Dissecting conserved cis-regulatory modules of Glu-1 promoters which confer the highly active endosperm-specific expression via stable wheat transformation

摘要

Wheat high-molecular-weight glutenin subunits (HMW-GS) determine dough elasticity and play an essential role in processing quality.HMW-GS are encoded by Glu-1 genes and controlled primarily at transcriptional level,implemented through the interactions between cis-acting elements and trans-acting factors.However,transcriptional mechanism of Glu-1 genes remains elusive.Here we made a comprehensive analysis of cis-regulatory elements within 1-kb upstream of the Glu-1 start codon (-1000 to-1) and identified 30 conserved motifs.Based on motif distribution pattern,three conserved cis-regulatory modules (CCRMs),CCRM1 (-300 to -101),CCRM2 (-650 to-400),and CCRM3 (-950 to-750),were defined,and their functions were characterized in wheat stable transgenic lines transformed with progressive 5'deletion promoter::GUS fusion constructs.GUS staining,qPCR and enzyme activity assays indicated that CCRM2 and CCRM3 could enhance the expression level of Glu-1,whereas the 300-bp promoter (-300 to-1),spanning CCRM1 and core region (-100 to-1),was enough to ensure accurate Glu-1 initiation at 7 days after flowering (DAF) and shape its spatiotemporal expression pattem during seed development.Further transgenic assays demonstrated that CCRM1-2 (-300 to-209) containing Complete HMW Enhancer (-246 to-209) was important for expression level but had no effect on expression specificity in the endosperm.In contrast,CCRM1-1 (-208 to -101) was critical for both expression specificity and level of Glu-1.Our findings not only provide new insights to uncover Glu-1 transcription regulatory machinery but also lay foundations for modifying Glu-1 expression.