Open Access

ARTICLE

Construction of a Yeast Hybrid Library and Identification of Proteins Regulating CaABI3/VP1-1 Expression in Capsicum annuum var. conoides

by Huiru Deng^#, Huan Tian^#, Liuyan Yang, Siyang Ou, Hui Wang, Guangdong Geng^*, Suqin Zhang^*

College of Agriculture, Guizhou University, Guiyang, 550025, China

* Corresponding Authors: Guangdong Geng. Email: ; Suqin Zhang. Email:
# These authors contributed equally to this work

(This article belongs to the Special Issue: Abiotic Stress Tolerance in Crop Plants: Physio-biochemical and Molecular Mechanisms)

Phyton-International Journal of Experimental Botany 2024, 93(12), 3273-3291. https://doi.org/10.32604/phyton.2024.058638

Received 17 September 2024; Accepted 11 December 2024; Issue published 31 December 2024

Abstract

Hot pepper (Capsicum annuum var. conoides) is a significant vegetable that is widely cultivated around the world. Currently, global climate change has caused frequent severe weather events, and waterlogging stress harms the pepper industry by affecting the planting period, growth conditions, and disease susceptibility. The gene CaABI3/VP1-1 could improve pepper waterlogging tolerance. In order to explore the upstream regulatory mechanism of CaABI3/VP1-1, a high-quality standardized yeast hybrid library was successfully constructed for yeast one-, two-, and three-hybrid screening using pepper ‘ZHC2’ as the experimental material, with a library recombinant efficiency of up to 100%. The length of inserted fragments varied from 650 to 5000 bp, the library titer was 5.18 × 10⁶ colony-forming units (CFU)·mL⁻¹, and the library capacity was 1.04 × 10⁷ CFU of cDNA inserts. The recombinant bait plasmid was used to successfully identify 78 different proteins through the yeast one-hybrid system, including one transcription factor within the ethylene-responsive factor family and the other within the growth-regulating factor family. The interaction happened between LOC124895848 and CaABI3/VP1-1 promoter by point-to-point yeast one-hybrid experiment. The expression level of the 12 selected protein-coding genes was then evaluated by quantitative real-time polymerase chain reaction. Results indicated the protein coding genes showed different responses to waterlogging stress and that the activity of the CaABI3/VP1-1 promoter could be inhibited or activated by up-regulating or down-regulating gene expression, respectively. The identification of these proteins interacting with the promoter provides a new perspective for understanding the gene regulatory network of hot pepper operating under waterlogging stress and provides theoretical support for further analysis of the complex regulatory relationship between transcription factors and promoters.

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