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. 2021 Mar 17;11:6086. doi: 10.1038/s41598-021-85486-x

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© The Author(s) 2021, corrected publication 2021

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Functional characterization of the enzyme activities of AmCYP76AD1, AmDODA, and AmcDOPA5GT by agroinfiltration for the heterologous engineering of betalain pigments in Nicotiana benthamiana. (a, b) N. benthamiana leaves coinfiltrated with Agrobacterium harboring plasmids for the expression of BvCYP76AD1-YFP (BvAD1), BvDODAα1-SFP (BvDAα1), MjcDOPA5GT-SFP (Mj5GT), AmCYP76AD1-YFP (AmAD1), AmDODA-SFP (AmDA), and AmcDOPA5GT-SFP (Am5GT). Both the adaxial (left) and abaxial (right) sides of leaves are presented in each panel. Bottom right corner indicated the betalain pigments produced in N. benthamiana. (c) Extracted betalain pigments were examined for betanin content by LC–MS/MS analysis. Shown are XICs of masses corresponding to betanin (m/z = 551). Time, retention time (min). (d) Western blotting assays were conducted with antibodies against the YFP- or FLAG (SFP)-tag to examine the expression levels of YFP-tagged CYP76AD1 (upper panel), SFP-tagged DODA (middle panel), and SFP-tagged cDOPA5GT (middle panel). As a loading control, the large subunit of Rubisco visualized by Coomassie brilliant blue staining is indicated by the arrowhead (lower panel).