Cloning and Function Analysis of Gene UGTPg17 and UGTPg36 in Lonicera macranthoides

doi:10.13560/j.cnki.biotech.bull.1985.2023-0194

Abstract

Abstract:

This work is to clone the genes of glycosyltransferases UGTPg17 and UGTPg36 from Lonicera macranthoides, and analyze the correlation between their expression and saponin content at different flowering stages. According to the Unigene sequence of L. macranthoides transcriptome, specific primers were designed to clone UGTPg17 and UGTPg36. The physical and chemical properties, protein structure and evolutionary relationship of the cloned UGTPg17- and UGTPg36-encoding proteins were analyzed using the bioinformatics websites; quantitative reverse-transcription polymerase chain reaction（RT-qPCR）was used to analyze gene expressions at different flowering stages. The content of L. macranthoides saponin was determined by HPLC; and the correlation between gene expression and saponin content was analyzed by SPSS. The open reading frame length of UGTPg17 and UGTPg36 genes was 1 410 bp and 1 428 bp, respectively, encoding 469 and 475 amino acids, containing 4 and 7 glycosylation sites, respectively, which belonged to unstable and hydrophilic proteins without transmembrane region. UGTPg36 protein did not contain signal peptide sequence, and the average S value of UGTPg17 protein was >0.5, suggesting that it may contain signal peptide. The expressions of UGTPg17 and UGTPg36 in different flowering stages showed an upward trend, and the saponin content fluctuated in different flowering stages. The correlation analysis between the two showed that the two UGT genes were significantly negatively correlated with the saponin content. In this study, UGTPg17 and UGTPg36 genes were successfully cloned from L. macranthoides, and their expressions variedt at different flowering stages. Correlation analysis showed that UGTPg17 and UGTPg36 genes were related to saponin biosynthesis of L. macranthoides, which lays a foundation for further exploring their specific functions.

Key words: Lonicera macranthoides, glycosyltransferase, gene cloning, bioinformatics, expression analysis

YANG Min, LONG Yu-qing, ZENG Juan, ZENG Mei, ZHOU Xin-ru, WANG Ling, FU Xue-sen, ZHOU Ri-bao, LIU Xiang-dan. Cloning and Function Analysis of Gene UGTPg17 and UGTPg36 in Lonicera macranthoides[J]. Biotechnology Bulletin, 2023, 39(10): 256-267.

