丹参SmJRB2基因的克隆与功能鉴定

Cloning and functional identification of SmJRB2 gene in Salvia miltiorrhiza

【目的】丹参Salvia miltiorrhiza是治疗心脑血管疾病的常用中药材。解析丹参药效物质合成代谢的分子调控机制能为丹参优质新品种的选育提供科学依据。【方法】基于比较转录组挖掘获得响应甲基茉莉酸(MeJA)诱导的转录因子SmJRB2。采用同源克隆技术克隆获得该基因的编码序列,并进行生物信息学分析。采用实时荧光定量PCR(RT-qPCR)分析SmJRB2基因的组织表达和MeJA诱导表达特征;基于农杆菌Agrobacterium tumefaciens介导的丹参遗传转化技术对SmJRB2基因的功能进行鉴定。【结果】SmJRB2共编码501个氨基酸,属于bHLH转录因子家族的MYC类转录因子。SmJRB2基因在丹参叶片和主根中的表达量最高。SmJRB2基因强烈响应MeJA的诱导,诱导4.0 h时表达量最高。超表达SmJRB2促进丹参酮的积累,抑制表达SmJRB2基因则降低丹参酮的合成。【结论】SmJRB2是丹参酮代谢合成的正向调节因子。

[Objective]Salvia miltiorrhiza is a traditional Chinese medicine used in clinical treatment of cardiovascular and cerebrovascular diseases.Elucidating the molecular regulation mechanism of metabolism and synthesis of pharmacophore of S.miltiorrhiza can provide scientific basis for breeding new varieties of S.miltiorrhiza with high quality.[Method]The transcriptional factor SmJRB2 in response to methyljasmonic acid(MeJA)induction was picked out based on comparative transcriptome mining.The coding sequence of this gene was cloned using homologous cloning technology and analyzed by bioinformatics.The tissue expression and MeJA induced expression of SmJRB2 gene were detected by quantitative real-time polymerase chain reaction(RT-qPCR).The function of SmJRB2 gene was identified based on the genetic transformation technology of S.miltiorrhiza mediated by Agrobacterium tumefaciens.[Result]The results showed that SmJRB2 encoded 501 amino acids and belonged to the MYC transcription factor of bHLH transcription factor family.The expression of SmJRB2 gene was the highest in leaves and principal root.SmJRB2 gene was intensively induced by MeJA and its highest expression level peaked at the induction time of 4.0 h.Overexpression of SmJRB2 promoted the accumulation of tanshinones and suppression of SmJRB2 gene decreased the biosynthesis of tanshinones.[Conclusion]SmJRB2 is a positive regulator of tanshinone metabolic synthesis.