Dissecting conserved cis-regulatory modules of Glu-1 promoters which confer the highly active endosperm-specific expression via stable wheat transformation
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 f...
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| Published in | The Crop journal Vol. 7; no. 1; pp. 8 - 18 |
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| Main Authors | , , , , , , , , , |
| Format | Journal Article |
| Language | English |
| Published |
Elsevier B.V
01.02.2019
KeAi Communications Co., Ltd |
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| Abstract | 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. |
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| AbstractList | 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. Keywords: Conserved cis-regulatory modules, Glu-1, Transcriptional regulation, Transgenic wheat, Triticum aestivum 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. |
| Author | Cao, Shuanghe Liu, Zhiyong Ye, Xingguo Xia, Xianchun Wang, Ke Li, Yulian He, Zhonghu Li, Jihu Li, Genying Zhang, Yong |
| Author_xml | – sequence: 1 givenname: Jihu surname: Li fullname: Li, Jihu organization: National Wheat Improvement Center, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China – sequence: 2 givenname: Ke surname: Wang fullname: Wang, Ke organization: National Wheat Improvement Center, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China – sequence: 3 givenname: Genying surname: Li fullname: Li, Genying organization: Crop Research Institute, Shandong Academy of Agricultural Sciences, Jinan 250100, Shandong, China – sequence: 4 givenname: Yulian surname: Li fullname: Li, Yulian organization: Crop Research Institute, Shandong Academy of Agricultural Sciences, Jinan 250100, Shandong, China – sequence: 5 givenname: Yong surname: Zhang fullname: Zhang, Yong organization: National Wheat Improvement Center, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China – sequence: 6 givenname: Zhiyong surname: Liu fullname: Liu, Zhiyong organization: College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, China – sequence: 7 givenname: Xingguo surname: Ye fullname: Ye, Xingguo organization: National Wheat Improvement Center, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China – sequence: 8 givenname: Xianchun surname: Xia fullname: Xia, Xianchun organization: National Wheat Improvement Center, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China – sequence: 9 givenname: Zhonghu surname: He fullname: He, Zhonghu email: hezhonghu02@caas.cn organization: National Wheat Improvement Center, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China – sequence: 10 givenname: Shuanghe surname: Cao fullname: Cao, Shuanghe email: caoshuanghe@caas.cn organization: National Wheat Improvement Center, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China |
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| Keywords | Transcriptional regulation Transgenic wheat Triticum aestivum Conserved cis-regulatory modules Glu-1 |
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| SubjectTerms | Conserved cis-regulatory modules Glu-1 Transcriptional regulation Transgenic wheat Triticum aestivum |
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| Title | Dissecting conserved cis-regulatory modules of Glu-1 promoters which confer the highly active endosperm-specific expression via stable wheat transformation |
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