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Open Access

ARTICLE

Construction of a Yeast cDNA Library from Chinese Cabbage and Identification of Upstream Regulators of the Key Tip-Burn Resistance Gene BrWRKY25

Yunduan Qin1,2,#, Kekang Su1,2,#, Meng Zhao3, Yu Xu1,2, Yuanyuan Zhang1,2, Chunyang Feng1,2, Xinlei Guo1,2, Chunhui Wu1,2, Changwei Shen3,*, Jingping Yuan1,2,*
1 School of Horticulture and Landscape Architecture, Henan Institute of Science and Technology, Xinxiang, China
2 Henan Engineering Research Center of the Development and Utilization of Characteristic Horticultural Plants, Xinxiang, China
3 School of Plant Protection and Environment/School of Bee Science, Henan Institute of Science and Technology, Xinxiang, China
* Corresponding Author: Changwei Shen. Email: email; Jingping Yuan. Email: email
# These authors contributed equally to this study

Phyton-International Journal of Experimental Botany https://doi.org/10.32604/phyton.2026.085158

Received 06 May 2026; Accepted 18 August 2026; Published online 20 August 2026

Abstract

Tip-burn in Chinese cabbage (Brassica rapa L. ssp. pekinensis) is a calcium-deficiency-induced physiological disorder, and BrWRKY25 functions as a negative regulator of tip-burn resistance. To elucidate the regulatory mechanisms underlying BrWRKY25-mediated tip-burn resistance, we constructed a yeast cDNA library from Chinese cabbage subjected to calcium-deficient treatment to identify its key upstream transcription factors. The constructed yeast cDNA library exhibited a titer of 9.5 × 106 colony forming unit (CFU)·mL−1 and an average insert size exceeding 1000 bp, meeting the quality criteria for downstream applications. Using the 1044-bp promoter of BrWRKY25 as bait, we screened several transcription factors, including zinc finger proteins, heat shock proteins, and ion transport proteins. Furthermore, conserved domain analysis of two zinc finger protein transcription factors, BrZFP4 (Bra039750) and BrZFP19 (Bra033588), revealed that BrZFP4 contains a conserved C2H2 zinc finger domain, while BrZFP19 contains two conserved B-box zinc finger domains. Yeast one-hybrid assays and dual-luciferase reporter gene assays demonstrated that both BrZFP4 and BrZFP19 directly bind to the BrWRKY25 promoter and activate its transcription. These findings suggest that the BrZFP4/19–BrWRKY25 regulatory module plays a pivotal role in tip-burn resistance, offering a theoretical framework for understanding the molecular mechanisms governing BrWRKY25-mediated resistance in Chinese cabbage.

Keywords

Chinese cabbage; library construction; promoter; transcription factors

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