大蓟O-糖基转移酶CjUGT10功能表征及酶学特性

doi:10.13560/j.cnki.biotech.bull.1985.2026-0180

摘要/Abstract

摘要：

目的克隆并鉴定大蓟（Cirsium japonicum Fisch. ex DC.）中参与黄酮苷生物合成的关键尿苷二磷酸糖基转移酶基因CjUGT10（GenBank：PX578358），明确其底物特异性、糖基化位点及催化特性。方法基于大蓟转录组数据克隆CjUGT10基因；通过多序列比对与系统发育分析确定其家族分类；利用RT-qPCR检测该基因在不同组织中的表达模式；构建pMAL-CjUGT10原核表达载体，纯化重组蛋白后进行体外酶促反应，结合HPLC和酶动力学分析其底物宽泛性、糖基化位点及催化效率。结果 CjUGT10属于UGT73家族，氨基酸序列含有高度保守的PSPG-box，第43位和第44位分别为E和Q。该基因在叶片中高表达。体外酶活表明，CjUGT10以UDP-葡萄糖为唯一糖供体，可催化柳穿鱼黄素、木犀草素和高车前素的7-OH或催化蓟黄素的4′-OH葡萄糖基化，生成4种关键黄酮苷。酶动力学参数显示，其对木犀草素的催化效率最高（k_cat/K_m =2.82 L/（μmol·s））。结论 CjUGT10是一个具有底物广谱性和位点灵活性的双功能黄酮糖基转移酶，参与大蓟主要活性成分黄酮苷的生物合成。

关键词: 大蓟, 糖基转移酶, 黄酮苷生物合成, 基因克隆, 功能鉴定, 酶动力学

Abstract:

Objective This study aimed to clone and functionally characterize CjUGT10 (GenBank: PX578358), a key uridine diphosphate-dependent glycosyltransferase (UGT) involved in flavonoid glycoside biosynthesis in the traditional Chinese medicinal herb Cirsium japonicum Fisch. ex DC., and to elucidate its substrate specificity, regioselectivity, and catalytic properties. Method The full-length coding sequence of CjUGT10 was isolated from C. japonicum based on transcriptome data. Phylogenetic and multiple sequence alignment analyses were performed to determine its family classification. Tissue-specific expression patterns were analyzed by RT-qPCR. The recombinant CjUGT10 protein was expressed in Escherichia coli using the pMAL system, purified by amylose affinity chromatography, and subjected to in vitro enzymatic assays with various flavonoid aglycones and UDP-sugars. Reaction products were identified by HPLC, and kinetic parameters (K_m, V_max, k_cat, k_cat/K_m ) were determined. Result CjUGT10 belongs to the UGT73 family and contains the highly conserved PSPG-box, with E and Q at positions 43 and 44, respectively. It is predominantly expressed in leaves. Enzyme assays demonstrated that CjUGT10 exclusively uses UDP-glucose as the sugar donor and exhibits dual regioselectivity. It catalyzes 7-OH glucosylation of pectolinarigenin, luteolin, and hispidulin, as well as 4′-OH glucosylation of cirsimaritin, yielding four major bioactive flavonoid glucosides. Kinetic analysis revealed the highest catalytic efficiency (k_cat/K_m =2.82 L/(μmol·s)) toward luteolin. Conclusion CjUGT10 is a multifunctional flavonoid UGT with broad substrate acceptance and flexible site selectivity. It plays a central role in the biosynthesis of pharmacologically important flavonoid glycosides in C. japonicum.

Key words: Cirsium japonicum Fisch. ex DC., glycosyltransferase, flavonoid glycoside biosynthesis, gene cloning, functional identification, enzyme kinetics

陈影虹, 曹先慧, 易佩琴, 詹若挺, 马东明. 大蓟O-糖基转移酶CjUGT10功能表征及酶学特性[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0180.

CHEN Ying-hong, CAO Xian-hui, YI Pei-qin, ZHAN Ruo-ting, MA Dong-ming. Functional Characterization and Enzymatic Properties of the O-Glycosyltransferase CjUGT10 from Cirsium japonicum Fisch. ex DC.[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0180.

图/表 12

图1 大蓟不同部位植物材料

Fig. 1 Samples from different tissues of C. japonicum

表1 本研究使用的引物序列

Table 1 Primer sequences used in this study

引物名称 Primer name	引物序列 Primer sequence（5′-3′）	用途 Purpose
CjUGT10-F	ATGATTGAACATAGATTCAATCACTCCACCCC	ORF序列扩增
CjUGT10-R	TTAGCTTAATTTCCCAGCACTGGCT	ORF序列扩增
pMAL-Cj10-F	GAAGGATTTCACATATGATGGCTTCCAATCTTCACTTTCTTGT （Ned Ⅰ）	ORF序列扩增
pMAL-Cj10-R	GTTTTATTTGAAGCTTTTAGCCAGCCAAGCCCTTATTTTTATG （Hind Ⅲ）	ORF序列扩增
qCjGAPDH-F	TCATGGCCAATGGAAGCACA	大蓟内参基因
qCjGAPDH-R	ACCACCCTTCAAGTGAGCAG	大蓟内参基因
qGlcT10-F	CTCTCTTGATCCGTGGGTGG	实时荧光定量PCR
qGlcT10-R	GCTCCTGAAACTGAGGCCAT	实时荧光定量PCR

