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

• Published in: The Crop journal Vol. 7; no. 1; pp. 8 - 18
• Main Authors: Li, Jihu, Wang, Ke, Li, Genying, Li, Yulian, Zhang, Yong, Liu, Zhiyong, Ye, Xingguo, Xia, Xianchun, He, Zhonghu, Cao, Shuanghe
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.02.2019; KeAi Communications Co., Ltd
• Subjects: Conserved cis-regulatory modules; Glu-1; Transcriptional regulation; Transgenic wheat; Triticum aestivum; Transcriptional regulation; Transgenic wheat; Triticum aestivum; Conserved cis-regulatory modules; Glu-1
• ISSN: 2214-5141; 2214-5141
• DOI: 10.1016/j.cj.2018.08.003

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 pattern 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.