Gene Cloning,Expression Analysis,and Subcellular Localization of ChXES1 and ChXES2 in Calibrachoa hybrida

SONG Junyan; ZHANG Yuanshan; SHEN Xiaohui; ZHAO Ying; CHEN Guanqun

doi:10.15933/j.cnki.1004-3268.2025.08.012

Abstract：Identification and cloning of the xanthophyll esterase gene XES from Calibrachoa hybrida,as well as investigation of its expression characteristics in different tissues and under stress conditions,and its subcellular localization,will lay the foundation for exploring the functions of the XES gene.Based on the transcriptome and genome databases of C.hybrida cv.Fengbaohuang,ChXES genes and promoters were identified and analyzed by DNAMAN and other softwares,and cloned using PCR technology.RT-qPCR was used to analyze the expression patterns of ChXESs in different tissues,flower developmental stages,and leaves under abiotic stress,and the subcellular localization was observed by injecting Agrobacterium into tobacco.The findings indicated that two ChXES genes were identified in C.hybrida,both encoding 710 amino acids and featuring ABH and LPLAT domains.Phylogenetic analysis revealed their closest relationship with Petunia hybrida.The promoter regions of ChXES1pro and ChXES2pro contained stress-responsive and hormone-responsive elements.ChXESs were expressed in all tested tissues.During flower development,carotenoid content increased,with ChXES1 showing higher expression levels than ChXES2.ChXES1 responded more significantly to cold and drought stress,reaching its highest transcription levels at 12 h and 6 h,respectively.In contrast,ChXES2 exhibited a stronger response to heat stress and ABA signaling,with peak expression at 18 h for both conditions.ChXES1 and ChXES2 were preliminarily localized to the chloroplast and plastoglobules.ChXES1 and ChXES2 demonstrate tissue-specific expression in C.hybrida,and their response patterns to stress and ABA are different,which provides guidance for further study on the functions of ChXES1 and ChXES2,regulation of flower color and resistance mechanism.