Abstract
4-coumarate: coenzyme A ligase (4CL) is a key enzyme in phenylpropanoid synthesis pathway. Bioinformatic analysis revealed that Pennisetum purpureum 4CL (Pp4CL) promoter contains TATA-box, CAAT-box, GC-box, TATC-box, ABRE, TGACG-motif and WBOXNTERF3. The Pp4CL promoter together with β-glucuronidase (GUS) reporter gene was cloned into pCAMBIA1305.1 vector, and transformed Nicotiana tabacum using Agrobacterium tumefaciens strain EHA105. The 5′-deleted promoter and histochemical assay were conducted on transformants to reveal the expression of GUS gene in the vascular tissues of roots, stems and leaves. The results demonstrated that Pp4CL promoter drove GUS expression in lignified tissues and particularly in xylem. The GUS activities of transgenic tobacco plants with 5′-deleted Pp4CL promoters in response to wounding were determined qualitatively by histochemical staining, and the results indicated that the longer of promoter fragment was, the bluer the wounding site detected was. In exogenous hormone treatments, the quantitative analysis of GUS activities illustrated that Pp4CL promoter of −228 bp was sufficient to respond to the ABA induction, while the Pp4CL promoters of −641 and −959 bp were sufficient to respond to the inductions of MeJA and GA to direct the expressions of GUS gene respectively. Furthermore, the electrophoretic mobility shift assays were carried out. The results indicated that GA-responsive element (from −913 to −907 bp) and ABRE cis-element (from −386 to −380 bp) as well as two MeJA-responsive elements (from −263 to −259 bp and from −388 to −384 bp) in the Pp4CL promoter were essential for hormone responsiveness. The findings further clarify the regulation mechanism of 4CL gene promoter in higher plants.
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Acknowledgments
This work was supported by the National Natural Science Foundation of China (Nos. 31272491, 30972138) and the Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20124404110011).
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XQP and SWK participated in the study design, carried out the data analysis and drafted the manuscript. They contributed equally to this work. JQL was responsible for the study design as well as carried out the experiments. SC took part in data analysis and manuscript editing. TXZ involved in data analysis. XMX participated in the study design and coordination as well as helped with manuscript editing and revision. All authors read and approved the final manuscript.
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Xiao-Qun Peng and Shan-Wen Ke contributed equally to this work.
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Peng, XQ., Ke, SW., Liu, JQ. et al. Deletion and hormone induction analyses of the 4-coumarate: CoA ligase gene promoter from Pennisetum purpureum in transgenic tobacco plants. Plant Cell Tiss Organ Cult 126, 439–448 (2016). https://doi.org/10.1007/s11240-016-1012-7
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DOI: https://doi.org/10.1007/s11240-016-1012-7