七叶一枝花糖基转移酶基因<em>PpUGT2</em>的克隆及原核表达

doi:10.3969/j.issn.1674-7968.2025.06.006

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摘要重楼皂苷是七叶一枝花(Parispolyphyllavar.chinensis)重要的活性成分,其生物合成途径尚未完全阐明,有研究表明尿苷二磷酸糖基转移酶(UDP-Glycosyltransferases,UGTs)是重楼皂苷下游生物合成途径的关键酶。本研究基于七叶一枝花转录组与重楼总皂苷含量关联分析,筛选出1个糖基转移酶候选基因PpUGT2(GenBankNo.PQ588702),可能参与七叶一枝花重楼皂苷的生物合成。利用PCR技术克隆PpUGT2基因全长,并对其进行生物信息学分析。在此基础上构建了PpUGT2的原核表达载体,IPTG诱导其表达,纯化重组蛋白。同时,结合qPCR技术,分析PpUGT2基因在七叶一枝花不同部位的表达模式。结果显示,PpUGT2基因全长1773bp,编码蛋白质由590个氨基酸组成,其蛋白相对分子质量为64.6kD,是不稳定且具有良好脂溶性的亲水蛋白。SDS-PAGE分析结果显示,在分子量约为64.6kD的位置上出现了蛋白条带,表明成功诱导PpUGT2重组蛋白的表达。此外,qPCR结果表明,PpUGT2基因在七叶一枝花的根、茎、叶和幼果中均有表达,表达模式为根>茎>叶>幼果,推测该基因主要在根中发挥作用。本研究丰富了UGT在七叶一枝花中的研究,为进一步解析重楼皂苷生物合成途径提供一定的理论基础。

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	王丽娅
	王靖涵
	周慧敏
	章瑶
	陈文强
	鲁志国
	朱丽娜
	唐其

关键词 ：七叶一枝花, 尿苷二磷酸糖基转移酶(UGTs), 生物信息学, 原核表达, qPCR

Abstract：Saponins are important active components of Paris polyphylla chinensis, but their biosynthetic pathway has not been fully elucidated. Research suggests that UPD-glycosyltransferases (UGTs) are key enzymes in the downstream biosynthetic pathway of polyphyllin. This study based on the correlation analysisbetween the transcriptome of P. polyphylla var. chinensis and the total saponins content, a glycosyltransferasecandidate gene PpUGT2 (GenBank No. pq588702) was screened out, which might be involved in the biosynthesis of polyphyllin. The PpUGT2 gene was cloned using PCR technology, and an expression vector was constructed for prokaryotic expression. Expression was induced using IPTG, followed by purification ofthe recombinant protein. Concurrently, qPCR was employed to analyze the expression patterns of PpUGT2across different parts of P. polyphylla var. chinensis. The results indicated that PpUGT2 had a total length of1 773 bp, encoded a protein consisting of 590 amino acids with a relative molecular weight of 64.6 kD. This hydrophilic protein was unstable and exhibited good lipid solubility. SDS-PAGE analysis confirmed the expression of the PpUGT2 recombinant protein at approximately 64.6 kD. Furthermore, qPCR resultsdemonstrated expression of the PpUGT2 gene in roots, stems, leaves, and young fruits of P. polyphylla var.chinensis, with a distinct expression hierarchy of roots>stems>leaves>young fruits, suggesting a predominant role of this gene in root tissues. This study enriches the study of UGT in P. polyphylla var. chinensis and provides a theoretical basis for further analysis of the biosynthesis pathway of polyphyllin.

Key words： Paris polyphylla var. chinensis UDP-glycosyltransferases (UGTs) Bioinformatics analysis Prokaryotic expression qPCR

收稿日期: 2024-09-19

中图分类号： S567.239

基金资助:湖南省教育厅项目(22C0106);湖南农业大学创新训练项目(XCX2023160)

通讯作者: *tangqi@hunau.edu.cn;zhulina_2021@163.com

引用本文:

王丽娅, 王靖涵, 周慧敏, 章瑶, 陈文强, 鲁志国, 朱丽娜, 唐其. 七叶一枝花糖基转移酶基因PpUGT2的克隆及原核表达[J]. 农业生物技术学报, 2025, 33(6): 1246-1258.
WANG Li-Ya, WANG Jing-Han, ZHOU Hui-Min, ZHANG Yao, CHEN Wen-Qiang, LU Zhi-Guo, ZHU Li-Na, TANG Qi. Cloning and Prokaryotic Expression of the UDP-glycosyltransferaseGene PpUGT2 from Paris polyphylla var. chinensis. 农业生物技术学报, 2025, 33(6): 1246-1258.

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https://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2025.06.006 或 https://journal05.magtech.org.cn/Jwk_ny/CN/Y2025/V33/I6/1246

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