中国农业科学Issue(23)：4724-4732,9.DOI:10.3864/j.issn.0578-1752.2014.23.020
拟南芥转录因子AtMYB73转录活性区域分析及互作蛋白的筛选
Regional Analysis of Transcription Activity and Screening of Interaction Proteins of AtMYB73 Transcription Factor in Arabidopsis
摘要
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.关键词
拟南芥/转录因子AtMYB73/转录活性/酵母双杂交/互作蛋白Key words
Arabidopsis thaliana/transcription factor AtMYB73/transcription activity/yeast two-hybrid/interaction protein引用本文复制引用
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,(23):4724-4732,9.基金项目
国家自然科学基金（31200203）、河北省自然科学基金（C2012204032）、高等学校博士学科点专项科研基金 （）
