灰毡毛忍冬UGTPg17、UGTPg36基因克隆及功能研究

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

生物技术通报 ›› 2023, Vol. 39 ›› Issue (10): 256-267.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0194

灰毡毛忍冬UGTPg17、UGTPg36基因克隆及功能研究

杨敏¹(), 龙雨青¹, 曾娟¹, 曾梅¹, 周新茹¹, 王玲¹, 付学森¹, 周日宝¹^,²^,³(), 刘湘丹¹^,²^,³()

1.湖南中医药大学药学院，长沙 410208
2.湘产大宗道地药材种质资源及规范化种植重点研究室，长沙 410208
3.湖南省普通高等学校中药现代化研究重点实验室，长沙 410208

收稿日期:2023-03-06 出版日期:2023-10-26 发布日期:2023-11-28
通讯作者: 周日宝，男，博士，教授，博士生导师，研究方向：中药资源与品质评价；E-mail: 1057323510@qq.com；
刘湘丹，女，博士，副教授，硕士生导师，研究方向：中药资源与品质评价；E-mail: paeonia_dd@126.com
作者简介:杨敏，女，硕士研究生，研究方向：中药资源与质量；E-mail: 2574467409@qq.com
基金资助:
国家现代农业产业技术体系(CARS-21);湖南省自然科学基金(2021JJ30497);湖南省自然科学基金(2021JJ30515);2022湖南省中医药科研计划项目(D2022134);2021湖南省教育厅科学研究项目(21C0245);2020年湖南省一流专业建设点：中药资源与开发，湖南中医药大学中药学一流学科项目(校行科字【2018】3号)

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

YANG Min¹(), LONG Yu-qing¹, ZENG Juan¹, ZENG Mei¹, ZHOU Xin-ru¹, WANG Ling¹, FU Xue-sen¹, ZHOU Ri-bao¹^,²^,³(), LIU Xiang-dan¹^,²^,³()

1. College of Pharmacy, Hunan University of Traditional Chinese Medicine, Changsha 410208
2. Key Laboratory for the Germplasm Resources and Standardized Planting of Hunan's Bulk Authentic Medicinal Materials, Changsha 410208
3. Hunan Provincial Key Laboratory of Traditional Chinese Medicine Modernization, Changsha 410208

Received:2023-03-06 Published:2023-10-26 Online:2023-11-28

摘要/Abstract

摘要：

克隆灰毡毛忍冬糖基转移酶UGTPg17、UGTPg36基因，并进行其不同花期表达量与皂苷含量相关性分析。根据灰毡毛忍冬转录组Unigene序列设计特异性引物进行UGTPg17、UGTPg36克隆；使用生物信息学网站对克隆的UGTPg17、UGTPg36编码蛋白进行理化性质、蛋白结构和进化关系等分析；实时荧光定量PCR（RT-qPCR）分析基因在不同花期表达情况；HPLC法测定灰毡毛忍冬皂苷含量；利用SPSS分析基因表达量与皂苷含量相关性。UGTPg17、UGTPg36基因开放阅读框长度分别为1 410 bp、1 428 bp，分别编码469、475个氨基酸，分别包含4、7个糖基化位点，均属于不稳定、亲水性蛋白，不具跨膜区域；UGTPg36蛋白不存在信号肽序列，UGTPg17蛋白平均S值>0.5，推测其可能含有信号肽。UGTPg17、UGTPg36在不同花期中表达均为上升趋势，皂苷含量在不同花期中存在波动，两者相关性分析得出，2个UGT基因与皂苷含量呈极显著负相关。成功从灰毡毛忍冬中克隆了UGTPg17、UGTPg36基因，其在不同花期表达存在差异性，且相关性分析表明UGTPg17、UGTPg36基因与灰毡毛忍冬皂苷生物合成相关，为进一步探索其具体功能奠定了基础。

关键词: 灰毡毛忍冬, 糖基转移酶, 基因克隆, 生物信息学, 表达分析

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

杨敏, 龙雨青, 曾娟, 曾梅, 周新茹, 王玲, 付学森, 周日宝, 刘湘丹. 灰毡毛忍冬UGTPg17、UGTPg36基因克隆及功能研究[J]. 生物技术通报, 2023, 39(10): 256-267.

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.

