黄草乌DXS基因克隆、生信学分析及表达载体构建
1. 昆明市农业农村局, 云南昆明 650500
2. 昆明理工大学, 生命科学与技术学院, 云南昆明 650500
3. 西南林业大学, 林学院, 云南昆明 650224
| 摘 要： | 黄草乌是一种重要的中药材，因其含有二萜类生物碱，具有具有祛风散寒、活血止痛、抗抑肿瘤、除湿解毒等功效。二萜类生物碱生物合成需要通过萜类骨架代谢得到中间产物，进而合成二萜类生物碱。萜类骨架合成包括MVA途径和MEP途径，1-脱氧木酮糖-5-磷酸合成酶（DXS）是MEP途径第一个限速酶基因，在萜类物质的合成中起到重要的调控作用。目的：为掌握黄草乌DXS基因的特性。方法：通过提取黄草乌叶片的RNA反转录为cDNA，依据黄草乌转录组数据，设计引物克隆基因全长，对编码蛋白序列进行生物信息学分析。构建过表达载体和抑制表达载体。结果：该基因编码区为1512 bp，有503个氨基酸，分子式为C2408H3822N652O713S22，分子量为54020.10，等电点pI为6.29，不稳定性指数为35.70，属稳定蛋白。成功构建了过表达载体和抑制表达载体，为进一步研究该基因在二萜生物合成途径中的功能提供科学的依据。 |
| 关 键 词： | 黄草乌; 二萜; DXS基因; 基因克隆; 表达载体 |
| DOI： | 10.57237/j.life.2023.02.004 |
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收稿日期: 2023-04-14
接受日期: 2023-05-23
在线出版日期: 2023-06-15
Cloning, Bioinformatics Analysis and Expression Vector Construction of DXS Gene from Aconitum vilmorinianum
1. Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China
2. Faculty of Forestry, Southwest Forestry University, Kunming 650224, China
| Abstract: | Aconitum vilmorinianum is an important Chinese medicinal herb, which contains diterpenoid alkaloids and has the functions of antirheumatic activity, improvement in human meridians, relief of swelling and pain, activation of the blood circulation, and inhibition of tumor growth. The biosynthesis of diterpenoid alkaloids requires intermediate products obtained through terpenoid skeleton metabolism, in order to synthesize diterpenoid alkaloids. The synthesis of terpenoid skeletons includes the MVA pathway and the MEP pathway. 1-deoxyglucose-5-phosphate synthase (DXS) is the first rate limiting enzyme gene in the MEP pathway and plays an important regulatory role in the synthesis of terpenoids. Objective: To understand the characteristics of the DXS gene in Polygonummultiflorum. Method: By extracting RNA from the leaves of A. vilmorinianumand reverse transcribing it into cDNA, according to the transcriptome data of A. vilmorinianum, primers were designed to clone the full length of the gene, and the sequence of the coding protein was analyzed by bioinformatics. Construct overexpression vectors and inhibitory expression vectors. Results: The results showed that the coding region of the gene is 1512 bp, with 503 amino acids, molecular formula C2408H3822N652O713S22, molecular weight 54020.10, the isoelectric point pI 6.29, the instability index 35.70, and belongs to the stable protein category. Successfully constructing overexpression vectors and inhibitory expression vectors can provide scientific basis for further research on the function of this gene in the diterpene biosynthesis pathway. |
| Keywords: | Aconitum vilmorinianum Kom.; Diterpenoids; DXS Gene; Gene Cloning; Expression Vector |
基金
| 1. | 国家自然科学基金地区科学基金项目 (编号 31960071; 31760349) |
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