拟南芥转录因子AtMYB73转录活性区域分析及互作蛋白的筛选

doi:10.3864/j.issn.0578-1752.2014.23.020

摘要/Abstract

摘要： 【目的】确定拟南芥抗逆转录因子AtMYB73的转录活性区域并筛选获得与其互作的蛋白，为进一步阐明AtMYB73调控拟南芥抗逆的分子机制奠定基础。【方法】构建转录因子AtMYB73的酵母诱饵表达载体pAS1-AtMYB73，检测pAS1-AtMYB73的自激活性及其对酵母Y190的细胞毒性。克隆AtMYB73的N端区域（含有R2R3结构域）和C端区域（不含R2R3结构域），检测AtMYB73的N端和C端的转录激活活性，分析AtMYB73自激活结构域的位置。利用酵母双杂交技术，以AtMYB73为诱饵筛选拟南芥的cDNA文库，将阳性克隆进行鉴定、测序；利用TAIR数据库对筛选获得的AtMYB73的候选互作蛋白进行分析。构建AtMYB73和F12F1.4的酵母双杂交载体pGBDT7-AtMYB73和pGADT7-F12F1.4，通过酵母双杂交技术，对其互作关系进行分析。【结果】含pAS1-MYB73的酵母菌在不同浓度的3-AT的SD/-His/-Trp/、SD/-Ade/-Trp培养基上均能生长，表明AtMYB73具有较高的自激活活性。含pAS1-AtMYB73的酵母菌在SD/-Trp/Amp液体培养基中培养24 h的OD₆₀₀值大于0.8，表明诱饵载体对酵母Y190没有细胞毒性。成功构建了pAS1-AtMYB73-N和pAS1-AtMYB73-C载体，获得了含有pAS1-AtMYB73-N和pAS1-AtMYB73-C的酵母；含pAS1-AtMYB73-N的酵母在含X-a-gal的YPAD培养基上呈现无色，而含pAS1-AtMYB73-C的酵母则呈现蓝色，表明AtMYB73的C端有明显的自激活性，而N端没有自激活性。以AtMYB73为诱饵，筛选拟南芥的cDNA文库，获得了与光合作用、防御反应途径、抗逆等相关的8个蛋白。利用酵母双杂交技术，确定了MYB73与F12F1.4之间存在一定的互作。【结论】拟南芥抗逆转录因子AtMYB73具有较高的转录激活活性，其活性区域位于C端；以AtMYB73为诱饵，筛选获得了与光合作用、防御反应途径、抗逆等相关的8个AtMYB73的候选互作蛋白；利用酵母双杂交技术，确定了AtMYB73与F12F1.4之间的互作关系。

关键词: 拟南芥, 转录因子AtMYB73, 转录活性, 酵母双杂交, 互作蛋白

Abstract: 【Objective】 The objective of this study is to analyze the transcription activity region and screen interaction proteins of Arabidopsis resistance related transcription factor AtMYB73 and the study will lay a foundation for clarifying the regulation mechanism of the AtMYB73 gene in Arabidopsis resistance in the future. 【Method】 The bait vector pAS1-AtMYB73 was constructed and introduced into yeast Y190 by PEG/LiAC mediated transformation method. The self-activation and cytotoxicity of pAS1-AtMYB73 were detected in this paper. The transcription activity domain of the AtMYB73 was analyzed through detecting the transcription activity of the N-terminus and C-terminus of the AtMYB73. The AtMYB73 was used as bait to screen Arabidopsis cDNA library by the yeast two-hybrid system. The positive clones were screened on SD/-Ade/-His/-Leu/-Trp plates, identified by PCR and sequenced, and function of the interaction proteins were analyzed using TAIR database. The pGBDT7-AtMYB73 and pGADT7-F12F1.4 vectors were constructed and co-transformed into yeast AH109. The interaction relationship between AtMYB73 and F12F1.4 was analyzed by yeast two-hybrid system.【Result】The bait vector of the AtMYB73, pAS1-AtMYB73, was successfully constructed and transformed into yeast Y190. The pAS1-AtMYB73 yeast could grow on SD/-His/-Trp/ and SD/-Ade/-Trp plates with different concentrations of 3-AT, suggesting that AtMYB73 has higher self-activation activity. The OD600 of the pAS1-AtMYB73 yeast cultured for 24 h in SD/-Trp/Amp liquid media was greater than 0.8, showing that the bait vector has no cytotoxicity. Vectors of pAS1-AtMYB73-N and pAS1-AtMYB73-C were successfully constructed and transformed into yeast Y190, respectively. The pAS1-AtMYB73-N yeast was colorless, but the pAS1-AtMYB73-C yeast was blue. These results indicated that the C-terminus of AtMYB73 has obvious self-activation activity and the N-terminus of AtMYB73 has no self-activation activity. Eight candidate interacting proteins of AtMYB73 were obtained by screening Arabidopsis cDNA library using yeast two-hybrid system. Function annotation showed that these candidate interacting proteins were related to photosynthesis, defense reaction and resistance. The interaction relationship between AtMYB73 and F12F1.4 was determined by yeast two hybrid system. 【Conclusion】 Transcription factor AtMYB73 has higher self-activation activity and the transcription activity region of AtMYB73 was localized in its C-terminus. Eight interaction proteins related to photosynthesis, defense reaction and resistance were obtained by screening Arabidopsis cDNA library. Transcription factor AtMYB73 interacting with F12F1.4 was determined by yeast two hybrid system.

Key words: Arabidopsis thaliana, transcription factor AtMYB73, transcription activity, yeast two-hybrid, interaction protein

樊锦涛，贾娇，蒋琛茜，王冠宇，张靖，邢继红，董金皋. 拟南芥转录因子AtMYB73转录活性区域分析及互作蛋白的筛选[J]. 中国农业科学, 2014, 47(23): 4754-4762.

FAN Jin-tao, JIA Jiao, JIANG Chen-xi, WANG Guan-yu, ZHANG Jing, XING Ji-hong, DONG Jin-gao. Regional Analysis of Transcription Activity and Screening of Interaction Proteins of AtMYB73 Transcription Factor in Arabidopsis[J]. Scientia Agricultura Sinica, 2014, 47(23): 4754-4762.

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