利用酵母双杂交筛选与丹参R2R3-MYB类转录因子SmPAP1互作的蛋白

Screening of the Proteins Interacting with SmPAP1 of R2R3-MYB Transcription Factor from Salvia miltiorrhiza Bunge by Yeast Two-Hybrid System

根据NCBI数据库公布的R2R3-MYB类转录因子Sm PAP1基因序列(Gen Bank登录号GU218694.2),构建丹参Sm PAP1的酵母双杂交诱饵表达载体p GBKT7-Sm PAP1,检测p GBKT7-Sm PAP1的自激活活性及其对酵母AH109细胞的毒性。以Sm PAP1为诱饵筛选构建到p GADK7中的丹参c DNA文库,挑取阳性克隆进行测序,并在NCBI数据库中进行BLAST比对分析。利用Uniprot在线网站,对筛选到的60个基因进行了功能注释。对其中4个候选互作蛋白,包括病程相关类蛋白AP2/ERF,氧化还原酶类蛋白Sm C4H,逆境胁迫相关类蛋白Sm WRKY及b HLH转录因子类蛋白Sm MYC2进行回转验证实验。通过酵母双杂交实验,确定Sm PAP1与AP2/ERF类、Sm C4H、Sm WRKY及Sm MYC2相互作用。这些数据为深入探究Sm PAP1调控丹参中酚酸类化合物合成的分子机理提供了参考。

The gene of SmPA P1, which belongs to R2R3-MYB transcription factor family, was cloned according to the published sequence （GenBank Accession No. GU218694.2）. The bait vector ofpGBKT7-SmPA P1 was con- structed. The self-activation and toxicity of pGBKT7-SmPA P1 were detected in this study. The interactive genes were screened from eDNA library of Salvia miltiorrhiza through the bait vector of pGBKT7-SmPA P1 and positive clones were sequenced and analyzed by BLAST alignment in the NCBI database. Using Uniprot online web site, sixty genes were annotated. Four candidate interacting protein were conducted verification experiments, including pathogenesis-related proteins （AP2/ERF）, oxidoreductases protein （SmC4H）, stress associated protein-like protein （SmWRKY） and transcription factor （SmMYC2）. The interaction relationship between SmPAP1 and AP2/ERF, SmC4H, SmWRKY and SmMYC2 was respectively determined by yeast two hybrid system. These date provides reference for further investigating the molecular mechanism of SmPAP1 in regulating the phenolic acid biosynthetic pathway of S. mihiorrhiza,