TY  - EJOU
AU  - Deng, Huiru 
AU  - Tian, Huan 
AU  - Yang, Liuyan 
AU  - Ou, Siyang 
AU  - Wang, Hui 
AU  - Geng, Guangdong 
AU  - Zhang, Suqin 

TI  - Construction of a Yeast Hybrid Library and Identification of Proteins Regulating <i>CaABI3/VP1-1</i> Expression in <i>Capsicum annuum</i> var. <i>conoides</i>
T2  - Phyton-International Journal of Experimental Botany

PY  - 2024
VL  - 93
IS  - 12
SN  - 1851-5657

AB  - Hot pepper (<i>Capsicum annuum</i> var. <i>conoides</i>) 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 <i>CaABI3/VP1-1</i> could improve pepper waterlogging tolerance. In order to explore the upstream regulatory mechanism of <i>CaABI3/VP1-1</i>, 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 × 10<sup>6</sup> colony-forming units (CFU)·mL<sup>−1</sup>, and the library capacity was 1.04 × 10<sup>7</sup> 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 <i>CaABI3/VP1-1</i> 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 <i>CaABI3/VP1-1</i> 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.
KW  - <i>Capsicum annuum</i> var. <i>conoides</i>; <i>CaABI3/VP1-1</i>; promoter; yeast one-hybrid; interacting protein; waterlogging stress

DO  - 10.32604/phyton.2024.058638