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| 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 |
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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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Received: 30 June 2025
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Corresponding Authors:
*crazysum41@163.com; chencunkun@126.com
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[1] 陈蒙, 杨铭慧, 刘月, 等. 2019. 药西瓜UDP-糖基转移酶基因的克隆与表达分析[J]. 中国农业大学学报, 24(11):70-77.
(Chen M, Yang M H, Liu Y, et al.2019. Cloning and expresion analysis of UDP-Glycosyl transferase gene from Citrulus colocynthis L.Shrad[J]. Journal of China Agricultural University, 24(11):70-77.)
[2] 陈锁英, 刘晓曼, 刘小刚, 等. 2021. 甜橙 UDP-糖基转移酶基因的筛选及其功能分析[J]. 植物生理学报, 57(09):1745-1754.
(Chen S Y, Liu X M, Liu X G, et al.2021. Screening and functional analysis of udp-glycosyltransferase genes in sweet orange (Citrus sinensis)[J]. Plant Physiology Journal, 57(09):1745-1754.)
[3] 齐书艺, 王璐暖, 何贝轩, 等. 2024. 红花黄酮生物合成途径糖基转移酶基因CtUGT25的功能研究[J]. 药学学报, 59(06):1854-1863.
(Qi S Y, Wang L N, He B X, et al.2024. Functional study of glycosyltransferase genes CtUGT25 in the flavone biosynthesis pathway of Carthamus tinctorius L.[J]. Acta Pharmaceutica Sinica, 59(06):1854-1863.)
[4] 郭思远, 尹艳, 石颖慧, 等. 2021. 滇重楼糖基转移酶基因的克隆和原核表达[J]. 中国实验方剂学杂志, 27(08):126-134.
(Guo S Y, Yin Y, Shi Y H, et al.2021. Cloning and prokaryotic expression of glycosyltransferase genes from paris polyphylla var. yunnanensis[J]. Chinese Journal of Experimental Traditional Medical Formulae, 27(08):126-134.)
[5] 李麒, 罗飞, 王成洋, 等. 2020. 葫芦科作物苦味物质葫芦素的研究进展[J]. 植物生理学报, 56(06):1137-1145.
(Li Q, Luo F, Wang C Y, et al.2020. Reseach status and advance of bitter compounds cucurbitacins in Cucurbitaceae crops[J]. Plant Physiology Journal, 56(06):1137-1145.)
[6] 李晓雪, 向蓓蓓, 孙继奇, 等. 2019. 藏药川西獐牙菜SmMK基因的克隆及生物信息分析[J]. 中草药, 50(13):3169-3177.
(Li X X, Xiang B B, Sun J Q, et al.2019. Cloning and bioinformatics analysis of SmMK gene in Swertia mussotii[J]. Chinese Traditional and Herbal Drugs, 50(13):3169-3177.)
[7] 彭素芳, 李晓波, 王圆圆, 等. 2025. 金平人参糖基转移酶的原核表达及功能验证[J]. 分子植物育种, 1-14.
(Peng S F, Li X B, Wang Y Y, et al.2025. Prokaryotic expression and functional verification of glycosyltransferase from P. vietnamensis var. fuscidiscus[J]. Molecular Plant Breeding, 1-14.)
[8] 邱果, 刘柳, 李小梅, 等. 2017. 甜瓜抗枯萎病和白粉病育种研究进展[J]. 生物技术通报, 33(08):14-19.
(Qiu G, Liu L, Li X M, et al.2017. Research progress on breeding for resistance to fusarium wilt and powdery mildew in muskmelon[J]. Biotechnology Bulletin, 33(08):14-19.)
[9] 尚轶, 黄三文. 2015.黄瓜苦味物质的代谢调控与合成生物学[J]. 生命科学, 27(08):1091-1094.
(Shang Y, Huang S W.2015. Bitterness metabolism in cucumber and synthetic biology[J]. Chinese Bulletin of Life Sciences, 27(08):1091-1094. )
[10] 唐岚, 赵亚, 单海峰, 等. 2012. 甜瓜蒂中葫芦素类成分分离及体外抗癌活性研究[J]. 浙江工业大学学报, 40(04):388-391+449.
(Tang L, Zhao Y, Shan H F, et al. 2012. Studies on the separation of cucurbitacins from the fruit base of Cucumis melo L. and their antitumor activities[J]. Journal of Zhejiang University of Technology, 40 (04):388-391+449.)
