这些结果说明AtAGO2可能通过JA途径参与植物抗病性的调控。
Abstract: Argonaute  （AGO） protein is a highlyconserved family with many members, containing three conserved domains, PAZ，MID and PIWI. Arabidopsis AGO comprises 10 proteins named from AtAGO1 to AtAGO10, which are grouped into three classes with AtAGO1, AtAGO5 and AtAGO10; AtAGO2, AtAGO3 and AtAGO7; and AtAGO4, AtAGO6, AtAGO8 and AtAGO9 as one class respectively. Some members of AGO family proteins have been found to bind small RNAs, which are generated through the function of DCLs, to form a RNAinduced silencing complex (RISC), leading to RNA silencing at either transcriptional or posttranscriptional level, thus results in alternation in development, abiotic and biotic stress resistance. However, the detailed molecular mechanism of AGO function is still largely unknown. To provide some insights into the mechanism of Arabidopsis AGO function, subcellular localization of all Arabidopsis AGO members
was  predicted using bioinformatics methods, and that of one member, AtAGO2, was further experimentally identified using a transient GFP reporting system in this study. Finally,  effect of jasmonic acid (JA) and benzothiodiazole （BTH）, two wellknown disease resistance regulatory molecules, on subcellular localization of AtAGO2 was examined in order to obtain a hint for possible role of this Arabidopsis AGO in regulation of plant disease resistance. Bioinformatics analyses using Signal P3.0 by algorithm of both Neural networks and Hidden Markov models revealed that all ten AtAGOs did not carry signal peptide, indicating that they were not secretory proteins. Analysis using PredictProtein software showed that AtAGO1, AtAGO2, AtAGO3, AtAGO4 and AtAGO9 contained nuclear localization signals (NLS), although differing in sequence, demonstrating that they might be localized in cell nucleus. To experimentally confirm the bioinformatics prediction, the fulllength cDNA sequence of AtAGO2 was cloned and the expression vector pCHF3eGFP∷AtAGO2 in which AtAGO2 was fused with eGFP reporter gene was constructed to identify the subcellular localization of AtAGO2. AtAGO2 was transiently expressed by Agrobacteriummediated method in leaves of Nicotiana benthamiana. Results of detection of green fluorescence signal by confocal laserscanning microscope showed that AtAGO2 was localized in cytoplasm, plasma membrane and nucleus. Comparative analysis revealed that treatment with 0.2 mmol/L JA, but not 0.38 mmol/L BTH, rendered AtAGO2 was distributed  discontinuously in plasma membrane and gathered  as bright “granules” in and around plasma membrane. Data in this study revealed that Arabidopsis AGOs were not secretory proteins; AtAGO1, AtAGO3, AtAGO4 and AtAGO9 might be localized in cell nucleus, while  AtAGO2 was localized in cytoplasm, plasma membrane and nucleus. Treatment with JA, but not BTH, altered localization of AtAGO2 in plasma membrane and cytoplasm, indicating that AtAGO2 might play a role in regulating disease resistance in JA pathway.
出版日期: 2013-01-20
CLC:  Q 945  
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赵丽
夏文强
蔡新忠*

引用本文:

赵丽, 夏文强, 蔡新忠*. 拟南芥AGO2的亚细胞定位分析[J]. 浙江大学学报(农业与生命科学版), 2013, 39(1): 1-.

ZHAO Li， XIA Wenqiang， CAI Xinzhong*. Identification of subcellular localization of Arabidopsis AGO2. Journal of Zhejiang University (Agriculture and Life Sciences), 2013, 39(1): 1-.

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http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2012.05.101    或    http://www.zjujournals.com/agr/CN/Y2013/V39/I1/1

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