秦艽1-羟基-2-甲基-2-(E)-丁烯基-4-焦磷酸还原酶基因(GmHDR)的克隆和表达分析

Cloning and expression analysis of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase gene(GmHDR)from Gentiana macrophylla

关键词:  秦艽  GmHDR  序列分析  表达模式

Key words:  Gentiana macrophylla  GmHDR  sequence analysis  expression pattern

摘要:

龙胆苦苷(gentiopicroside)等裂环烯醚萜苷类化合物是中药秦艽中主要的有效成分,属于萜类化合物的衍生物,1-羟基-2-甲基-2-(E)-丁烯基-4-焦磷酸还原酶(1-hydroxy-2-methyl-2-(E)-butenyl- 4-diphosphate reductase, HDR)是植物萜类物质合成的关键酶之一。该研究在实验室高通量测序的基础上,采用RT-PCR方法克隆了秦艽HDR基因(即GmHDR基因),利用生物信息学的方法分析了GmHDR编码氨基酸序列的理化性质、信号肽、转运肽、亚细胞定位、保守结构域和高级结构等特征,并采用实时定量PCR分析了HDR的表达模式。结果表明:秦艽GmHDR基因包含一个完整的长1 392 bp的ORF框,编码463个氨基酸; GmHDR与萝芙木、艾菊等植物HDR蛋白具有很高的一致性(≥84%),无跨膜结构域,有叶绿体转运肽等结构,主要定位于叶绿体中。实时定量PCR结果显示,GmHDR基因在秦艽的花中进行表达量高,在叶、茎和根等部位表达较低。GmHDR在序列上与其他植物的HDR蛋白序列特征上存在很大的相似性; GmHDR基因主要在秦艽花中表达,可能主要参与花中萜类物质的合成。该研究结果可为今后研究秦艽环烯醚萜类化合物的生物合成机制提供依据。

Abstract:

Secoiridoids,such as gentiopicroside, are the main active compounds in “Qinjiao”, a traditional Chinese herbal medicine derived from the dried roots of Gentiana macrophylla. These compounds have widely biological and pharmacological effects, such as stomachic, choleretic, anti-hepatotoxic activities, anti-inflammatory, antifungal and antihistamine activities. Secoiridoids belonged to monoterpenoid, were biosynthesized via the secoiridoid pathway(sometimes also called “ridoid pathway”)in high plant. 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase(HDR)is one of the key enzymes in the pathway of iridoid biosynthesis. In this paper, we cloned the gene sequence of HDR from G. macrophylla, and analyzed the characteristic of sequence and expression patterns in order to know its roles in secoiridoid biosynthesis. Based on our library generated by high-through sequencing of G. macrophylla transcriptome, we cloned HDR gene from G. macrophylla(named as GmHDR)by RT-PCR. And the GmHDR coding amino acid sequence characterization, such as physicochemical characteristics, signal peptide, transit peptide, subcellular localization, conserved domain and secondary structure, analyzed with bioinformatics methods. Then we also detected the expression patterns of GmHDR in different parts of G. macrophylla by real time PCR. One 1 630-bp length sequence of GmHDR gene was obtained from G. macrophylla. GmHDR contains a completed open reading frame(ORF)of 1 392 bp, which encoded a polypeptide with 463 amino acids. GmHDR, the encoding protein by GmHDR, has high homology(identities ≥ 84%)to HDR proteins from Rauvolfia verticillata, Tanacetum parthenium and other plants. One neighbor joining tree was constructed to show evolution ship between GmHDR and HDR proteins from other plants using MEGA5.2 soft. The phylogenetic tree also gave a same conclusion that GmHDR had a closed relation with HDR proteins from Catharanthus roseus and Rauvolfia verticillata. Further analysis with bioinformatics methods showed that GmHDR was one protein without transmembrane domain,and had one transit peptide with 36 amino acids predicted with ChloroP server. These indicated that GmHDR protein might be located in the chloroplast. Real time quantitative PCR results showed that GmHDR had a very high expression level in the flowers of G. macrophylla, and GmHDR gene expressed lower in roots, stems and leaves of Gentiana macrophylla. Conclusion: The sequence of GmHDR had a lot of similar features with HDR proteins from other plants, such as conserved four-cysteine sites, transit peptide in their N terminal. GmHDR mainly expressed in the flowers of G. macrophylla. These results indicated that GmHDR may be principal involved in terpenoid biosynthesis, which are accumulated in flowers of G. macrophylla. The research is not only very helpful for research on HDR roles in G. macrophylla,but also will lay the foundation for the further study on biosynthetic pathway of secoiridoid compounds in G. macrophylla.