马铃薯StSuSy4基因转录调控因子的筛选及互作验证

Screening and Verification of Transcriptional Regulators Interacted with Potato StSuSy4 Gene

【目的】探究马铃薯块茎高表达基因StSuSy4的转录调控机制。【方法】克隆StSuSy4的启动子,构建pAbAi-StSuSy4pro诱饵载体,并将其转化至酵母细胞Y1HGold中,获得诱饵酵母菌株;利用诱饵载体筛选马铃薯幼嫩块茎组织酵母文库,并对转录因子进行组织表达模式分析。【结果】经酵母单杂交筛选,获得1个候选转录因子StBBX25,该基因的开放阅读框为774 bp,编码258个氨基酸。生物信息分析显示:StBBX25的N端含有B-box蛋白的特征结构域B-box1。候选转录因子与启动子点对点的酵母单杂交验证结果显示:在200 ng/mL金担子素条件下,pGADT7-AD空载对照组酵母不能正常生长,而pGADT7-StBBX25试验组能正常生长,表明StBBX25与StSuSy4基因启动子存在互作。组织表达模式分析显示:StBBX25和StSuSy4均为在马铃薯块茎中表达较高的基因。【结论】StBBX25与StSuSy4启动子互作,暗示StBBX25可能在马铃薯叶片和块茎淀粉合成以及产量形成中发挥作用。研究结果为深入理解马铃薯淀粉合成调控网络提供了重要的理论依据。

[Purpose]To explore the transcriptional regulation mechanism of StSuSy4 gene,which is highly expressed in potato tuber.[Methods]The promoter of StSuSy4 was cloned,the pAbAiStSuSy4pro bait vector was constructed,and then transformed into yeast cell Y1HGold to obtain the bait yeast strain.Yeast one-hybrid(Y1H) screen was carried out based on the yeast library built by potato young tuber tissues.[Results] Y1H screening yielded a potential transcription factor StBBX25.The open reading frame of StBBX25 was 774 bp,and it encoded a protein of 258 amino acids.Bioinformatic analysis showed that its N-terminal contained the characteristic domain B-box1of the B-box proteins.Y1H was conducted with the candidate transcription factor and the protomer,and the results showed that under the condition of 200 ng/mL aureobasidin A,yeast cells in pGADT7-AD control group could not grow,while that in PGADT7-StBBX25 experimental group grew well,showing that StBBX25 and StSuSy4 gene promoter interacted.The expression pattern analysis indicated that both StBBX25 and StSuSy4 were highly expressed genes in potato tubers.[Conclusion]StBBX25 interacts with StSuSy4 promoter,suggesting that StBBX25 may play a role in starch synthesis and yield formation in potato leaves and tubers.The results provide an important theoretical basis for further understanding of the regulatory network of potato starch synthesis.