Fig. 6

DNA pull-down assay screened the transcription regulator of Tgmst1. A 2 kb upstream of Tgmst1 was amplified as a probe by primers and biotin-primers respectively. Note that the products of biotin primers (right) were slightly larger than those of normal primers (left). B Pulled-down proteins were separated by SDS-PAGE and stained by Coomassie brilliant blue (R250). C Venn showed the number of differential/common identified proteins in EXP and CON. 79 common proteins and 37 differential proteins of EXP. D 37 differential proteins were ranked according to mass spectrum signal intensity. Low signal intensity and substance synthase were filtered, and 5 proteins were considered as the possible transcription factors of Tgmst1. E DNA–Protein interaction between the 5 proteins and Tgmst1 promoter was verified by Y1H assay, see Additional file 1: Fig. S6A for original image. Bait-reporter could not grow in SD-Ura medium containing AbA (400 ng mL⁻¹). Victors pGADT7-ATPeF1B pGADT7-GRP, pGADT7-KatG, pGADT7-ATPeF1A, pGADT7-EF5A, and pGADT7-EV were respectively transformed into the bait-reporter. GRP and Tgmst1 promoter have strong interaction; ATPeF1B and ATPeF1A have no interaction with Tgmst1 promoter; KatG and EF5A have weak interaction with Tgmst1 promoter. (F) ChIP-qPCR assay verified the interaction between GRP and Tgmst1 promoter. DNA obtained by immunoprecipitation was detected by qPCR. Percent Input was counted, Tgmst1 promoter accounts for 17.6% in IP, 0.296% in Mock (IgG); (G) Fold enrichment was counted, and the immune coprecipitation enrichment folds of IP was 61.6 folds relative to Mock (IgG). H Y1H assay of Tgmst2 promoter region and GRP, see Additional file 1: Fig. S6B for original image; Bait-reporter (pGADT7-EV + pABAi-Tgmst2P) could not grow in SD medium containing AbA (800 ng mL⁻¹); the transformant (pGADT7-GRP + pABAi-Tgmst2P) could grow in the SD medium containing AbA (800 ng mL⁻¹)