Transcriptional Regulation of Deastringency Gene

The in vivo regulatory effects of the previously characterized high-CO₂/hypoxia-responsive transcription factors on the newly identified deastringency-related gene (DkPK1) were investigated via dual-luciferase assays. The promoter of DkPK1 was cloned into the pGreen II 0800-LUC vector (LUC) (primers are listed in Supplementary Table 28), and the transcription factors were previously cloned into the pGreen II 002962-SK vector^{17 (link),18 (link)}. All constructs were electroporated into Agrobacterium tumefaciens GV1301. The constructed SK and LUC plasmids were transiently expressed in tobacco (Nicotiana benthamiana) leaves as described by Min et al.^{9 (link)}. The Dual-luciferase® Reporter Assay System 10-Pack kit (Promega, USA) was employed to analyze firefly luciferase and Renilla luciferase in tobacco leaves at 3 d after infiltration by using a GLOMAX^TM 96 Microplate Luminometer (Promega).

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Zhu Q.G., Xu Y., Yang Y., Guan C.F., Zhang Q.Y., Huang J.W., Grierson D., Chen K.S., Gong B.C, & Yin X.R. (2019). The persimmon (Diospyros oleifera Cheng) genome provides new insights into the inheritance of astringency and ancestral evolution. Horticulture Research, 6, 138.

Publication 2019

GLOMAXTM 96 Microplate Luminometer

Agrobacterium tumefaciens Assay Firefly luciferase Hypoxia Luciferase Plasmids Primers Promega Renilla luciferase Tobacco Transcription factors Vector

Corresponding Organization : Chinese Academy of Forestry

Other organizations : Northwest A&F University, University of Nottingham

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Variable analysis

independent variables

Transcription factors previously cloned into the pGreen II 002962-SK vector

dependent variables

Firefly luciferase and Renilla luciferase activities in tobacco leaves

control variables

Agrobacterium tumefaciens GV1301 used for transient expression
Tobacco (Nicotiana benthamiana) leaves used as the host for transient expression
Dual-luciferase® Reporter Assay System 10-Pack kit (Promega, USA) used to analyze luciferase activities
GLOMAX™ 96 Microplate Luminometer (Promega) used to measure luciferase activities

controls

No positive or negative controls were explicitly mentioned in the protocol.

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