Figures/Tables 16

References 27

[1]	中国科学院中国植物志编辑委员会. 中国植物志-第三十九卷[M]. 北京: 科学出版社, 1988.
	Editorial Committee of Flora of Traditional Chinese Medicine, Chinese Academy of Sciences. Flora of China: Vol 72[M]. Beijing: Science Press, 1988.
[2]	国家药典委员会. 中华人民共和国药典-一部: 2020年版[M]. 北京: 中国医药科技出版社, 2020.
	National Pharmacopoeia Commission. People's republic of China(PRC)pharmacopoeia-part I: 2020 edition[M]. Beijing: China Pharmaceutical Science and Technology Press, 2020.
[3]	Long CM, Zhou XH, Wang QH, et al. Dietary supplementation of Lonicera macranthoides leaf powder improves amino acid profiles in serum and longissimus thoracis muscle of growing-finishing pigs[J]. Anim Nutr, 2016, 2(4): 271-275. doi: 10.1016/j.aninu.2016.08.006 URL
[4]	Hu X, Chen L, Shi SY, et al. Antioxidant capacity and phenolic compounds of Lonicerae macranthoides by HPLC-DAD-QTOF-MS/MS[J]. J Pharm Biomed Anal, 2016, 124: 254-260. doi: S0731-7085(16)30126-1 pmid: 26970594
[5]	Wang YD, He Y, Dai Z, et al. Simultaneous determination of five triterpenoid saponins in different parts of Lonicera macranthoides by RRLC-MS/MS method[J]. Pharmazie, 2016, 71(6): 306-310. pmid: 27455548
[6]	Wang J, Zhao XZ, Qi Q, et al. Macranthoside B, a hederagenin saponin extracted from Lonicera macranthoides and its anti-tumor activities in vitro and in vivo[J]. Food Chem Toxicol, 2009, 47(7): 1716-1721. doi: 10.1016/j.fct.2009.04.034 pmid: 19406196
[7]	管福琴, 冯煦, 彭峰, 等. 灰毡毛忍冬次皂苷乙抑制白血病细胞HL-60的增殖及其机制研究[J]. 天然产物研究与开发, 2010, 22(5): 765-768, 811.
	Guan FQ, Feng X, Peng F, et al. Antiproliferative effect of macranthoside B on leukemia cell lines HL-60 and its mechanism[J]. Nat Prod Res Dev, 2010, 22(5): 765-768, 811.
[8]	Chen S, Li MZ, Jiang W, et al. The role of Neu1 in the protective effect of dipsacoside B on acetaminophen-induced liver injury[J]. Ann Transl Med, 2020, 8(13): 823. doi: 10.21037/atm-19-3850 pmid: 32793668
[9]	管福琴, 刘敏, 单宇, 等. 灰毡毛忍冬中五环三萜类化合物的抗氧化活性研究[J]. 时珍国医国药, 2013, 24(6): 1315-1317.
	Guan FQ, Liu M, Shan Y, et al. The antioxidant activity evaluations of the triterpene saponins from Lonicera macranthoides in vitro[J]. Lishizhen Med Mater Med Res, 2013, 24(6): 1315-1317.
[10]	Oh SR, Jung KY, Son KH, et al. In vitro anticomplementary activity of hederagenin saponins isolated from roots of Dipsacus asper[J]. Arch Pharm Res, 1999, 22(3): 317-319. pmid: 10403139
[11]	Kim YJ, Zhang DB, Yang DC. Biosynthesis and biotechnological production of ginsenosides[J]. Biotechnol Adv, 2015, 33(6 Pt 1):717-735. doi: 10.1016/j.biotechadv.2015.03.001 URL
[12]	Augustin JM, Kuzina V, Andersen SB, et al. Molecular activities, biosynthesis and evolution of triterpenoid saponins[J]. Phytochemistry, 2011, 72(6): 435-457. doi: 10.1016/j.phytochem.2011.01.015 pmid: 21333312
[13]	周宸, 巩婷, 陈晶晶, 等. 三萜皂苷生物合成相关糖基转移酶研究进展[J]. 生物工程学报, 2022, 38(3): 1004-1024.
	Zhou C, Gong T, Chen JJ, et al. The glycosyltransferases involved in triterpenoid saponin biosynthesis: a review[J]. Chin J Biotechnol, 2022, 38(3): 1004-1024.
[14]	Paquette S, Møller BL, Bak S. On the origin of family 1 plant glycosyltransferases[J]. Phytochemistry, 2003, 62(3): 399-413. doi: 10.1016/s0031-9422(02)00558-7 pmid: 12620353
[15]	Augustin JM, Drok S, Shinoda T, et al. UDP-glycosyltransferases from the UGT73C subfamily in Barbarea vulgaris catalyze sapogenin 3-O-glucosylation in saponin-mediated insect resistance[J]. Plant Physiol, 2012, 160(4): 1881-1895. doi: 10.1104/pp.112.202747 pmid: 23027665
