Zhang Zhi-Li, Lv Hai-Zhou, Guo Xu, He Liu, Song Jing-Yuan, Sun Chao, Luo Hong-Mei
Chinese Academy of Medical Sciences and Peking Union Medical College, Institute of Medicinal Plants/Endangered Medicinal Breeding National Engineering Laboratory, Beijing 100193, China.
College of Horticulture and Gardening, Yangtze University, Jingzhou 434025, China.
Zhongguo Zhong Yao Za Zhi. 2016 Nov;41(22):4169-4174. doi: 10.4268/cjcmm20162214.
The open reading frame of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR) was cloned from Phlegmarirus carinatus by RT-PCR method and the sequence was analyzed by bioinformatics tools. After searching the transcriptome dataset of P. carinatus, one unique sequence encoding 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase was discovered. The primers were designed according to the cDNA sequence of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase from the dataset. And then, the open reading frame (ORF) of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase, named as PcHDR1 (GenBank Accession number：JQ957845), was cloned by RT-PCR strategy with the template of mixed RNA extracted from roots, stem and leaf of P. carinatus. The bioinformatic analysis of this gene and its corresponding protein was performed. The ORF of PcHDR1 consisted of 1 437 base pairs (bp), encoding one polypeptide with 478 amino acids. The sequence comparison showed that PcHDR1 is closest with GbHDR (Ginkgo biloba),and the sequence homology was up to 78%. Bioinformatics prediction and analysis indicated that PcHDR1 protein contained a conserved domain of LytB, without transmembrane region and signal peptides. This study cloned and analyzed 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase from P. carinatus. The result will provide a foundation for exploring the function of PcHDR1 involved in terpene biosynthesis in P. carinatus plants.
通过RT-PCR方法从龙骨风（Phlegmarirus carinatus）中克隆了1-羟基-2-甲基-2-（E）-丁烯基-4-二磷酸还原酶（HDR）的开放阅读框，并利用生物信息学工具对其序列进行了分析。在搜索龙骨风的转录组数据集后，发现了一个编码1-羟基-2-甲基-2-（E）-丁烯基-4-二磷酸还原酶的独特序列。根据数据集中1-羟基-2-甲基-2-（E）-丁烯基-4-二磷酸还原酶的cDNA序列设计引物。然后，以从龙骨风的根、茎和叶中提取的混合RNA为模板，通过RT-PCR策略克隆了1-羟基-2-甲基-2-（E）-丁烯基-4-二磷酸还原酶的开放阅读框，命名为PcHDR1（GenBank登录号：JQ957845）。对该基因及其相应蛋白质进行了生物信息学分析。PcHDR1的开放阅读框由1437个碱基对（bp）组成，编码一个含有478个氨基酸的多肽。序列比较表明，PcHDR1与银杏（Ginkgo biloba）的GbHDR亲缘关系最近，序列同源性高达78%。生物信息学预测与分析表明，PcHDR1蛋白含有LytB保守结构域，无跨膜区和信号肽。本研究对龙骨风中的1-羟基-2-甲基-2-（E）-丁烯基-4-二磷酸还原酶进行了克隆和分析。该结果将为探索PcHDR1在龙骨风植物萜类生物合成中的功能奠定基础。
