Dual Luciferase Transcriptional Assays

Dual luciferase transcriptional activity assays were performed as previously described (Guan et al., 2014 (link)). Fragments corresponding to 1,600 bp upstream of the TSS of TaGlu-1Bx, TaGlu-1By, TaGlu-1Dx, TaGlu-1Dy, TaSuSy1, and TaSSIIa were PCR-amplified from CS genomic DNA and cloned into the pGreenII 0800-LUC vector as reporter plasmids (Hellens et al., 2005 (link)). The ORFs of TaNAC019 and TaGAMyb were cloned into the pHB vector as effectors. Young leaves of 4-week-old N. benthamiana plants were co-infiltrated with Agrobacterium (strain GV3101) harboring different combinations of these plasmids. Firefly luciferase activity derived from the TaGlu-1Bx (TaGlu-1Bxpro:LUC), TaGlu-1Dy (TaGlu-1Dypro:LUC), TaGlu-1By (TaGlu-1Bypro:LUC), TaGlu-1Dx (TaGlu-1Dxpro:LUC), TaSuSy1 (TaSuSy1pro:LUC), or TaSSIIa (TaSSIIapro:LUC) promoters and Renilla luciferase under the control of the 35S promoter (35Spro:REN) were quantified using the Dual-Luciferase Reporter Assay system (Promega) with a Synergy 2 Multi-Detection Microplate Reader (BioTek Instruments). Normalized data are presented as the ratio of luminescent signal intensity for reporter versus internal control reporter (35Spro:REN) from three independent biological samples.

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Gao Y., An K., Guo W., Chen Y., Zhang R., Zhang X., Chang S., Rossi V., Jin F., Cao X., Xin M., Peng H., Hu Z., Guo W., Du J., Ni Z., Sun Q, & Yao Y. (2021). The endosperm-specific transcription factor TaNAC019 regulates glutenin and starch accumulation and its elite allele improves wheat grain quality. The Plant Cell, 33(3), 603-622.

Publication 2021

Dual-Luciferase Reporter Assay system Synergy 2 Multi-Detection Microplate Reader

Agrobacterium Assay Biological Firefly luciferase Genomic Luciferase Luminescent Orfs Plants leaves Plasmids Promega Renilla luciferase Strain Transcriptional Vector

Corresponding Organization : China Agricultural University

Other organizations : Qingdao Agricultural University, Cereal Research Centre, Shandong Academy of Agricultural Sciences

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Variable analysis

independent variables

TaNAC019
TaGAMyb

dependent variables

Firefly luciferase activity derived from the TaGlu-1Bx (TaGlu-1Bxpro:LUC), TaGlu-1Dy (TaGlu-1Dypro:LUC), TaGlu-1By (TaGlu-1Bypro:LUC), TaGlu-1Dx (TaGlu-1Dxpro:LUC), TaSuSy1 (TaSuSy1pro:LUC), or TaSSIIa (TaSSIIapro:LUC) promoters

control variables

35Spro:REN (Renilla luciferase under the control of the 35S promoter)

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

Annotations

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