[16]	Tang QY, Chen G, Song WL, et al. Transcriptome analysis of Panax zingiberensis identifies genes encoding oleanolic acid glucuronosyltransferase involved in the biosynthesis of oleanane-type ginsenosides[J]. Planta, 2019, 249(2): 393-406. doi: 10.1007/s00425-018-2995-6
[17]	Nomura Y, Seki H, Suzuki T, et al. Functional specialization of UDP-glycosyltransferase 73P12 in licorice to produce a sweet triterpenoid saponin, glycyrrhizin[J]. Plant J, 2019, 99(6): 1127-1143. doi: 10.1111/tpj.14409
[18]	Yano R, Takagi K, Tochigi S, et al. Isolation and characterization of the soybean Sg-3 gene that is involved in genetic variation in sugar chain composition at the C-3 position in soyasaponins[J]. Plant Cell Physiol, 2018, 59(4): 792-805. doi: 10.1093/pcp/pcy019 pmid: 29401289
[19]	Naoumkina MA, Modolo LV, Huhman DV, et al. Genomic and coexpression analyses predict multiple genes involved in triterpene saponin biosynthesis in Medicago truncatula[J]. Plant Cell, 2010, 22(3): 850-866. doi: 10.1105/tpc.109.073270 URL
[20]	陈勋, 刘畅宇, 陈娅, 等. 灰毡毛忍冬LmPAL1基因的克隆及表达分析[J]. 中草药, 2019, 50(1): 178-187.
	Chen X, Liu CY, Chen Y, et al. Cloning and expression analysis of phenylalanine ammonia-lyase gene in Lonicera macranthoides[J]. Chin Tradit Herb Drugs, 2019, 50(1): 178-187.
[21]	陈娅, 王安琪, 刘聪, 等. 灰毡毛忍冬LmC3H1基因的克隆及表达模式与绿原酸含量相关性分析[J]. 中国实验方剂学杂志, 2020, 26(9): 167-175.
	Chen Y, Wang AQ, Liu C, et al. Cloning and expression patterns of LmC3H1 gene in Lonicera macranthoides and its correlation with chlorogenic acid content[J]. Chin J Exp Tradit Med Formulae, 2020, 26(9): 167-175.
[22]	曾娟, 龙雨青, 李灿, 等. 灰毡毛忍冬CHI和CHS基因的克隆及功能研究[J]. 中国中药杂志, 2022, 47(9): 2419-2429.
	Zeng J, Long YQ, Li C, et al. Cloning and function analysis of chalcone isomerase gene and chalcone synthase gene in Lonicera macranthoides[J]. China J Chin Mater Med, 2022, 47(9): 2419-2429.
[23]	王志辉, 周新茹, 成惠珍, 等. 基于多元统计分析的山银花抗氧化与抑菌质量标志物预测[J]. 湖南中医药大学学报, 2022, 42(7): 1105-1111.
	Wang ZH, Zhou XR, Cheng HZ, et al. Prediction of antioxidant and antibacterial quality markers of Flos lonicerae based on multivariate statistical analysis[J]. J Hunan Univ Chin Med, 2022, 42(7): 1105-1111.
[24]	Wang PP, Wei YJ, Fan Y, et al. Production of bioactive ginsenosides Rh2 and Rg3 by metabolically engineered yeasts[J]. Metab Eng, 2015, 29: 97-105. doi: S1096-7176(15)00029-4 pmid: 25769286
[25]	Wei W, Wang PP, Wei YJ, et al. Characterization of Panax ginseng UDP-glycosyltransferases catalyzing protopanaxatriol and biosyntheses of bioactive ginsenosides F1 and Rh1 in metabolically engineered yeasts[J]. Mol Plant, 2015, 8(9): 1412-1424. doi: 10.1016/j.molp.2015.05.010 pmid: 26032089
[26]	Lu J, Yao L, Li JX, et al. Characterization of UDP-glycosyltransferase involved in biosynthesis of ginsenosides Rg1 and Rb1 and identification of critical conserved amino acid residues for its function[J]. J Agric Food Chem, 2018, 66(36): 9446-9455. doi: 10.1021/acs.jafc.8b02544 URL
[27]	Yang CS, Li CJ, Wei W, et al. The unprecedented diversity of UGT94-family UDP-glycosyltransferases in Panax plants and their contribution to ginsenoside biosynthesis[J]. Sci Rep, 2020, 10(1): 15394. doi: 10.1038/s41598-020-72278-y pmid: 32958789

