滇龙胆香叶醇-10-羟化酶基因克隆、生物信息学分析和表达

目的克隆滇龙胆Gentiana rigescens香叶醇-10-羟化酶(geraniol-10-hydroxylase,G10H)基因,对其进行定量表达以及生物信息学分析。方法采用PCR技术获得G10H的c DNA序列,对G10H蛋白进行理化性质、二级结构和三级结构等生物信息学分析,并利用实时荧光定量PCR方法检测G10H基因在滇龙胆根、茎、叶中的表达情况。结果克隆得到滇龙胆G10H基因,全长为1 400 bp,ORF 1 248 bp,编码415个氨基酸。生物信息学预测该基因编码蛋白质分子式为C_(2131)H_(3390)N_(586)O_(615)S_(17),等电点为7.62,不稳定系数为44.20,疏水性系数GRAVY为-0.245。G10H基因在滇龙胆根、茎、叶中均有表达,其中在根中表达量最高,茎中最低。结论首次从滇龙胆中克隆得到了G10H基因,为进一步阐明该基因的羟基化功能在滇龙胆龙胆苦苷生物合成途径中的重要作用奠定基础。

Molecular cloning, differential expression, and bioinformatics analysis of G10H gene in Gentiana rigescens

Objective To clone the geraniol-10-hydroxylase (G10H) gene from Gentiana rigescens and analyze the gene expression. Methods The cDNA sequence of G10H was obtained from G. rigescens using PCR technique. The physical and chemical properties, secondary structure and tertiary structure of G10H protein were forecasted and analyzed using related software. The expression of G10H gene was detected using real-time PCR in roots, stems, and leaves of G. rigescens. Results The cloned G10H gene was 1 400 bp including 1 248 bp open reading frame and encoding a predicted protein of 415 amino acids. Bioinformatics predicted that the gene encoding protein molecular formula was C₂₁₃₁H₃₃₉₀N₅₈₆O₆₁₅S₁₇, the isoelectric point was 7.62, the instability coefficient was 44.20, and the hydrophobic coefficient was -0.245. RT-PCR showed that G10H gene expressed in all organs. And the highest expression level was found in roots, while the lowest in the stems. Conclusion This G10H gene is cloned from G. rigescens for the first time. The results provide a foundation for exploring the mechanism of gentiopicroside biosynthesis in G. rigescens.