CAJ | 학술논문

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1876 bp,含有1个1464 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.
1-간기-2-갑기-2-E-정희기-4-초린산환원매(HDR)시갑기-D-적선순-4-린산(MEP)도경중적최후일개매,재식물첩류생물합성중기주공작용.해연구근거사모송(Pinus kesiya var．langbianensis )수피전록조수거분석결과,수선획득료사모송HDR기인편단,연후근거소획득적기인편단설계특이인물,제취수상후적사모송수피적RNA,병운용RT-PCR화RACE기술종사모송수피중극륭득도완정적HDR 기인(PkHDR).생물신식학분석표명：극륭획득적PkHDR1기인cDNA전장서렬위1876 bp,함유1개1464 bp적개방열독광(ORF),편마487개안기산.동원성분석결과표명：사모송 HDR단백여적송(Pinus densiflora)HDR단백적상사성고체99％.아세포정위급결구역분석결과표명：사모송PkHDR안기산서렬중포함전운태서렬(A1-A61)급식물 HDR단백다개보수적공능위점(A143,A234,A288,A371).계통진화분석결과표명：PkHDR단백여적송 HDR단백적친연관계최위접근.반정량PCR검측결과표명：수피적창상촉진사모송HDR기인적표체.해연구성공극륭획득HDR기인,병학정기여송지대사밀절상관,위천명사모송송지생물합성궤제화분자육충제공료삼고.
1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase(HDR)catalyzes the last step of the 2C-meth-yl-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.langbianen-sis was sequenced by Next-Generation Sequencing.First,a fragment of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphos-phate 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)-bute-nyl-4-diphosphate reductase.RNA of inj ured 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 se-quence 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 anal-ysis 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.