[11] 吴一凡, 张睿琪, 董泽安, 等. 2021. 植物糖基转移酶的分类、分布及在果实发育中作用研究进展[J]. 食品科技, 46(07):1-6.
(Wu Y F, Zhang R Q, Dong Z A, et al.2021. Classification and distribution of glycosyltransferases in plant and its roles in the fruit development[J]. Food Science and Technology, 46(07):1-6.)
[12] 徐欢欢, 李逸, 邢嘉怡, 等. 2024. 大葱类黄酮3-O-葡萄糖基转移酶AfUFGT基因的克隆与表达分析[J]. 分子植物育种, 22(23):7678-7683.
(Xu H H, Li Y, Xing J Y, et al.2024. Cloning and expression analysis of flavonoid-3-o-glucosyltransferase AFUFGT from welsh onion[J]. Molecular Plant Breeding, 22(23):7678-7683.)
[13] 钟匀, 林春, 刘正杰, 等. 2024. 芦笋皂苷合成相关糖基转移酶基因克隆及原核表达分析[J]. 生物技术通报, 40(04):255-263.
(Zhong Y, Lin C, Liu Z J, et al.2024. Cloning and prokaryotic expression analysis of asparagus saponin synthesis related glycosyltransferase genes[J]. Biotechnology Bulletin, 40(04):255-263.)
[14] Chomicki G, Schaefer H, Renner S.2020. Origin and domestication of Cucurbitaceae crops:Insights from phylogenies, genomics and archaeology[J]. New Phytologist, 226(5):1240-1255.
[15] Dai L, Liu C, Zhu Y, et al.2015. Functional characterization of cucurbitadienol synthase and triterpene glycosyltransferase involved in biosynthesis of mogrosides from Siraitia grosvenorii[J]. Plant and Cell Physiology, 56(6):1172-1182.
[16] Hua D, Fu J, Liu L, et al.2019. Change in bitterness, accumulation of Cucurbitacin B and expression patterns of CuB biosynthesis-related genes in melon during fruit development[J]. The Horticulture Journal, 88(2):253-262.
[17] Kim Y C, Choi D, Cha A, et al.2020. Critical enzymes for biosynthesis of cucurbitacin derivatives in watermelon and their biological significance[J]. Communications Biology, 3(1):444.
[18] Luo F, Li Q, Yu L, et al.2020. High concentrations of CPPU promotes cucurbitacin B accumulation in melon (Cucumis melo var. makuwa Makino) fruit by inducing transcription factor CmBt[J]. Plant Physiology and Biochemistry, 154:770-781.
[19] Morikawa T, Inoue N, Nakanishi Y, et al.2020. Collagen synthesis-promoting and collagenase inhibitory activities of constituents isolated from the rhizomes of Picrorhiza kurroa Royle ex Benth[J]. Fitoterapia, 143:104584.
[20] Wang M, Ji Q, Lai B, et al.2023. Structure-function and engineering of plant UDP-glycosyltransferase[J]. Computational and Structural Biotechnology Journal, 21:5358-5371.
[21] Wang Q, Li X, Zhang C, et al.2023. Discovery and identification of the key contributor to the bitter taste in oriental melon after forchlorfenuron application[J]. Journal of Agricultural and Food Chemistry, 71(16):6415-6423.
[22] Xu Y, Liu S, Bian L, et al.Engineering of a UDP-glycosyltransferase for the efficient whole-cell biosynthesis of siamenoside I in Escherichia coli[J]. Journal of Agricultural and Food Chemistry, 70(5):1601-1609.
[23] Zhong Y, Xue X, Liu Z, et al.Developmentally regulated glucosylation of bitter triterpenoid in cucumber by the UDP-glucosyltransferase UGT73AM3[J]. Molecular Plant, 10(7):1000-1003.
[24] Zhong Y, Xun W, Wang X, et al.2022. Root-secreted bitter triterpene modulates the rhizosphere microbiota to improve plant fitness[J]. Nature Plants, 8(8):887-896.
[25] Zhou Y, Ma Y, Zeng J, et al.2016. Convergence and divergence of bitterness biosynthesis and regulation in Cucurbitaceae[J]. Nature Plants, 2:16183. |
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