阳春砂与海南砂BPPS启动子比较及GCN4-motif正调控作用的鉴定

Comparison of BPPS promoters between Amomum villosum and Amomum longiligulare and identification of GCN4 motif positive regulation

目的龙脑基二磷酸合酶(bornyl diphosphate synthase,BPPS)是砂仁药效萜类合成途径中的关键酶,获得海南砂AlBPPS的启动子并与阳春砂AvBPPS启动子进行调控元件和瞬时表达的比较,以期解析AvBPPS在种子中高表达的分子机制。方法通过FPNI-PCR的方法从海南砂gDNA中克隆AlBPPS的启动子,并与阳春砂AvBPPS启动子进行序列比较,对启动子相似区域进行截短,获得其相应的截短片段;对AvBPPS启动子截短片段中的GCN4-motif进行突变;构建由上述启动子驱动β-葡萄糖苷酸酶基因(β-glucuro-nidase gene,GUS)的重组表达载体,利用农杆菌介导法注射侵染本氏烟草叶片进行瞬时表达,验证启动子的活性和GCN4-motif的功能。结果克隆获得365 bp的AlBPPS启动子序列,3’端约300 bp的序列与AvBPPS启动子的相应序列相似度高达93%,但AlBPPS启动子不含有AvBPPS启动子的GCN4-motif。构建成功与GUS报告基因融合的系列启动子重组载体——VBP∷GUS(AvBPPS启动子全长)、VBPT∷GUS(AvBPPS启动子截短至320 bp)、VBPT-GM∷GUS(截短AvBPPS启动子且突变GCN4-motif)和LBP∷GUS(AlBPPS启动子全长)、LBPT∷GUS(AlBPPS启动子截短至305 bp)。通过GUS染色发现虽然上述启动子均具有驱动GUS基因转录的活性,然而,含有GCN4-motif的启动子,包括全长或截短的AvBPPS启动子(VBP∷GUS和VBPT∷GUS)的活性均高于GCN4-motif突变(VBPT-GM∷GUS)或无GCN4-motif的启动子(LBP∷GUS和LBPT∷GUS)。结论GCN4-motif对基因转录具有正调控作用,为进一步探究AvBPPS启动子的调控机制奠定了基础。

Objective Bornyl diphosphate synthase(BPPS)is an important enzyme in the synthetic pathway of Amomum medicinal terpenoids.To investigate the molecular mechanism of AvBPPS high expression in seed,the promoter of AlBPPS was obtained from Amomum longiligulare and was compared with the promoter of AvBPPS from A.villosum.Methods The promoter of AlBPPS was cloned from A.longiligulare gDNA by FPNI-PCR,and its sequence was compared with the promoter of AvBPPS;The similar regions of the promoter were truncated,and the corresponding truncated sequences were obtained.Meanwhile,the GCN4 motif was mutated from the truncated sequence of AvBPPS promoter.The recombinant expression vectors ofβ-glucuronidase gene(GUS)driven by the above-mentioned promoter were constructed,and the Agrobacterium-mediated method was used to infect the leaves of Nicotiana benthamiana for transient expression to verify the promoter activities and the functions of GCN4 motif.Results The 365bp AlBPPS promoter sequence was cloned.The 3'end of 300 bp sequences was 93%similarity to the corresponding sequence of the AvBPPS promoter.In this sequence,only the AvBPPS promoter contained GCN4 motif.We successfully constructed a series of vectors fused with the reporter gene of GUS:VBP::GUS(AvBPPS promoter full length),VBPT::GUS(AvBPPS promoter truncated to 320bp),VBPT-GM::GUS(the mutant AvBPPS promoter on GCN4 motif),LBP::GUS(AlBPPS promoter full length)and LBPT::GUS(AlBPPS promoter truncated to 305 bp).Through GUS staining,it was found that all of the above-mentioned promoters have the activity of driving the transcription of GUS.However,promoters containing GCN4 motif,including full-length or truncated AvBPPS promoters(VBP::GUS and VBPT::GUS)are more active than GCN4 motif mutation(VBPT-GM::GUS)or the promoters lack of GCN4 motif(LBP::GUS and LBPT::GUS).Conclusion This study improves GCN4-motif has a positive regulatory effect on AvBPPS and provides a basis for further exploration of the regulation mechanisms of AvBPPS promoter.