石仙桃中天麻素合成相关糖基转移酶基因的克隆与表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-1297

摘要/Abstract

摘要：

目的发掘石仙桃中天麻素生物合成最后关键步骤的糖基转移酶基因，为利用工程菌高效生产天麻素奠定基础。方法采用双酶切法，克隆候选基因transcript28360/f2p0/1592（1592）和transcript17418/f3p0/2174（2174），以AsUGT为阳性对照，在大肠杆菌和酵母中进行表达。结果基因2174由1 413个核苷酸组成，编码470个氨基酸，大小约为51.12 kD，主要位于叶绿体中。基因1592由1 395个核苷酸组成，编码464个氨基酸，大小约为50.85 kD，主要位于细胞质中。3个酶催化天麻苷元的活性中心均有亮氨酸、苯丙氨酸、苏氨酸、谷氨酸等残基，可能是催化天麻苷元形成天麻素的关键活性残基。在大肠杆菌和酵母中表达表明，基因2174具有较高的催化天麻苷元生成天麻素的活性，是对照基因AsUGT的2.31-3.39倍，而基因1592具有较弱的催化活性。结论石仙桃中糖基转移酶基因2174和1592催化天麻苷元生成天麻素过程中存在活性差异，基因2174可作为工程菌生产天麻素关键酶的候选基因。

关键词: 石仙桃, 天麻素, 糖基转移酶, 大肠杆菌, 酵母

Abstract:

Objective This study aimed to discover the glycosyltransferase gene involved in the final key step of gastrodin biosynthesis in Pholidota chinensis, providing a basis for the high-yield production of gastrodin using engineered microbial systems. Method The candidate genes transcript28360/f2p0/1592 (1592) and transcript17418/f3p0/2174 (2174) were cloned by double restriction enzyme digestion. Using AsUGT as a positive control, the three genes were expressed in Escherichia coli and yeast. Result Gene 2174 consists of 1 413 nucleotides, encoding 470 amino acids with a molecular weight of approximately 51.12 kD, and is primarily localized in the chloroplast. Gene 1592 consists of 1 395 nucleotides, encoding 464 amino acids with a molecular weight of approximately 50.85 kD, and is primarily localized in the cytoplasm. The active sites of all three enzymes catalyzing gastrodianin conversion contained residues such as leucine, phenylalanine, threonine, and glutamic acid, which may represent key active residues for the conversion of gastrodianin to gastrodin. Expression studies in E. coli and yeast revealed that gene 2174 exhibited higher catalytic activity in converting gastrodigenin to gastrodin, demonstrating 2.31 - to 3.39-fold higher activity than the control gene AsUGT. However, gene 1592 showed weaker catalytic activity. Conclusion The glycosyltransferase genes 2174 and 1592 from P. chinensis exhibit differential catalytic activities in the conversion of gastrodigenin to gastrodin. Gene 2174 is a promising candidate for use as a key enzyme in the engineered production of gastrodin.

Key words: Pholidota chinensis, gastrodin, glycosyltransferase, Escherichia coli, yeast

刘保财, 胡学博, 赵云青, 黄颖桢, 张武君, 陈菁瑛. 石仙桃中天麻素合成相关糖基转移酶基因的克隆与表达分析[J]. 生物技术通报, 2026, 42(11): 164-173.

LIU Bao-cai, HU Xue-bo, ZHAO Yun-qing, HUANG Ying-zhen, ZHANG Wu-jun, CHEN Jing-ying. Cloning and Expression Analysis of Glycosyltransferase Genes Involved in Gastrodin Biosynthesis in Pholidota chinensis Lindl.[J]. Biotechnology Bulletin, 2026, 42(11): 164-173.

