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. 2023 Aug 25;14:5194. doi: 10.1038/s41467-023-40838-1

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a Subcellular localization of free GFP and BnA09MYB47a-GFP fusion proteins in B. napu protoplasts. Scale bars, 20 μm. Assays were performed three times using protoplasts from three independent experiments. b Word cloud of top 20 significant metabolic pathways (q-value ≤ 0.05) for the DEGs between control (ZY821) and RNAi samples. The figure was generated using https://wordart.com/. c The relative expression of BnPAL, BnTT3, BnTT4, BnTT6, BnAHA10, and BnTT18 in developing seed coats of RNAi transgenic plants compared to those of ZY821, as determined by RT-qPCR. Relative gene expression was normalized to the expression values in ZY821 at 10 days after flowering (DAF). P values were calculated using multiple t tests without adjustments. Data are presented as average ± SEM of 3 biological repeats. d Dual-luciferase reporter assay showing that BnA09MYB47a^ZY821 rather than BnA09MYB47a^GH06 regulates BnTT18 promoter activity. Values are normalized to the level of the blank effector and presented as mean ± SEM from three biological repeats. P values were calculated using multiple t tests without adjustments. e Probes used in the electrophoretic mobility shift assay (EMSA). f EMSA was used to assess the binding of BnA09MYB47a^ZY821 and BnA09MYB47a^GH06 to the promoter element in BnTT18. g BnTT18 is a downstream target of BnA09MYB47a. Upper panel, diagram of the BnTT18 promoter fragments (P1 and P2) used in chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR). The red and black boxes in P1 and P2, respectively, indicate the detection probes used in the EMSA, as shown in (e). Lower panel, BnA09MYB47a-mediated ChIP-qPCR enrichment. Chromatin prepared from transgenic leaves using an anti-Flag antibody (IP) was detected by qPCR with IgG as the control. BnActin7 and BnTT18-P2 were used as nonspecific targets. Data are presented as means ± SD (n = 6). P values were calculated using the two-tailed Student’s t test. Source data are provided as a Source Data file.