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. 2015 Nov 19;6:1015. doi: 10.3389/fpls.2015.01015

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Copyright © 2015 Li, Shao, Tang, Shen, Wang, Chen and Hong.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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BnWRI1 binding to proximal upstream regions of genes involved in lipid anabolism. (A) The AW-box [CnTnG](n)₇[CG] in the proximal upstream regions of genes involved in glycolysis, FA synthesis, and lipid assembly. (B) Consensus [CnTnG](n)₇[CG] of the AW-box and its mutant AW-box [TnCnA](n)₇[CG] of the KASI promoter or [CnTnG](n)₇ [TA] of GPAT9 promoter. (C) Recombinant BnWRI1 protein (50 kD) expressed in E. coli cells. (D) BnWRI1 specifically binds to the promoter region of KASI and GPAT9 containing the AW-box as assayed by EMSA. The binding was abolished when the conserved sequence was mutated into the [TnCnA](n)₇[CG] of the KASI promoter or the [CnTnG](n)₇[TA] of the GPAT9 promoter. The purified BnWRI1 protein (0.4 μg) was incubated with the target DNA fragment for 1 h. The resulting complex was visualized under UV light. The lanes from left to right: M, DNA ladder; KASI promoter only; BnWRI1 (0.4 μg) + KASI promoter; KASI mutant promoter only; BnWRI1 (0.4 μg) + KASI mutant promoter; GPAT9 promoter only; BnWRI1 (0.4 μg) + GPAT9 promoter; GPAT9 mutant promoter only; BnWRI1 (0.4 μg) + GPAT9 mutant promoter; BnWRI1 only. (E) The binding activity of BnWRI1 to the AW-box or its mutant AW-box (mAW) in the promoter regions of KASI and GPAT9 by EMSA using the DNA probes labeled with biotin. The shifted band is indicated by the arrow.