中药材

目的：对川续断中川续断皂苷Ⅵ合成关键酶基因DaAACT进行克隆和表达分析，为进一步研究川续断皂苷Ⅵ合成机制奠定基础。方法：克隆川续断中DaAACT基因并进行生物信息学分析；分析DaAACT基因在川续断不同组织及低温诱导时的表达模式；构建DaAACT基因的过表达载体并异源转化拟南芥，并分析其功能。结果：克隆得到的川续断DaAACT基因全长1 780 bp,编码406个氨基酸；生物信息学分析表明DaAACT为稳定的亲水性膜蛋白；DaAACT与唇形科糙苏PuAACT聚为一支，同源性较高；DaAACT基因在川续断不同组织均有表达，在根中表达量最高，且受低温诱导表达；过表达DaAACT基因能促进拟南芥三萜化合物合成途径中AtHMGS、AtHMGCR、AtSE等基因上调表达，激活三萜代谢路径。结论：成功克隆得到川续断DaAACT基因，DaAACT在川续断的根、低温处理样品中表达量高，过表达DaAACT拟南芥中三萜皂苷合成关键酶基因表达上调，表明DaAACT基因具有促进川续断皂苷Ⅵ合成的潜在功能。

Objective: To clone and analyze the expression of DaAACT gene, a key enzyme gene for the synthesis of asperosaponin Ⅵ in Dipsacus asper,so as to lay the foundation for further study on the synthesis mechanism of asperosaponin Ⅵ.Methods: The DaAACT gene in Dipsacus asper was cloned and bioinformatic analysis was performed.The expression patterns of DaAACT gene in different tissues and low temperature induction were analyzed.The overexpression vector of DaAACT gene was constructed and allogenetically transformed into Arabidopsis thaliana,and its function was analyzed.Results: The total length of the cloned DaAACT gene was 1 780 bp, encoding 406 amino acids.Bioinformatics analysis showed that DaAACT was a stable hydrophilic membrane protein.DaAACT and PuAACT of Phlomis umbrosa in Labiaceae were clustered into one branch with high homology.DaAACT gene was expressed in different tissues of Dipsacus asper.The expression of DAAACT gene was high in root and was induced by low temperature.Overexpression of DaAACT gene could promote the up-regulated expressions of AtHMGS,AtHMGCR,AtSE and other genes in the triterpene synthesis pathway of Arabidopsis thaliana,and activate the triterpene metabolic pathway.Conclusion: DaAACT gene in Dipsacus asper has been successfully cloned.The expression of DaAACT gene is high in Dipsacus asperoides root and low temperature treated samples.Overexpression of DaAACT can up-regulate the expressions of key enzyme genes of triterpenoid saponin synthesis in Arabidopsis thaliana,indicating that DaAACT gene has potential function to promote the synthesis of asperosaponin Ⅵ.