龙骨马尾杉PcHDR1基因克隆及序列分析

Cloning and bioinformatics analysis of 1-hydroxy-2-methyl-2-( E)-butenyl4-diphosphate reductase(PcHDR1) gene in Phlegmarirus carinatus

该研究采用RT-PCR方法对龙骨马尾杉Phlegmarirus carinatus(Desv.)Ching 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]基因Pc HDR1的编码区进行克隆并利用生物信息学方法进行序列分析。根据实验室已获得的龙骨马尾杉转录组数据,从中获得1条编码HDR的转录本,采用RT-PCR方法获得该基因的全长c DNA序列,所克隆的Pc HDR1基因编码区长为1 437 bp,编码478个氨基酸残基,Gen Bank登录号为JQ957845。Pc HDR1与银杏Ginkgo biloba的HDR序列同源性最高,达78%。生物信息学预测Pc HDR1蛋白没有跨膜区,具有Lyt B保守结构域,不含信号肽。该研究克隆并获得了龙骨马尾杉Pc HDR1基因的编码区序列,并对其编码的蛋白进行了序列分析及结构域预测,为进一步研究HDR的功能奠定基础。

The open reading frame of l-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 transeriptome 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）-buteny] 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 （Gen- Bank 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 consis- ted of 1 437 base pairs （bp） , encoding one polypeptide with 478 amino acids. The sequence comparison showed that PcHDR1 is clo- sest 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 ana- lyzed 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.