试剂名称Reagents name	编号No.	生产厂家Manufacturer
多糖多酚植物总RNA提取试剂盒	BSC65S1	杭州博日科技有限公司
DNA凝胶回收试剂盒	GE0101	北京擎科新业生物技术有限公司
RevertAid First Strand cDNA Synthesis Kit	K1622	美国Thermo Fisher Scientific
TranStart® Green qPCR SuperMix UDG	AQ111	北京全式金生物科技有限公司
2×Pfu MasterMix（Dye）	CW0686	康为世纪科技有限公司
2×Taq Master Mix（Dye）	CW0682	康为世纪科技有限公司
TS-Gel Red Ver.2 10000 in Water	TSJ003	北京擎科新业生物技术有限公司
DM 2 000 DNA Marker	CW0632	康为世纪科技有限公司
pEASY®-T1载体	CT101	北京全式金生物科技有限公司
Trelief^TM5α感受态细胞	TSC-C01	北京擎科新业生物技术有限公司
TranStart® Green qPCR SuperMix UDG	AQ111	北京全式金生物科技有限公司

试剂名称Reagents name	编号No.	生产厂家Manufacturer
多糖多酚植物总RNA提取试剂盒	BSC65S1	杭州博日科技有限公司
DNA凝胶回收试剂盒	GE0101	北京擎科新业生物技术有限公司
RevertAid First Strand cDNA Synthesis Kit	K1622	美国Thermo Fisher Scientific
TranStart® Green qPCR SuperMix UDG	AQ111	北京全式金生物科技有限公司
2×Pfu MasterMix（Dye）	CW0686	康为世纪科技有限公司
2×Taq Master Mix（Dye）	CW0682	康为世纪科技有限公司
TS-Gel Red Ver.2 10000 in Water	TSJ003	北京擎科新业生物技术有限公司
DM 2 000 DNA Marker	CW0632	康为世纪科技有限公司
pEASY®-T1载体	CT101	北京全式金生物科技有限公司
Trelief^TM5α感受态细胞	TSC-C01	北京擎科新业生物技术有限公司
TranStart® Green qPCR SuperMix UDG	AQ111	北京全式金生物科技有限公司

引物名称Primer	序列 Primer sequence（5'-3'）	用途Purpose
UGTPg17-F	ATGGCCGGCGGCAGCAAGCTCCACG	基因克隆 Gene clone
UGTPg17-R	TCACTCAACTTTTTCAGACGCTTCATTC
UGTPg36-F	ATGTCTCAACACCACCACCGCGGCGA
UGTPg36-R	CTACCTAATAATGTGAGCAATGAAAGAA
Q-UGTPg17-S	GGCGCCTCAGTTGAAGATATTA	实时荧光定量PCR RT-qPCR
Q-UGTPg17-A	CATAATCAACGGCTGTCCAAAC
Q-UGTPg36-S	GGTTCTGCGAGATGCTGATAA
Q-UGTPg36-A	GCCCAATCTCTCACCACTATTC
18S-F	CTTCGGGATCGGAGTAATGA	内参基因 Reference gene
18S-R	GCGGAGTCCTAGAAGCAACA	内参基因 Reference gene

引物名称Primer	序列 Primer sequence（5'-3'）	用途Purpose
UGTPg17-F	ATGGCCGGCGGCAGCAAGCTCCACG	基因克隆 Gene clone
UGTPg17-R	TCACTCAACTTTTTCAGACGCTTCATTC
UGTPg36-F	ATGTCTCAACACCACCACCGCGGCGA
UGTPg36-R	CTACCTAATAATGTGAGCAATGAAAGAA
Q-UGTPg17-S	GGCGCCTCAGTTGAAGATATTA	实时荧光定量PCR RT-qPCR
Q-UGTPg17-A	CATAATCAACGGCTGTCCAAAC
Q-UGTPg36-S	GGTTCTGCGAGATGCTGATAA
Q-UGTPg36-A	GCCCAATCTCTCACCACTATTC
18S-F	CTTCGGGATCGGAGTAATGA	内参基因 Reference gene
18S-R	GCGGAGTCCTAGAAGCAACA	内参基因 Reference gene

用途Purpose	网址Website
理化性质分析	ProtParam（https://web.expasy.org/protparam/）
糖基化位点预测	NetOGly 4.0（https://services.healthtech.dtu.dk/service.php?NetOGlyc-4.0）
亲/疏水性预测	ProtScale（https://web.expasy.org/protscale/）
亚细胞定位预测	WOLF PSORT（https://wolfpsort.hgc.jp/）
跨膜结构预测	TMHMM 2.0（https://services.healthtech.dtu.dk/service.php?TMHMM-2.0）
信号肽预测	SignalP 4.1 Server（https://services.healthtech.dtu.dk/service.php?SignalP-4.1）
结构域分析	NCBI（https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi）
二级结构预测	SOPMA（https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html）
三级结构预测	SWISS-MODEL（https://swissmodel.expasy.org/）