图/表 14

表1 基因克隆PCR引物序列

Table 1 Primer sequences of PCR for gene clone

引物名称 Primer name	序列 Sequence（5′-3′）	用途 Purpose
2174-SpeI-F	GGACTAGTATGGAAATCGCTGGAT	Gene clone
2174-BglII-R	GAAGATCTCTAAAGTGCTACGCTCTT
1592-SpeI-F	GGACTAGTATGGCTGATGGCAACCCA
1592-BglII-R	GAAGATCTTCACTTAGTAGCCGTTGAATCCTT
2174-F	GTTTAACTTTAAGAAGGAGATATACATATGCACCATCATCATCATCATATGGAAATCGCCG	Seamless gene clone
2174-R	GGTGGTGGTGCTCGAGTGCGGCCGCAAGCTTTTACAGTGCCACACTTTTCCACTTAT	Seamless gene clone
1592-F	GTTTAACTTTAAGAAGGAGATATACATATGCACCATCATCATCATCATATGGCTGATGGTAATCC
1592-R	GGTGGTGGTGCTCGAGTGCGGCCGCAAGCTTTTATTTGGTTGCGGTGCTATCTTTCAGA
AsUGT-F	GTTTAACTTTAAGAAGGAGATATACATATGCACCATCATCATCATCATATGGAACATACCCCG
AsUGT-R	GGTGGTGGTGCTCGAGTGCGGCCGCAAGCTTTTAGGTACTACTAATCTTATTTTCCCA

图1 石仙桃中RNA和克隆基因的凝胶电泳A：RNA凝胶电泳；B：克隆基因的凝胶电泳。M：DL2 000TM DNA marker；A1：基因1592；A2：基因AsUGT；A3：基因2174

Fig. 1 Gel electrophoresis of RNA and cloned genes in P. chinensisA: Gel electrophoresis of RNA. B: Gel electrophoresis of cloned genes. M: DL2 000TM DNA marker; A1:gene 1592; A2: gene AsUGT; A3: gene 2174

图2 pESC370与目的基因的双酶切及菌液验证A：1592和2174双酶切（A1：线性化pESC370；A2：未线性化pESC370；A3：未酶切1592；A4：已酶切1592；A5：已酶切2174；A6：未酶切2174）。B：pESC370-1592-Ura大肠杆菌菌液验证（B1-B8：pESC370-1592-Ura单克隆）。C：pESC370-AsUGT-Ura和pESC370-2174-Ura大肠杆菌菌液验证（C1-C6：pESC370-AsUGT-Ura单克隆；C7-C12：pESC370-2174-Ura单克隆）。M：DL2 000TM DNA marker

Fig. 2 Dual enzyme digestion of pESC370 with target gene and bacterial culture validationA: Double enzyme digestion of 1592 and 2174 (A1: linearized pESC370; A2: unlinearized pESC370; A3: unenzymatically digested 1592; A4: enzymatically digested 1592; A5: enzymatically digested 2174; A6: unenzymatically digested 2174). B: The pESC370-1592-Ura validation in E. coli bacterial fluid (B1-B8: The pESC370-1592-Ura monoclonal). C: pESC370-AsUGT-Ura and pESC370-2174-Ura validation in E. coli bacterial suspension (C1-C6: pESC370-AsUGT-Ura monoclonal; C7-C12: pESC370-2174-Ura monoclonal). M: DL2 000TM DNA marker

图3 表达载体pET30a酶切验证

Fig. 3 Enzymatic validation of expression vector pET30a

图4 三个基因的氨基酸序列比较

Fig. 4 Comparison of amino acid sequences of three genes

图5 三个基因的保守结构域

Fig. 5 Conserved motifs of three genes

表2 三个基因的理化特性

Table 2 Physicochemical properties of three genes

基因Gene

氨基酸数Amino acid quantity （aa）

相对分子质量Relative molecular weight （kD）

等电点 PI

不稳定指数Instability index

脂肪族氨基酸指数Aliphatic index

亲水性平均系数GRAVY

α螺旋 Alpha-helix （%）

延长链Extended strand

（%）

β转角 Beta-turn （%）

无规则卷曲Random coil （%）

亚细胞定位Subcellular localization

2174

chlo： 6， mito： 2， extr： 2， E.R.： 2， nucl： 1

AsUGT

cyto： 7， chlo： 4， plas： 2

1592

cyto： 8， chlo： 3， nucl： 2

图6 预测的3个基因的空间结构和与天麻苷元对接的活性中心A和D：2174；B和E：1592；C和F：AsUGT

Fig. 6 Predicted spatial structure of the three genes and active centers docked to gastrodigeninA and D: Gene 2174; B and E: gene 1592; C and F: gene AsUGT

