鱼腥草1-脱氧-D-木酮糖-5-磷酸合成酶1基因克隆与表达分析

Cloning and expression analysis of 1-deoxy-D-xylulose-5-phosphate synthase 1 gene in Houttuynia cordata

目的克隆鱼腥草1-脱氧-D-木酮糖-5-磷酸合成酶1(DXS1)基因并分析其表达差异。方法根据已经获得的鱼腥草DXS1转录本序列设计1对引物,采用RT-PCR方法获得DXS1基因cDNA序列并对DXS1蛋白进行理化性质、蛋白二级结构及三维结构预测分析,并预测了该蛋白功能;利用实时荧光定量PCR方法检测了DXS1基因在鱼腥草的地下茎、地上茎、叶、花中的表达情况。结果克隆获得的DXS1基因长为2 172 bp,编码723个氨基酸。生物信息学预测DXS1蛋白不含跨膜区,不含信号肽,具有定位肽。DXS1基因在鱼腥草的花中表达丰度最高,其次是叶片,再次是地下茎,地上茎中表达量最低。结论首次从鱼腥草中克隆了DXS1基因,为进一步阐明该基因在鱼腥草萜类化合物代谢途径中的重要作用奠定基础。

Objective To clone the 1-deoxy-D-xylulose-5-phosphate synthase 1（DXS1） gene from Houttuynia cordata and to analyze the expression difference.Methods The cloning primers were designed based on the transcriptome dataset of H.cordata,one unique sequence encoding DXS1 was discovered.The sequence of DXS1 was cloned from H.cordata by RT-PCR.The physicochemical properties,secondary structure,and three-dimensional structure of the DXS1 protein were forecasted and analyzed,and its structure and function were predicted.And the different expression levels of DXS1 gene in rhizome,stems,leaves,and flowers were analyzed by fluorescent quantitative PCR.Results The cDNA（named as DXS1） contains a 2 172 bp open reading frame and encodes a predicted protein of 723 amino acids.No transmembrane region and signal peptide were present in DXS1.The conserved domain of DXS was present in DXS1.Relative real-time PCR analysis indicated that DXS1 showed the highest transcript abundance in the flowers,moderate level in the leaves,lower level in the rhizomes,and the lowest level in the stems.Conclusion This study cloned the DXS1 gene from H.cordata for the first time.The results will lay a foundation for exploring the mechanism of terpenoid biosynthesis in H.cordata plants.