地黄环烯醚萜氧化酶基因的克隆与功能分析
Gene cloning and functional analysis of an iridoid oxidase gene in Rehmannia glutinosa
宋词 1高俊鸽 1杨雅贺 1丁宁 1王丰青 1袁媛2
作者信息
- 1. 河南农业大学 农学院,河南 郑州 450002
- 2. 中国中医科学院 中药资源中心,北京 100700
- 折叠
摘要
地黄为我国传统的常用大宗中药材,具有清热凉血、养阴生津之功效.环烯醚萜苷是地黄药材的主要活性成分,环烯醚萜氧化酶是环烯醚萜苷生物合成途径的关键限速酶.该研究基于转录组数据,筛选到 1 条环烯醚萜氧化酶基因RgIO,对其进行了系统的生物信息学、表达特性和亚细胞定位分析.结果表明,RgIO的编码区为 1 536 bp,编码 511 个氨基酸,相对分子质量 58 258.01.RgIO的蛋白序列包含细胞色素P450 氧化酶的保守结构域和基序,与列当科 3 个植物独脚金、黄独脚金和大花胡麻草的同源蛋白序列一致性最高,与长春花CrIO的序列一致性也高达 77.28%.RgIO在地黄的叶中特异性高表达,而且响应茉莉酸甲酯(MeJA)的诱导,MeJA处理3、6 h,在叶和块根中其表达量分别增加了 3.15、1.3 倍.亚细胞定位结果显示RgIO分布在内质网上.利用根癌农杆菌介导法在地黄叶片中瞬时表达RgIO,梓醇的含量较瞬时表达空载体的对照增加了 0.82倍.该研究为地黄梓醇的分子调控和生物合成提供了关键的靶基因,也为完整解析地黄梓醇的生物合成途径奠定了基础.
Abstract
Rehmannia glutinosa is one of the commonly used Chinese herbal medicines,which has activities of heat-clearing,blood-cooling,Yin-nourishing,and body fluid-promoting.Iridoid glycosides are the main bioactive in R.glutinosa.Iridoid oxidase is a key rate-limiting enzyme in the biosynthetic pathway of iridoid glycosides.In this study,an iridoid oxidase gene RgIO was screened based on the transcriptome data,followed by bioinformatics analysis,expression characteristic detection,and subcellular localization analysis.The results show that the coding region of RgIO is 1 536 bp,with 511 amino acids encoded,and the molecular weight is about 58 258.01.The protein sequence of RgIO contains the conserved domains and motifs of cytochrome P450 oxidases.RgIO has the highest sequence identities with its ortholog proteins in Striga asiatica,Striga hermonthica,and Centranthera grandiflora and has good sequence identities(77.28% )with Catharanthus roseus CrIO.RgIO shows specific expression in the leaf of R.glutinosa.In response to MeJA induction,the expression of MeJA in leaves and roots after treatment increases by 3.15 and 1.3 times at 3 h and 6 h,respectively.The result of subcellular localization shows that RgIO is distributed in the endoplasmic reticulum.Agrobacterium-mediated transient expression of RgIO gene in leaves of R.glutinosa makes the content of catalpol increase by 0.82 times compared with the transient expression of the empty vector.This study provides a key target gene for the molecular regulation and biosynthesis of catalpol in R.glutinosa and lays a foundation for revealing the complete biosynthetic pathway of catalpol.
关键词
地黄/环烯醚萜氧化酶/基因克隆/表达/亚细胞定位Key words
Rehmannia glutinosa/iridoid oxidase/gene cloning/expression/subcellular localization引用本文复制引用
基金项目
国家自然科学基金(82373985)
中央本级重大增减支项目(2060302)
出版年
2024
NETL
NSTL
万方数据