思茅松HDR基因全长cDNA克隆与序列分析

Cloning and sequence analysis of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase gene cDNA from Pinus kesiya var. langbianensis

关键词:  思茅松  HDR  cDNA 克隆  基因功能分析  半定量PCR

Key words:   Pinus kesiya var. langbianensis  1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase gene  cDNA clone  gene function analysis  RT-PCR

摘要:

1-羟基-2-甲基-2-E-丁烯基-4-焦磷酸还原酶(HDR)是甲基-D-赤藓醇-4-磷酸(MEP)途径中的最后一个酶,在植物萜类生物合成中起主控作用。该研究根据思茅松(Pinus kesiya var. langbianensis)树皮转录组数据分析结果,首先获得了思茅松HDR基因片段,然后根据所获得的基因片段设计特异引物,提取受伤后的思茅松树皮的RNA,并运用RT-PCR和RACE技术从思茅松树皮中克隆得到完整的HDR基因(PkHDR)。生物信息学分析表明:克隆获得的PkHDR1基因cDNA全长序列为1 876 bp,含有1个1 464 bp的开放阅读框(ORF),编码487个氨基酸。同源性分析结果表明:思茅松HDR蛋白与赤松(Pinus densiflora)HDR蛋白的相似性高达99%。亚细胞定位及结构域分析结果表明:思茅松PkHDR氨基酸序列中包含转运肽序列(A1-A61)及植物HDR蛋白多个保守的功能位点(A143,A234,A288,A371)。系统进化分析结果表明:PkHDR蛋白与赤松HDR蛋白的亲缘关系最为接近。半定量PCR检测结果表明:树皮的创伤促进思茅松HDR基因的表达。该研究成功克隆获得HDR基因,并确定其与松脂代谢密切相关,为阐明思茅松松脂生物合成机制和分子育种提供了参考。

Abstract:

1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase(HDR)catalyzes the last step of the 2C-methyl-D-erythritol-4-phosphate(MEP)pathway,1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase plays an important role in regulation of terpenes biosynthesis. To explore the function of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase in Pinus kesiya var. langbianensis,and to study the role of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase in regulation of resin biosynthesis,the transcriptome of bark of Pinus kesiya var. langbianensis was sequenced by Next-Generation Sequencing. First,a fragment of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase gene was obtained from Pinus kesiya var. langbianensis transcriptome after gene assemble and gene function annotation. The special primers were designed according to the fragment of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase. RNA of injured bark was extracted by Trizol method. The full length gene of PkHDR was cloned from Pinus kesiya var. langbianensis by Reverse Transcription-Polymerase Chain Reaction(RT-PCR)and rapid-amplification of cDNA ends(RACE). Bioinformation analysis showed that the obtained full cDNA sequence of PkHDR had 1 876 bp. It was consisted of 1 464 bp open reading frame(ORF)which encoded 487 amino acid. Homology analysis indicated that the deduced PkHDR protein shared 99% identities with the 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase came from Pinus densiflora. Subcellular localization and structural domain analysis showed that the transit peptide sequence(A1-A61)and multiple conserved functional sites(A143,A234,A288,A371)of plant HDR protein were found in the deduced coding sequence of PKHDR. Phylogenetic analysis revealed that the evolutionary relationship of PkHDR protein was the closest to Pinus densiflora HDR protein. Reverse transcription polymerase chain reaction(RT-PCR)detection showed that PkHDR gene expression was up-regulated by wounding treatment. The full cDNA of PkHDR from Pinus kesiya var. langbianensis was cloned and the reverse transcription polymerase chain reaction(RT-PCR)showed that PkHDR was involved in regulation of resin biosynthesis in Pinus kesiya var. langbianensis. These results would provide important information to reveal the resin biosynthesis in Pinus kesiya var. langbianensis. And this study also can be applied in the research of the high yield of resin variety molecular breeding.