广藿香转录因子PcFBA-1的克隆及与FPPS启动子的互作调控分析

Cloning of transcription factor PcFBA-1 in Pogostemon cabin and its interaction with FPPS promoter

法尼基焦磷酸合酶(farnesyl diphosphate synthase,FPPS)是倍半萜生物合成途径分支点的关键酶,但目前尚未有广藿香FPPS启动子的转录调控研究相关报道。该研究前期挖掘获得广藿香FPPS基因启动子的结合蛋白PcFBA-1,为了探究PcFBA-1参与调控广藿香醇生物合成的可能机制,对PcFBA-1进行PCR克隆和测序分析;采用荧光定量PCR分析PcFBA-1在不同组织的表达模式;通过原生质体转化实验对PcFBA-1进行亚细胞定位分析;通过酵母单杂交系统检测PcFBA-1蛋白与FPPS启动子的结合情况,利用双荧光素酶报告实验验证转录调节功能。结果表明:克隆的PcFBA-1的开放阅读框(ORF)为1131 bp,编码376个氨基酸,包含F-box-like superfamily和FBA-1 superfamily 2个保守结构域,属于F-box家族蛋白,且PcFBA-1不含信号肽,无跨膜域,属于不稳定亲水性蛋白;此外,荧光定量PCR结果显示PcFBA-1在叶中表达量最高,在根、茎中的表达无显著性差异;PcFBA-1蛋白主要定位于细胞质;酵母单杂交实验和双荧光素酶实验结果表明PcFBA-1在体外和体内均能够结合FPPS启动子,并且增强FPPS启动子的活性。综上,该研究鉴定得到广藿香全新的转录因子PcFBA-1,通过直接结合FPPS基因启动子,增强启动子活性,为广藿香醇等活性成分的生物合成提供科学依据,为广藿香代谢工程和遗传改良提供基础。

Farnesyl diphosphate synthase(FPPS)is a key enzyme at the branch point of the sesquiterpene biosynthetic pathway,but there are no reports on the transcriptional regulation of FPPS promoter in Pogostemon cabin.In the early stage of this study,we obtained the binding protein PcFBA-1 of FPPS gene promoter in P.cabin.In order to explore the possible mechanism of PcFBA-1 involved in the regulation of patchouli alcohol biosynthesis,this study performed PCR-based cloning and sequencing analysis of PcFBA-1,analyzed the expression patterns of PcFBA-1 in different tissues by fluorescence quantitative PCR and its subcellular localization using the protoplast transformation system,detected the binding of PcFBA-1 protein to the FPPS promoter in vitro with the yeast one-hybrid system,and verified its transcriptional regulatory function by dual-luciferase reporter gene assay.The findings demonstrated that the cloned PcFBA-1 had an open reading frame(ORF)of 1131 bp,encoding a protein of 376 amino acids,containing two conserved domains named F-box-like superfamily and FBA-1 superfamily,and belonging to the F-box family.Moreover,neither signal peptide nor transmembrane domain was contained,implying that it was an unstable hydrophilic protein.In addition,as revealed by fluorescence quantitative PCR results,PcFBA-1 had the highest expression in leaves,and there was no significant difference in expression in roots or stems.PcFBA-1 protein was proved mainly located in the cytoplasm.Furthermore,yeast one-hybrid screening and dual-luciferase reporter gene assay showed that PcFBA-1 was able to bind to FPPS promoter both in vitro and in vivo to enhance the activity of FPPS promoter.In summary,this study identifies a new transcription factor PcFBA-1 in P.cabin,which directly binds to the FPPS gene promoter to enhance the promoter activity.This had laid a foundation for the biosynthesis of patchouli alcohol and other active ingre-dients and provided a basis for metabolic engineering and genetic improvement of P.cabin.