芦笋皂苷合成相关糖基转移酶基因克隆及原核表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2023-1074

生物技术通报 ›› 2024, Vol. 40 ›› Issue (4): 255-263.doi: 10.13560/j.cnki.biotech.bull.1985.2023-1074

芦笋皂苷合成相关糖基转移酶基因克隆及原核表达分析

钟匀¹(), 林春¹^,², 刘正杰¹^,², 董陈文华¹^,², 毛自朝¹^,²(), 李兴玉¹^,²^,³()

1.云南农业大学农学与生物技术学院，昆明 650201
2.云南农业大学特色小宗作物研究中心，昆明 650201
3.云南农业大学理学院农业化学研究中心，昆明 650201

收稿日期:2023-11-14 出版日期:2024-04-26 发布日期:2024-04-30
通讯作者: 李兴玉，男，教授，硕士生导师，研究方向：天然产物及生物防治；E-mail: lixingyu@ynau.edu.cn；
毛自朝，男，教授，博士生导师，研究方向：生物信息学；E-mail: zmao@ynau.edu.cn
作者简介:钟匀，男，硕士研究生，研究方向：药用植物合成生物学；E-mail: 1292726737@qq.com
基金资助:
云南省外专引智项目(202105AO130001);云南省农业联合专项重点项目(202101BD070001-027);云南省农业联合专项重点项目(202301BD070001-018);云南省基础研究计划面上项目(202201AT070254)

Cloning and Prokaryotic Expression Analysis of Asparagus Saponin Synthesis Related Glycosyltransferase Genes

ZHONG Yun¹(), LIN Chun¹^,², LIU Zheng-jie¹^,², DONG Chen-wen-hua¹^,², MAO Zi-chao¹^,²(), LI Xing-yu¹^,²^,³()

1. College of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming 650201
2. Institute of Improvement and Utilization of Characteristic Resource Plants, Yunnan Agricultural University, Kunming 650201
3. Research Center of Agricultural Chemistry, Yunnan Agricultural University, Kunming 650201

Received:2023-11-14 Published:2024-04-26 Online:2024-04-30

摘要/Abstract

摘要：

【目的】克隆芦笋（Asparagus officinalis）甾醇糖基转移酶基因AoSGT1，分析其潜在催化活性，为解析芦笋皂苷合成途径及代谢调控机制提供科学依据。【方法】基于芦笋转录组数据设计特异性引物，扩增AoSGT1基因的完整开放阅读框（open reading frame, ORF），经测序验证获得目标基因序列并进行生物信息学分析；实时荧光定量PCR（quantitative real-time PCR, RT-qPCR）测定各组织基因表达量；构建pGEX-4T-3-AoSGT1原核表达载体，并转入大肠杆菌（Escherichia coli）BL21（De3），随后诱导实现重组蛋白表达。【结果】AoSGT1长1 800 bp，编码599个氨基酸，其相对分子质量为66.72 kD，属于亲水性蛋白，无跨膜域和信号肽。系统发育结果表明，AoSGT1与盾叶薯蓣（Dioscorea zingiberensis）Dz3GT2有较高同源性，同属UGT80B1亚家族。多序列比对揭示了该蛋白序列包含甾醇糖基转移酶保守结构域PSBD Box和PSPG Box，预示其具有对甾体化合物3β-OH位点潜在糖基化活性。RT-qPCR结果显示，AoSGT1在芦笋根中高表达，而在茎和花中低表达。此外，SDS-PAGE结果表明，目标蛋白在大肠杆菌中以可溶性形式高效表达，其大小与预测值相符。【结论】成功克隆AoSGT1基因，并确认其在芦笋中呈现组织特异性表达，推测其可能参与芦笋甾体皂苷生物合成。同时，成功在大肠杆菌中实现了目标蛋白的异源表达。

关键词: 芦笋, 甾体皂苷, 甾醇糖基转移酶, 基因克隆, 生物信息学分析, RT-qPCR, 原核表达, 转录组

Abstract:

【Objective】This study aims to clone the sterol glycosyltransferase gene AoSGT1 from Asparagus officinalis, to assess its catalytic properties, and to elucidate its role in the biosynthesis of asparagus saponins and their metabolic regulation.【Method】We designed specific primers based on asparagus transcriptome data to amplify the complete open reading frame（ORF）of AoSGT1. The gene was sequenced and analyzed through bioinformatics, and its expression profile was investigated via quantitative real-time PCR（RT-qPCR）. We also constructed a pGEX-4T-3-AoSGT1 expression vector, expressed it in Escherichia coli BL21（DE3）, and induced the recombinant protein production.【Result】AoSGT1 has a length of 1 800 base pairs, encoding 599 amino acids, with a relative molecular weight of 66.72 kD. It is a hydrophilic protein with no transmembrane domains or signal peptides. The phylogenetic analysis revealed a close homology between AoSGT1 and Dioscorea zingiberensis Dz3GT2, indicating their membership in the UGT80B1 subfamily. Multiple sequence alignment revealed that the protein sequence contained conserved domains “PSBD Box” and “PSPG Box” of sterol glycosyltransferase, indicating its potential glycosylation activity at the 3β-OH position of steroidal compounds. RT-qPCR results showed that AoSGT1 was highly expressed in the roots, while expression in stems and flowers was relatively low. Additionally, SDS-PAGE results revealed that the target protein was efficiently expressed in a soluble form within E. coli, matching the predicted size.【Conclusion】The AoSGT1 gene was successfully cloned and demonstrated to exhibit tissue-specific expression in asparagus, suggesting its potential involvement in the biosynthesis of steroidal saponins in asparagus. Additionally, heterologous expression of the target protein in E. coli was successfully achieved.