图/表 16

图1 灰毡毛忍冬不同花期样品

Fig. 1 Flower samples at different flowering stages of L. macranthoides

表1 实验试剂

Table 1 Reagents for experiment

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

表2 引物序列

Table 2 Primer sequences

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

表3 生物信息学分析网站

Table 3 Websites on bioinformatics analysis

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/）

图2 灰毡毛忍冬各花期总RNA提取 1：花期1；2：花期2；3：花期3；4：花期4；5：花期5；6：花期6；7：花期7；M：2K DNA Marker

Fig. 2 Extraction of total RNA from the L macranthoides in different flowering stages 1: Flowering stage 1; 2: flowering stage 2; 3: flowering stage 3; 4: flowering stage 4; 5: flowering stage 5; 6: flowering stage 6; 7: flowering stage 7; M: 2K DNA marker

图3 灰毡毛忍冬UGTPg17、UGTPg36 PCR产物

Fig. 3 PCR product of UGTPg17 and UGTPg36 gene cloned from L. macranthoides

图4 LmUGTPg17（A）、LmUGTPg36（B）蛋白结构域预测

Fig. 4 Domain prediction of LmUGTPg17（A）and LmUGTPg36（B）proteins

图5 LmUGTPg17（A）、LmUGTPg36（B）蛋白二级结构

Fig. 5 Secondary structure of LmUGTPg17（A）and LmUGTPg36（B）proteins

图6 LmUGTPg17（A）、LmUGTPg36（B）蛋白三级结构预测

Fig. 6 Prediction of tertiary structure of LmUGTPg17（A）and LmUGTPg36（B）protein

图7 LmUGTPg17蛋白与多种植物UGT蛋白序列比对红框为UGT家族PSPG保守结构域

Fig. 7 Multiple sequences alignment of LmUGTPg17 with UGT protein of other plants The red box indicates PSPG conserved domains of the UGT family

图8 LmUGTPg36蛋白与多种植物UGT蛋白序列比对红框为UGT家族PSPG保守结构域

Fig. 8 Multiple sequences alignment of LmUGTPg36 with UGT protein of other plants The red box represents PSPG conserved domains of he UGT family

图9 LmUGTPg17、LmUGTPg36蛋白与多种植物UGT家族蛋白系统进化树

Fig. 9 Phylogenetic tree of LmUGTPg17 and LmUGTPg36 with UGT protein from other plants

图10 LmUGTPg17（A）、LmUGTPg36（B）基因在不同花期中表达模式

Fig. 10 Expression patterns of LmUGTPg17（A）and LmUGTPg36（B）in different flowering stages of L. macranthoides

图11 对照品溶液（A）和灰毡毛忍冬样品溶液（B）的 HPLC图谱1：灰毡毛忍冬皂苷乙；2：灰毡毛忍冬皂苷甲；3：川续断皂苷乙

Fig. 11 HPLC profiles of reference substance（A）and L. macranthoides sample solution（B） 1: Macranthoides saponin B. 2: Macranthoides saponin A. 3: Dipsacoside B

图12 灰毡毛忍冬不同花期皂苷含量趋势

Fig. 12 Trend of saponins at different flowering stages of L. macranthoides

表4 灰毡毛忍冬UGTPg17、UGTPg36基因表达量与皂苷含量相关性法分析

Table 4 Correlation analysis between the gene expression of UGTPg17 and UGTPg36 and the content of saponins of L. macranthoides

基因 Gene	灰毡毛忍冬皂苷乙 Macranthoides saponin B		灰毡毛忍冬皂苷甲 Macranthoides saponin A		川续断皂苷乙 Dipsacoside B		总皂苷 Total saponin
基因 Gene	相关系数Correlation coefficient（r）	显著性 Significance（P）	相关系数Correlation coefficient（r）	显著性 Significance（P）	相关系数Correlation coefficient（r）	显著性 Significant（P）	相关系数Correlation coefficient（r）	显著性 Significant（P）
UGTPg17	-0.955^**	0.001	-0.617	0.140	-0.294	0.522	-0.918^**	0.004
UGTPg36	-0.997^**	0.000	-0.606	0.149	-0.295	0.521	-0.935^**	0.002

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灰毡毛忍冬UGTPg17、UGTPg36基因克隆及功能研究

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

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