图2 CjUGT10 的结构域组成（A）和保守基序分布（B）A图中紫色区域为UGT家族特征结构域PF00201；B图中红框为PSPG保守序列

Fig. 2 Domain architecture (A) and conserved motifs (B) of CjUGT10The purple region indicates the characteristic domain PF00201 of the UGT family in figure A; and the red box indicates PSPG-box in figure B

图3 CjUGT10 与 UGT73 家族成员多序列比对（A）和系统发育树分析（B）A图中黑框为UGT73家族PSPG保守结构域

Fig. 3 Multiple sequence alignment (A) and phylogenetic tree analysis (B) of CjUGT10 and UGT73 familyThe black box in figure A represents PSPG conserved domains of UGT73 family

图4 CjUGT10二级结构预测（A）和三级结构预测（B）

Fig. 4 Secondary structure prediction (A) and tertiary structure prediction (B) of CjUGT10

图5 CjUGT10组织表达模式

Fig. 5 Tissue expression pattern of CjUGT10

图6 CjUGT10基因克隆（A）与载体构建（B）A：CjUGT10 基因PCR扩增产物的琼脂糖凝胶电泳图，M为DL2000 DNA标记；B：pMAL-CjUGT10重组表达载体的菌落PCR鉴定结果，M为DL2000 DNA标记；C：pMAL-CjUGT10重组质粒的结构图

Fig. 6 Cloning of CjUGT10 gene (A) and vector construction (B)A: Agarose gel electrophoresis of PCR-amplified CjUGT10 gene, M: DL2000 DNA marker. B: Colony PCR results of the pMAL-CjUGT10 recombinant expression vector. M: DL2000 DNA marker. C: The plasmid map of pMAL-CjUGT10

图7 CjUGT10重组蛋白的SDS-PAGE分析M：蛋白质分子量标准；1：上清蛋白；2：流穿液；3：洗杂液；4：MBP 洗脱液；5：PD10 脱盐后蛋白

Fig. 7 SDS-PAGE analysis of recombinant CjUGT10 proteinM: Protein marker; 1: supernatant protein; 2: flow-through fraction; 3: wash fraction; 4: MBP-tagged protein elution; 5: protein after PD10 desalting

图8 CjUGT10对8种黄酮底物的体外酶活反应HPLC谱图A-H：分别为8种黄酮底物的酶促反应HPLC谱图。黑线：对照组；红实线：标准品；蓝实线：反应组。Peak 1：底物；Peak 2：7-O-或4'-O-葡萄糖苷；Peak 3：山萘酚-3-O-葡萄糖苷

Fig. 8 HPLC profiles of in vitro enzymatic reactions of CjUGT10 with eight flavonoid substratesA-H: HPLC chromatograms of enzymatic reactions with eight flavonoid substrates, respectively. Black: Control; red: standard; blue: reaction. Peak 1: Substrate; Peak 2: 7-O- or 4'-O-glucoside; Peak 3: kaempferol-3-O-glucoside

图9 CjUGT10对8种黄酮底物的转化率

Fig. 9 Conversion rates of CjUGT10 for eight flavonoid substrates

图10 CjUGT10对四种黄酮底物的酶动力学曲线A-D：分别为CjUGT10对柳穿鱼黄素、蓟黄素、高车前素和木犀草素的酶动力学曲线；Vmax：最大反应速率（μmol/L·s）；Km：米氏常数（μmol/L）；kcat：催化常数（1/s）；kcat/Km：催化效率（L/（μmol·s））

Fig. 10 Kinetic curves of CjUGT10 with four flavonoid substratesA-D: The kinetic curves of CjUGT10 with pectolinarigenin, cirsimaritin, hispidulin, and luteolin, respectively. Vmax: Maximum reaction rate (μmol/L·s); Km: Michaelis constant (μmol/L); kcat: turnover number (1/s); kcat/Km: catalytic efficiency (L/(μmol·s)

图11 CjUGT10介导的大蓟4种重要黄酮苷生物合成路径红色星号标示CjUGT10催化的葡萄糖基化位点。该图展示了CjUGT10在大蓟中参与合成柳穿鱼黄素-7-O-葡萄糖苷、蓟黄素-4'-O-葡萄糖苷、木犀草素-7-O-葡萄糖苷和高车前素-7-O-葡萄糖苷的4条路径。上游反应参考已报道黄酮通路［31-32］，CjUGT10催化步骤经本研究验证

Fig. 11 Proposed biosynthetic routes of four key flavonoid glucosides mediated by CjUGT10 in C. japonicumRed stars indicate CjUGT10-catalyzed glucosylation sites. The schematic shows four biosynthetic pathways in C. japonicum leading to the production of pectolinarigenin-7-O-glucoside, cirsimaritin-4'-O-glucoside (Cirsimarin), luteolin-7-O-glucoside, and hispidulin-7-O-glucoside. Upstream steps are based on previously reported flavonoid biosynthesis pathways[31-32]; and CjUGT10-mediated reactions were validated in this study

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