Key words: Asparagus officinalis, steroidal saponins, sterol glycosyltransferase, gene cloning, bioinformatics analysis, RT-qPCR, prokaryotic expression, transcriptome

钟匀, 林春, 刘正杰, 董陈文华, 毛自朝, 李兴玉. 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, 2024, 40(4): 255-263.

ZHONG Yun, LIN Chun, LIU Zheng-jie, DONG Chen-wen-hua, MAO Zi-chao, LI Xing-yu. Cloning and Prokaryotic Expression Analysis of Asparagus Saponin Synthesis Related Glycosyltransferase Genes[J]. Biotechnology Bulletin, 2024, 40(4): 255-263.

图/表 8

表1 本研究涉及PCR引物序列

Table 1 PCR primer sequences used in this study

引物Primer	引物序列Primer sequence（5'-3'）	用途Purpose
UAoSGT1	F：TCTTGATGCGATTACAGCGG R：GAGATGGCTACAAGGTGAAAGG	基因克隆 Gene clone
PGEX-AoSGT1	F：GTGGATCCCC GAATTCCATGAGGAGCGGAGATTTCGAAG R：GGCCGCTCGA GTCGACCTACAAGGTGAAAGGAAGACAACACC	原核表达 Prokaryotic expression
qRT-AoSGT1	F：ATCCTTTGGGTGGTGATCCG R：AGAGCTTCAGCAACATGCGT	实时荧光定量PCR RT-qPCR
EF1A	F：CTGGCCAGGGTGGTTCATGAT R：TAAGTCTGTTGAGATGCACC	内参基因 Reference gene

图1 芦笋RNA及AOSGT1基因扩增产物凝胶电泳 A：芦笋各组织总RNA琼脂糖凝胶电泳；B：AoSGT1基因扩增；M：DNA marker 2000；1：根；2：茎；3：花；4：AoSGT1

Fig. 1 Agarose gel electrophoresis of asparagus RNA and AOSGT1 gene amplification products A: Agarose gel electrophoresis of total RNA from various tissues of asparagus. B: Amplification of the AoSGT1 gene. M: DNA marker 2000. 1: Root. 2: Stem. 3: Flower. 4: AoSGT1

图2 AoSGT1的生物信息学分析 A：NCBI保守结构域预测；B：跨膜域预测；C：信号肽分析

Fig. 2 Bioinformatics analysis of AoSGT1 A: NCBI conservative structural domain prediction. B: Transmembrane domain prediction. C: Signal peptide analysis

图3 芦笋AoSGT1蛋白的结构特征 A：AoSGT1二级结构（蓝：α-螺旋；绿：β-转角；橙：无规则卷曲；紫：无二级结构）；B：AoSGT1三维结构预测（蓝：螺旋；粉红：折叠；橙：环）

Fig. 3 Structural characteristics of asparagus AoSGT1 protein A: AoSGT1 secondary structure（Blue: α- Spiral; green: β- Corner; orange: irregular curl; purple: no secondary structure）. B: AoSGT1 3D structure prediction（Blue: helix; pink: sheet; orange: loop）

图4 芦笋AoSGT1与其他植物SGTs的系统进化树 GhSGT1: AHX00584; AtUGT80A2: OAP05643.1; SiSGT1: ATO74554.1; SiSGT2: ATO74555.1; Dz3GT1: AVI57699; ZmUGT80A2: PWZ44786.1; OsSGT1: AWW17242; UGT80A2: XP_015647949.1; WsSGTL1: ABC96116; AtUGT80B1: OAP13743.1; GhUGT80B1: NP: 001314026.1; ZmUGT80B1: PWZ33516.1; OsUGT80B1: XP_015622442.1; Dz3GT2: AVI57700

Fig. 4 Phylogenetic tree of asparagus AoSGT1 and SGTs from other plants

图5 芦笋AoSGT1多序列比对分析黑色框区域：PSBD Box结构域；蓝色框区域：PSPG Box结构域

Fig. 5 Multiple sequence alignment analysis of asparagus AoSGT1 Black box area: PSBD Box structural domain; blue box area: PSPG Box structure domain

图6 AoSGT1在芦笋不同组织部位的相对表达量 ****P<0.000 1

Fig. 6 Relative expression levels of AoSGT1 in different tissue parts of asparagus

图7 AoSGT1原核表达的 SDS-PAGE凝胶电泳 M：蛋白marker；1：空载上清液；2：空载沉淀；3：AoSGT1上清液；4：AoSGT1沉淀

Fig. 7 SDS-PAGE gel electrophoresis of prokaryotic expression of AoSGT1 M: Protein marker. 1: Empty supernatant. 2: Empty sediment. 3: AoSGT1 supernatant. 4: AoSGT1 sediment

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芦笋皂苷合成相关糖基转移酶基因克隆及原核表达分析

Cloning and Prokaryotic Expression Analysis of Asparagus Saponin Synthesis Related Glycosyltransferase Genes

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