Functional analysis of the 5' regulatory region of the maize GALACTINOL SYNTHASE2 gene
【摘要】：正Maize(Zea mays)GALACTINOL SYNTHASE(GolS) is a key enzyme in the raffinose biosynthetic pathway.We have previously characterized that the maize GolS2 (ZmGolS2)gene is heat shock induced in maize germinating seeds and cultured cells.Here we report the identification,isolation and characterization of the 1.5 Kb,5' regulatory region of the ZmGolS2 gene.Several cis-acting motifs,such as CCAAT/enhancer binding protein elements (C/EBP);Sugar Response Elements(SRE)and Heat Shock Responsive Elements (HSEs),were predicted in ZmGolS2 promoter fragment by Online Softwares.The 1.5 Kb fragment and its deletions were tested for promoter activity by their regulation of Renilla(Renilla reniformis) luciferase reporter gene expression in maize protoplasts cultured at either 25℃or 42℃for 24 h.The expression of a constitutively expressed firefly(Photinus ssp.)luciferase gene in the same vector was used as a reference.This dual luciferase reporter vector is more accurate and reliable than the mono-luciferase reporter for testing promoter activity.One of two HSEs that have been predicted to reside in ZmGolS2 promoter was identified to be functional by comparing the promoter activity of each fragment under normal and heat shock conditions. Deletion or triplication of this HSE motif,abolished or enhanced the heat shock response of the ZmGolS2 promoter,respectively.This HSE motif is specifically bound by proteins in the nuclear extracts of heat shock stressed,but not unstressed maize cells as confirmed by DNAEMSA. Replacing the 5'UTR of ZmGolS2 5' regulatory region with human beta globin 5'UTR did not affect its response to heat shock stress but inhibit the downstream ORF translation,indicating that the 5'UTR of ZmGolS2 affects translation,but not transcription under heat shock stress.This work helps to understand the regulatory mechanism of the ZmGolS2 gene under stress conditions.