Bioinformatic and Prokaryotic Expression Analysis of UDP-Glycosyltransferase CmUGT6 in Oriental Melon (Cucumis melo)
LI Xiao-Xue1, YAO Xue-Yang2, REN Jie3, ZHANG Hong-Yan3, ZHANG Na1, LIU Ning4, BAI Ji-Xin5, JI Hai-Peng1,*, CHEN Cun-Kun1,5,*
1 Institute of Agricultural Products Preservation and Processing Technology (National Agricultural Products Preservation Engineering and Technology Research Center (Tianjin))/State Key Laboratory of Vegetable Biological Breeding/Key Laboratory of Agricultural Products Storage and Preservation, Ministry of Agriculture and Rural Affairs/Key Laboratory of Agricultural Products Post-harvest Physiology and Storage and Preservation, Tianjin Academy of Agricultural Sciences, Tianjin 300384, China; 2 School of Life Sciences, Nankai University, Tianjin 300071, China; 3 School of Food Science and Bioengineering, Tianjin Agricultural College, Tianjin 300392, China; 4 Liaoning Tungsheng Plastics Co., Ltd., Yingkou 115002, China; 5 Yunnan Haofeng Agricultural Technology Development Co., Ltd., Dali 671604, China
Abstract：Cucurbitacin B-type compounds in melon (Cucumis melo) possess important pharmacological effects such as anticancer and anti-inflammatory activities. Due to the high costs associated with their chemical synthesis and extraction, the mining and functional characterization of key genes involved in their biosynthetic pathways have attracted significant attention. To date, the key gene responsible for catalyzing the conversion of cucurbitacin B to cucurbitacin B-O-β-D-glucoside in melon remains unidentified. In this study, using the amino acid sequence of glycosyltransferases UDP-glycosyltransferase 74F2 (UGT74F2), which catalyzed the glycosylation of cucurbitacin E in watermelon (Citrullus lanatus), as a probe, the glycosyltransferases CmUGT6 gene in the melon genome with relatively high similarity was identified. Using the oriental melon variety 'Yumeiren' as experimental material, a 1 401 bp fragment of the CmUGT6 gene was cloned, encoding 467 amino acids. Bioinformatics analysis revealed that CmUGT6 contained a specific domain of the glycosyltransferase family, carried a negative charge, and possessed 46 phosphorylation sites, 3 N-glycosylation sites, and 4 O-glycosylation sites, with no transmembrane domain. The promoter region contained several cis-acting elements such as hormone responses, light responses, anaerobic induction, and metabolic regulation. Meanwhile, the CmUGT6 gene expressed in both the roots and fruits of the thin-skinned melon, with the highest expression level observed in the roots. Additionally, prokaryotic expression and purification of the CmUGT6 gene yielded a fusion protein band of approximately 100 kD. Molecular docking simulations between CmUGT6 and cucurbitacin B showed a binding energy of -8.9 kcal/mol. This study provides a reference for the subsequent in-depth exploration of the function of the CmUGT6 gene and the mechanism of glycosylation modification of cucurbitacin B in melon.
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