穿心莲ApbHLH80基因克隆及表达分析

Cloning and expression analysis of ApbHLH80 gene in Andrographis paniculata

目的:克隆穿心莲转录因子ApbHLH80基因,并对其进行生物信息学分析和表达分析。方法:根据穿心莲转录组数据库设计引物,采用PCR扩增等方法克隆ApbHLH80全长序列,使用生物信息学在线分析工具,分析和预测该基因编码蛋白的理化性质,采用实时荧光定量PCR分析该基因在茉莉酸甲酯处理下的表达模式,并采用高效液相色谱法测定穿心莲内酯含量。结果:生物信息学分析表明,该基因开放阅读框长度包含1 095 bp,共编码364个氨基酸;该基因的氨基酸序列分子量约为39.96 ku,理论等电点为5.73,无信号肽和无跨膜结构,属亲水性核蛋白;该蛋白与苏麻、丹参、一串红和芡欧鼠尾草等物种亲缘关系较为接近,都具有b HLH_AtbHLH_like保守结构域。实时荧光定量PCR分析结果表明,ApbHLH80的表达量在叶片中最高,并且响应茉莉酸甲酯的诱导,其表达模式与穿心莲内酯合成存在密切联系。结论:成功克隆得到的ApbHLH80基因可能参与调控穿心莲内酯合成,为进一步探讨其潜在的调控作用提供理论支撑。

Objective:To clone the transcription factor gene ApbHLH80 of Andrographis paniculata(A.paniculata)and analyze its bioinformatics and expression.Methods:Primers were designed according to the transcriptome database of A.paniculata,and the full-length sequence of ApbHLH80 was cloned by PCR amplification and other methods.The physical and chemical nature of the protein encoded by this gene was analyzed and predicted through bioinformatics online tools.Reverse transcription-quantitative PCR(RT-qPCR)was used to analyze the expression pattern of this gene under methyl jasmonate treatment.And the content of andrographolide was determined by high-performance liquid chromatography(HPLC).Results:Bioinformatics analysis showed that the open reading frame length of the gene contained 1,095 bp and encoded 364 amino acids.The amino acid sequence encoded by this gene without signal peptide and transmembrane structure had a molecular weight of approximately 39.96 ku and a theoretical isoelectric point of 5.73,and it’s a hydrophilic protein in the nucleus.This protein was closely related to many species,such as Perilla frutescens var.Frutescens,Salvia miltiorrhiza,Salvia splendens and Salvia hispanica,all of which had the bHLH_AtbHLH_like conserved domain.The results of RTqPCR analysis showed that the gene expression of ApbHLH80 was the highest in leaves,and its expression pattern was closely related to andrographolide synthesis in response to the induction of methyl jasmonate.Conclu sion:The successful cloning of ApbHLH80 gene may regulate the andrographolide synthesis,providing theoretical support for further exploration of its potential regulatory effects.