月季RhNAC31互作蛋白的筛选及分析

Screening and Analysis of Rose(Rosa hybrida) RhNAC31 Interacting Proteins by Yeast Two-Hybrid System

为了进一步研究RhNAC31在胁迫响应中的作用机制,以RhNAC31蛋白为诱饵,利用酵母双杂交系统在切花月季‘萨曼莎’失水胁迫cDNA文库中进行了互作蛋白的筛选和分析。根据RhNAC31的转录激活区域(C端,157~286 aa)的序列特征,将其划分为C1(157~250 aa,RhNAC31-C1)和C2(251~286 aa,RhNAC31-C2)两段,分别构建到pGBKT7上检测其自激活活性及对酵母Y2H Gold细胞的毒性。结果显示,pGBKT7-RhNAC31-C1和pGBKT7-RhNAC31-C2对酵母Y2H Gold细胞均没有毒性,能激发金担子素A(Aureobasidin A,AbA)报告基因的表达,具有自激活活性,在含有400 ng·mL-1 Ab A的SD缺陷型培养基上其活性受到抑制,以含该浓度AbA的培养基作为筛选文库培养基。在此筛选条件下,在酵母双杂交文库中筛选到21个阳性克隆,对其测序并用Blast比对获得了16个与RhNAC31互作的候选蛋白。利用Uniprot网站对互作蛋白进行基因本体学分析,结果显示RhNAC31能够与RZFP34、MIEL1、RUB泛素连接酶E3、SGT1蛋白、DnaJ蛋白等多种蛋白相互作用,可能参与糖脂生物合成、蛋白质泛素化、植物防御、叶绿素生物合成、模式识别受体信号通路、蛋白质折叠、乙烯生物合成、胁迫响应等生物学过程。

To further understand the molecular mechanism of RhNAC31 in stress response,we used the RhNAC31 protein as bait through cut rose’Samantha’water deficit stress yeast two-hybrid(Y2H)cDNA libraries and screened its interacting proteins.RhNAC31 transactivation domain was located in its C-terminal(157–286 aa).Two different deletion regions,including RhNAC31-C1(157–250 aa)and RhNAC31-C2(251–286 aa),were constructed to pGBKT7 vector,respectively.Self-activation activity and toxicity were also examined in Y2H Gold yeast cells.The results showed that both p GBKT7-Rh NAC31-C1 and pGBKT7-RhNAC31-C2 had no toxicity in Y2H Gold cells and could stimulate Aureobasidin A(AbA)reporter gene with auto-activation activity.The activities were obviously inhibited when supplemented with 400 ng?mL-1 AbA under SD deficient medium.The SD deficient medium plus 400 ng·mL-1 AbA was used as Y2H screening library medium.Twenty-one positive colonies were screened by Y2H,and 16 of them may be candidate interacting proteins of RhNAC31 which were further sequenced.In addition,these interacting proteins were also functional associations based on gene ontology through Uniprot website.RhNAC31 may interact with RZFP34,MIEL1,RUB E3 ubiquitin ligase,SGT1,DnaJ and other proteins.These different proteins may participate in glycolipid biosynthesis,protein ubiquitination,plant defense,chlorophyll biosynthesis,pattern recognition receptor signaling pathway,protein folding,ethylene biosynthesis,stress response and other biological processes.