三七PnUGT1基因的全长cDNA克隆和生物信息学分析

Full-length cDNA cloning and bioinformatics analysis of PnUGT1 gene in Panax notoginseng

本研究通过对三七的转录组高通量测序结果进行分析,采用5'-RACE和RT-PCR方法,对其中一条可能参与三萜皂苷合成的UDP-糖基转移酶基因转录本(Pn02086)进行全长克隆,预测了该基因编码蛋白的理化性质、保守结构域等,并对其进行了进化分析。通过全长克隆,得到一条开放阅读框为1 488 bp的cDNA序列,命名为PnUGT1,GenBank登录号JX018210。该基因编码495个氨基酸,蛋白分子量为55.453 kD,属于不稳定蛋白。二级结构中α-螺旋占36.16%、β-折叠占11.31%、无规卷曲占52.53%。InterProScan预测该蛋白具有7个保守结构域,包括在植物次生代谢产物中相关糖基转移酶特有的PSPG保守基序。该蛋白不具有信号肽和跨膜区,最有可能定位在细胞质中。序列比对和进化关系分析表明,该蛋白和蒺藜苜蓿三萜合成相关的UDP-糖基转移酶AAW56092的亲缘关系较近,PSPG保守区域的相似性为66%。该基因在三七叶中表达量较在花、茎和根中的表达量高。推测PnUGT1基因可能参与了三七皂苷的合成。

After searching the transcriptome dataset of Panax notoginseng,one unique sequence Pn02086 encoding UDP-glucosyltransferase（UGT）,which may be involved in triterpene saponin biosynthesis,was discovered.The open reading frame of the UGT gene,named as PnUGT1,was cloned by 5’-RACE and RT-PCR method from P.notoginseng.The GenBank accession number for this gene is JX018210.The bioinformatic analysis of this gene and its corresponding protein was performed.The PnUGT1 gene contains a 1 488 bp open reading frame and encodes a predicted protein of 495 amino acids.The molecular weight is 55.453 kD and the protein is unstable.In the secondary structure,the percentage of alpha helix,beta turn,random coil were 36.16%,11.31%,52.53%,respectively.The PnUGT1 contains 7 conserved domains predicted by InterProScan,including PSPG-box which is a unique consensus sequence of glycosyltransferases involved in plant secondary metabolism.The PnUGT1 was most likely to be located in the cytoplasm,without signal peptide and transmembrane region.Sequence alignment and phylogenetic analysis demonstrated that PnUGT1 had relative close relationship to the triterpene UDP-glucosyltransferase of Medicago truncatula（AAW56092）,with the 66% similarity of conserved domain PSPG-box.PnUGT1 was more abundant in P.notoginseng leaf than in flower,stem and root.Therefore,PnUGT1 gene may be involved in notoginsenoside biosynthesis.