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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。
In this study, the high-throughput sequencing results of the Panax notoginseng transcriptome were analyzed. One of the UDP-glycosyltransferase transcripts (Pn02086), which may participate in the synthesis of triterpene saponins, was amplified by 5’-RACE and RT- Full-length cloning was carried out to predict the physicochemical properties and conserved domains of the protein encoded by the gene and to carry out evolutionary analysis. Through full-length cloning, a 1 488 bp open reading frame cDNA sequence, named PnUGT1, GenBank accession number JX018210. The gene encoding 495 amino acids, the molecular weight of 55.453 kD, belonging to unstable protein. In the secondary structure, α-helix accounted for 36.16%, β-sheet accounted for 11.31% and random coil accounted for 52.53%. InterProScan predicts that this protein has seven conserved domains, including the PSPG-conserved motifs unique to related glycosyltransferases in plant secondary metabolites. The protein does not have a signal peptide and a transmembrane region, most likely localized in the cytoplasm. Sequence alignment and phylogenetic analysis showed that the protein was closely related to the UDP-glycosyltransferase AAW56092, a tricholinergic synthesis of Medicago truncatula, with a similarity of 66% in the conserved region of PSPG. The expression level of the gene in the thirty-seven leaves was higher than that in the flowers, stems and roots. It is speculated that PnUGT1 gene may be involved in the synthesis of notoginseng saponins.