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. 2019 Oct 31;9:15725. doi: 10.1038/s41598-019-51749-x

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© The Author(s) 2019

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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Knock-down of PcGSS1 and PcGSS2 gene expression in P. chrysocephala using RNA interference. Adults were injected with double-stranded RNA (dsRNA) targeting the 3′-UTR of PcGSS1 (GSS1), PcGSS2 (GSS2), and a lepidopteran-specific inducible metalloproteinase inhibitor gene from the greater wax moth, Galleria mellonella (IMPI; AY330624.1) as a control, respectively. After seven days, guts were dissected in RNAlater solution for subsequent gene expression analysis (a–d) or in protein extraction buffer for enzyme activity assays (e–h), respectively. Copy number estimates of PcGSS1, PcGSS2, PcGSS4, and PcGSS5 are given per 1,000 copies of mRNA for the reference gene RPL13a. For GSS activity assays, gut tissue homogenates were incubated with a mixture of eight different glucosinolates (GLS) at 35 °C for 2 h, and desulfo-GLS produced were quantified by LC-MS/MS via external standard curves. Bars labeled with different letters are significantly different (details of statistical analyses are summarized in Table S3). Means + SE of n = 9–11 biological replicates. I3M, indol-3-ylmethyl; 4MSOB, 4-methylsulfinylbutyl.