Song Ci, Gao Jun-Ge, Yang Ya-He, Ding Ning, Wang Feng-Qing, Yuan Yuan
College of Agronomy, Henan Agricultural University Zhengzhou 450002, China.
National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences Beijing 100700, China.
Zhongguo Zhong Yao Za Zhi. 2024 Jun;49(11):2897-2905. doi: 10.19540/j.cnki.cjcmm.20240317.105.
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 Rg IO 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 Rg IO is 1 536 bp, with 511 amino acids encoded, and the molecular weight is about 58 258. 01. The protein sequence of Rg IO contains the conserved domains and motifs of cytochrome P450 oxidases. Rg IO 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 Cr IO. Rg IO 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 Rg IO is distributed in the endoplasmic reticulum. Agrobacterium-mediated transient expression of Rg IO 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.
地黄是常用的中药材之一，具有清热、凉血、滋阴、生津的功效。环烯醚萜苷是地黄中的主要生物活性成分。环烯醚萜氧化酶是环烯醚萜苷生物合成途径中的关键限速酶。本研究基于转录组数据筛选出一个环烯醚萜氧化酶基因RgIO，随后进行了生物信息学分析、表达特性检测和亚细胞定位分析。结果表明，RgIO的编码区为1536bp，编码511个氨基酸，分子量约为58258.01。RgIO的蛋白质序列包含细胞色素P450氧化酶的保守结构域和基序。RgIO与其在亚洲独脚金、非洲独脚金和大花胡麻草中的直系同源蛋白具有最高的序列同一性，与长春花CrIO也具有良好的序列同一性（77.28%）。RgIO在地黄叶片中特异性表达。响应茉莉酸甲酯（MeJA）诱导，处理后3h和6h时，叶片和根中MeJA的表达分别增加3.15倍和1.3倍。亚细胞定位结果表明，RgIO分布在内质网中。农杆菌介导的RgIO基因在地黄叶片中的瞬时表达使梓醇含量比空载体瞬时表达增加了0.82倍。本研究为地黄中梓醇的分子调控和生物合成提供了关键靶基因，为揭示梓醇完整的生物合成途径奠定了基础。
