丹参转录因子SmMYC2转录自激活区域鉴定及互作蛋白的筛选  

Identification of Transcription Activity Region and Screening of Interaction Proteins of SmMYC2 Transcription Factor in Salvia miltiorrhiza

作　　者：王怀琴[1,2] 李莎莎 曹晓燕 WANG Huai-Qin;LI Sha-Sha;CAO Xiao-Yan(Key Laboratory of the Ministry of Education for Medicinal Resources and Natural Pharmaceutical Chemistry/National Engineering Laboratory for Resource Development of Endangered Crude Drugs in Northwest of China,Shaanxi Normal University,Xi＇an 710062,China;Jining Middle School Attached JiNing Normal University,Jining 012000,China)

机构地区：[1]陕西师范大学,药用资源与天然药物化学教育部重点实验室/西北濒危药材资源开发国家工程实验室,西安710062 [2]集宁师范学院附属实验中学,集宁012000

出　　处：《农业生物技术学报》2018年第10期1670-1677,共8页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31670299); 陕西省社发类重点项目(No.2017ZDXM-SF-005); 中央高校基本科研业务费专项资金(No.GK201706004)

摘　　要：MYC2(myelocytomatosis protein2)是茉莉酸信号通路上的核心转录因子,参与植物防御反应、次生代谢调控及生长发育等多个方面。筛选丹参(Salvia miltiorrhiza)转录因子SmMYC2的互作蛋白,有助于深入研究丹参SmMYC2的生物学功能。克隆丹参中SmMYC2基因并构建到pGBKT7-GW(BD)上获得BD-SmMYC2诱饵表达载体,检测BD-SmMYC2对酵母(Saccharomyces cerevisiae)AH109细胞的毒性以及其自激活特性。结果表明,SmMYC2具有强烈的自激活活性。为进一步确定自激活区域,分别克隆SmMYC2的N端区域(含bHLH-MYC-N superfamily结构域)和C端区域(含HLH结构域),并将其构建到酵母表达载体BD上,检测SmMYC2-N-BD和SmMYC2-C-BD的转录激活特性,结果显示,含SmMYC2-NBD的酵母细胞在SD/-Trp/X-α-gal培养基上变蓝,并且在SD-Trp-His、SD-Trp-Ade培养基上均有不同程度的生长,而含SmMYC2-C-BD的酵母细胞在SD/-Trp/X-α-gal培养基上无变蓝现象,也不能在SD-Trp-His、SD-Trp-Ade培养基上生长,表明SmMYC2的N端有强烈的自激活活性,而C端没有。以SmMYC2-C-BD为诱饵筛选构建到AD载体的丹参cDNA文库,利用酵母双杂交技术,获得了与逆境胁迫相关的含WRKYGQK典型结构域的WRKY转录因子、过氧化物还原酶(peroxiredoxins, Prxs)家族蛋白以及琥珀酸半醛脱氢酶。本实验结果为进一步开展MYC2的互作蛋白研究提供了基础数据。Myelocytomatosis protein2（MYC2） is the core transcription factor of jasmonic acid signaling pathway and involves in plant defense response, secondary metabolism regulation, growth and development.Screening interaction proteins of Salvia miltiorrhiza transcription factor SmMYC2 is helpful to further study the biological function of SmMYC2. The MYC2 gene in S. miltiorrhiza was cloned and constructed into pGBKT7-GW（BD） to obtain the yeast（Saccharomyces cerevisiae） bait expression vector of BD-SmMYC2. The cytotoxicity and self-activation of BD-SmMYC2 were detected. The results showed that SmMYC2 has strong self-activation activity. In order to analyze the self-activation region, the N-terminal region（including the bHLH-MYC-N superfamily domain） and the C-terminal region（including the HLH domain） of SmMYC2 were cloned and constructed into the yeast expression vector BD. The transcription activity domain of the SmMYC2 was analyzed through detecting the transcription activity of the N-terminus and C-terminus of the SmMYC2. The SmMYC2-N-BD yeast turned blue on SD/-Trp/X-α-gal medium and could grow on SD-Trp-His and SD-Trp-Ade mediums, However, the SmMYC2-C-BD yeast was colorless on SD/-Trp/X-α-gal medium and no growth on SD-Trp-His and SD-Trp-Ade medium. These results indicated that the N-terminus of SmMYC2 has obvious self-activation activity and the C-terminus of SmMYC2 had no self-activation activity.The interactive proteins were screened from cDNA library of S. miltiorrhiza using yeast two-hybrid system through the bait vector of SmMYC2-C-BD. Function annotation showed that these candidate interaction proteins included WRKY protein containing the WRKYGQK domain, the peroxiredoxin reductase of Prxs protein family and succinate semialdehyde dehydrogenase. The experimental results provide basic date for further studying on the interaction proteins of MYC2.

关 键 词：丹参 转录因子SmMYC2 自激活活性 酵母双杂交 互作蛋白 

分 类 号：S184[农业科学—农业基础科学]