《植物生理学报》 2017, 53(4): 563-571

地黄RgDXR基因的克隆及表达分析

朱畇昊, 董诚明, 俎梦航, 李攀登, 赵乐^*

河南中医药大学药学院, 呼吸疾病诊疗与新药研发河南省协同创新中心, 郑州450046

通信作者：赵；E-mail: zhaole1983@126.com

摘　要：

本研究采用RT-PCR法对地黄DXR基因进行克隆, 并用生物信息学方法分析其功能结构域、系统进化等; 将地黄DXR基因克隆到原核表达载体pET-32a上, 使用大肠杆菌BL21菌株进行RgDXR蛋白的原核表达及纯化, 实时荧光定量PCR方法分析RgDXR在不同组织、不同内生菌诱导下的表达特性。获得的RgDXR基因cDNA全长为1 425 bp, 编码474个氨基酸。生物信息学分析发现RgDXR基因编码的蛋白质具有典型的DXR功能结构域, 属于植物DXR基因家族。系统进化树分析表明, 地黄RgDXR与芝麻亲缘关系最近。荧光定量PCR结果显示, 栽培品种‘北京3号’根中RgDXR表达量最高。内生真菌诱导实验表明, RgDXR基因的转录水平与梓醇等环烯醚萜类物质的含量成正相关。本研究克隆所得的地黄RgDXR基因属于DXR基因家族, 推测其可能与地黄中环烯醚萜类物质的生物合成有关。

关键词：地黄; RgDXR基因; 生物信息学分析; 原核表达; 表达分析

收稿：2016-10-08   修定：2017-01-24

资助：国家自然科学基金(81603232)、国家“十二五”科技支撑计划(2011BAl06802)和河南中医学院博士科研基金(BSJJ2011-07)。

Cloning and expression analysis of 1-deoxy-D-xylulose 5-phosphate reductoisomerase gene in Rehmannia glutinosa

ZHU Yun-Hao, DONG Cheng-Ming, ZU Meng-Hang, LI Pan-Deng, ZHAO Le^*

School of Pharmacy, Henan University of Traditional Chinese Medicine, Collaborative Innovation Center for Respiratory Disease Diagnosis and Treatment & Chinese Medicine Development of Henan Province, Zhengzhou 450046, China

Corresponding author: ZHAO Le; E-mail: zhaole1983@126.com

Abstract:

The RgDXR cDNA was amplified from Rehmannia glutinosa by reverse transcription PCR. Bioinformatics analysis was conducted to analyze the properties, functional domains and evolutionary relationships of the gene. RgDXR was inserted into the prokaryotic expression vector pET-32a, and then transformed into E. coli BL21. Gene expression in different organs and inducing conditions were detected by real-time quantitative PCR. The RgDXR cDNA sequence contained a 1 425 bp open reading frame and encoded a predicted protein of 474 amino acids. Bioinformatic analyses revealed that RgDXR exhibited typical features of the DXR domain and belonged to the plant DXR superfamily. Phylogenetic tree analysis showed that RgDXR had the closest genetic relationship with Sesamum indicum. Tissue expression pattern analysis revealed that the expression of Rg-DXR was tissue-specific, and accumulation of transcripts was highest in roots of cultivated varieties ‘Beijing No.3’. After induced by endophyte fungi, the mRNA expression level of RgDXR was tightly correlated with the abundance of iridoid, such as catalpol. It is speculated that RgDXR might participated in the iridoid biosynthesis in R. glutinosa.

Key words: Rehmannia glutinosa; 1-deoxy-D-xylulose 5-phosphate reductoisomerase gene; bioinformatics analysis; prokaryotic expression; expression analysis