图7 三个基因诱导表达产物的SDS-PAGE分析A：AsUGT；B：1592；C：2174。M：SDS-PAGE Protein marker；0：对照；1：15 ℃诱导16 h；2：37 ℃诱导16 h；3：15 ℃诱导上清；4：15 ℃诱导沉淀；5：37 ℃诱导上清；6：37 ℃诱导沉淀

Fig. 7 SDS-PAGE analysis of three genes induced expression productsA: AsUGT; B: 1592; C: 2174. M: SDS-PAGE Protein marker; 0: control; 1: induced at 15 ℃ for 16 h; 2: induced at 37 ℃ for 16 h; 3: induced at 15 ℃ for supernatants; 4: induced at 15 ℃ for precipitations; 5: induced at 37 ℃ for supernatants; 6: induced at 37 ℃ for precipitations

图8 三个基因的表达蛋白分离后SDS-PAGE分析A： AsUGT； B： 1592； C： 2174。M： SDS-PAGE Protein marker； 1：包涵体溶解后离心上清；2：上清同Ni-IDA孵育流出液； 3-5： 50 mmol/L咪唑的洗脱组分；6-8： 100 mmol/L咪唑的洗脱组分；9-13： 500 mmol/L咪唑的洗脱组分

Fig. 8 SDS-PAGE analysis of three genes isolated target proteinsA: AsUGT; B: 1592; C: 2174. M: SDS-PAGE Protein marker; 1: centrifugation supernatant after inclusion body lysis; 2: supernatant with Ni-IDA incubation effluent; 3-5: elution fractions of 50 mmol/L imidazole; 6-8: elution fractions of 100 mmol/L imidazole; 9-13: elution fractions of 500 mmol/L imidazole

图9 三个重组目标蛋白纯化后SDS-PAGE分析M： SDS-PAGE marker； 1：BSA 1.0 μg； 2：BSA 2.0 μg； 3：蛋白2174 1.0 μg； 4：蛋白2174 2.0 μg； 5：蛋白2174 3.0 μg； 6：蛋白1592 1.0 μg； 7：蛋白1592 2.0 μg； 8：蛋白1592 3.0 μg； 9：蛋白AsUGT 1.0 μg； 10：蛋白AsUGT 2.0 μg； 11：蛋白AsUGT 3.0 μg

Fig. 9 SDS-PAGE analysis of three purified recombinant target proteinsM: SDS-PAGE marker; 1: BSA 1.0 μg; 2: BSA 2.0 μg; 3: 2174 protein 1.0 μg; 4: 2174 protein 2.0 μg; 5: 2174 protein 3.0 μg; 6: 1592 protein 1.0 μg; 7: 1592 protein 2.0 μg; 8: 1592 protein 3.0 μg; 9: AsUGT protein 1.0 μg; 10: AsUGT protein 2.0 μg; 11: AsUGT protein 3.0 μg

图10 重组蛋白催化天麻苷元生成天麻素峰图

Fig. 10 Peak diagram of recombinant protein catalyzing the GAS generation from HBA

图11 重组蛋白的催化活性

Fig. 11 Catalytic activity of recombinant protein

图12 酵母菌液加入底物后催化活性差异A：底物；B：产物，红色的虚线是0-48 h之间，基因2174和AsUGT催化天麻苷元生成天麻素的回归直线，方程是以0-40 h和0-48 h所产生天麻素含量分别做的回归方程

Fig. 12 Differences in catalytic activity after adding substrates to yeast solutionsA: Substrate; B: product. The red dashed line indicates the regression line of gastrodin generation from gastrodigenin catalyzed by gene 2174 and AsUGT in 0-48 h. The equations were regression equations based on the gastrodin content produced from 0 to 40 h and 0 to 48 h, respectively

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