滇龙胆GrHDR基因的克隆与表达分析

Cloning and expression analysis of GrHDR gene in Gentiana rigescens

关键词:  滇龙胆, GrHDR基因, 序列分析, 组织表达分析, 龙胆苦苷

Key words:   Gentiana rigescens, GrHDR gene, sequence analysis, tissue expression analysis, gentiopicroside

摘要:

龙胆苦苷(gentiopicroside)是中药龙胆中的主要药效成分,属于萜类化合物的衍生物。1-羟基-2-甲基-2-(E)-丁烯基-4-二磷酸还原酶[1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase,HDR]是萜类物质合成途径中的关键酶。为探讨不同光照条件下滇龙胆HDR(GrHDR)基因的表达与龙胆苦苷含量之间的关系,该文以滇龙胆叶片cDNA为模板,采用PCR和TA克隆技术获得GrHDR基因序列,对该序列进行生物信息学分析和表达分析,并采用高效液相色谱法测定龙胆苦苷含量,对该基因表达与龙胆苦苷含量进行比较。结果表明:(1)GrHDR基因(GenBank登录号: KJ917165.1)全长1 398 bp,编码465个氨基酸,推定GrHDR蛋白是亲水且稳定的,相对分子质量是52 281.25 Da,理论等电点是5.32;(2)该蛋白属于LYTB蛋白家族,可能定位于叶绿体上,无信号肽,二级结构主要由α-螺旋(45.16%)、β-转角(6.24%)、无规卷曲(33.98%)、延伸链(14.62%)构成;(3)GrHDR蛋白序列与同属植物秦艽的HDR蛋白相似性最高(95.71%);(4)实时荧光定量PCR结果显示GrHDR基因在滇龙胆中的表达量为根 > 叶 > 茎,而在10%、30%、100%全光光照条件下各组织的表达量有很大差异;(5)高效液相色谱法结果显示,不同光照条件下龙胆苦苷含量一致,均为根 > 叶 > 茎,其中100%全光光照下,药用部位根中龙胆苦苷含量达到7.141%,约是30%、10%全光光照条件的两倍,但该结果与同一光照条件下GrHDR基因表达规律不完全一致。该研究为阐述HDR基因功能及其与龙胆苦苷含量的关系提供参考。

Abstract:

Gentiopicroside, a derivative of terpenoids, is the main medicinal ingredient in traditional Chinese medicine “Long Dan”. 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase(HDR)is a key enzyme in the synthesis of terpenoids. In order to explore the relationship between the expression of Gentiana rigescens HDR(GrHDR)gene and contents of gentiopicroside under different light conditions, the cDNA from the leaves of G. rigescens was used as template, the GrHDR gene sequence was obtained by PCR and TA cloning technologies, and then bioinformatics analysis and tissue expression analysis were carried out. Finally, high performance liquid chromatography(HPLC)was used to determine the contents of gentiopicroside, and the expression of GrHDR gene were compared to the contents of gentiopicroside. The results were as follows:(1)The GrHDR gene(GenBank accession number: KJ917165.1)was 1 398 bp in length and encodes 465 amino acids, its putative protein was hydrophilic and stable, the relative molecular mass was 52 281.25 Da, and the theoretical isoelectric point was 5.32.(2)The putative protein that belonged to the LYTB protein family may be located on the chloroplast and had no signal peptide, and its secondary structure was mainly composed of α-helix(45.16%), β-turn(6.24%), random coil(33.98%), and extended chain(14.62%).(3)The GrHDR protein sequence had the highest similarity to the HDR protein of the G. macrophylla(95.71%).(4)The results of real-time PCR showed that the expression levels of GrHDR gene in G. rigescens were in the order of root > leaf > stem, its tissue expressions were significantly different under the condition of 10%, 30% and 100% of full-light.(5)The results of HPLC showed that the contents of gentiopicroside under different light conditions were in the order of root > leaf > stem; among them, the content of gentiopicroside in root(7.141%)under 100% of full light, which was about twice that of 30% and 10% of full light, but the results were not completely consistent with the GrHDR gene expression pattern under the same light condition. This study provides references for explaining the function of HDR gene and its relationship with gentiopicroside.