竹节参达玛烷合成酶基因的克隆和生物信息学分析

Cloning of Dammarenediol Synthase Gene from Panax japonicus C.A. Mey and Its Bioinformatics Analysis

达玛烷合成酶(dammarenediol synthase,DS)是三萜类皂苷化合物进入达玛烷型皂苷合成途径的首个关键酶,对人参属植物三萜皂苷的生物合成具有重要的调控作用。本实验以6年生栽培种竹节参为实验材料,基于竹节参转录组数据,利用RT-PCR方法克隆得到DS基因的全长cDNA序列,命名为pjDS。该基因序列长度为2556 bp,其中包含一段完整开放阅读框,长达2310 bp,一共编码了769个氨基酸。经生物信息学分析后得出,该基因编码的蛋白质分子量为88.40 kD,理论等电点为5.84,为亲水性蛋白质,并含有跨膜区,具有56处磷酸化位点。其二级结构主要由α-螺旋(44.21%)和无规则线圈(34.72%)构成,并具有QW(QXXXXXW)和DCTAE这2种氧化鲨烯环化酶(OCS)家族的特征性保守基序。pjDS蛋白序列与人参(ACZ71036.1)、西洋参(AGK62449.1)、三七(AIK21788.1)、锡金三七(ANB82450.1)和越南人参(AGS16975.1)氨基酸序列的相似性分别为99%、99%、99%、99%和98%。实时荧光定量PCR的结果表明,pjDS基因在竹节参的根状茎、茎、叶和花等各器官组织均有所表达,进一步发现该基因在花中表达量最高,而在根状茎中的表达量最低。本研究为进一步研究竹节参中三萜皂苷生物合成机制提供理论依据。

Dammarenediol synthase(DS)is considered as the first key enzyme in the process of synthesis pathway of dammarane saponin,which plays a critical regulating role in ginsenoside biosynthesis pathway.In this study,A6-year-old Panax japonicas was used as experimental material.Based on the transcriptome data of Panax japonicus.the full-length cDNA sequence of DS gene was cloned by RT-PCR and named pjDS.The length of the gene is2556 bp containing a complete ORF(open reading frame)of 2310 bp,which encodes 769 amino acids.The bioinformatics analysis showed that the putative molecular weight and theory isoelectric point of the protein encoded by pjDS were 88.40 k D and 5.84,respectively.This hydrophilic protein contained a transmembrane region and 56 phosphorylation sites.Its secondary structure was mainly composed ofα-helix(44.21%)and random coil(34.72%).And two conserved motifs of oxidosqualene cyclase(OSC)-QW(QXXXXXW)and DCTAE were also found in the gene.The protein sequence similarity of DS genes was analyzed between P.japonicus and P.ginseng(ACZ71036.1)P.quinquefolius(AGK62449.1),P.notoginseng(AIK21788.1),P.sokpayensis(ANB82450.1)and P.vietnamensis(AGS16975.1)respectively,which showed the high similarities(99%,99%,99%,99%and 98%).Moreover,q RT-PCR analysis indicated that different expressions in the rhizome,stem,leaves and flowers of P.japonicus,in which pjDS was significantly up-regulated in the flower but not obviously increased in the rhizome.This study provided a theoretical basis for further study on the mechanism of triterpenoid saponin biosynthesis in Panax japonicus C.A.Mey.