基于丹参功能基因的道地品质形成的分子机制

Molecular Mechanism of Salvia miltiorrhiza Quality Formation Based on Single Nucleotide Polymorphism of Functional Genes

目的:探讨丹参功能基因单核苷酸多态性(SNP)和丹参有效成分之间的关系以及丹参药材产地特异性品质形成的分子机制。方法:以8个不同产地及变型的丹参为研究对象,采用高效液相色谱(HPLC)方法得到不同产地丹参成分指纹图谱,同时对丹参酮代谢途径中的3个功能基因丹参乙酰CoA酰基转移酶(SmAACT),4-diphosphocytidyl-2-C-methyl-Derythritol kinas(SmCMK),丹参异戊烯焦磷酸异构酶(SmIPPI)的cDNA全长进行聚合酶链式反应(PCR)扩增、克隆测序以及生物信息学分析。结果:获得了8个产地及变型共23株丹参样品中3个功能基因SmAACT,SmCMK,SmIPPI的cDNA全长序列。通过对3个功能基因的SNP位点及氨基酸多态性序列分析,分别发现18,16和14个SNP位点。HPLC指纹图谱可以将8个产地的丹参聚为两大类,北京、湖北、山东白花、山西、河南、山东紫花样品聚为一支,河北与内蒙古样品聚为另一支,这也说明丹参成分变化趋势与地理距离关联不大,而变型及品种因素对质量差异的贡献率较高。结论:不同产地丹参具有明显的遗传分化趋势,并形成了各自的产地特异性基因型。通过探讨不同产地丹参药材特异性品质形成的分子机制,为保证临床用药的稳定有效奠定基础,同时为丹参优良品种选育提供指导。

Objective: To investigate the relationship between the single nucleotide polymorphism(SNP)of function genes and effective components of Salvia miltiorrhiza and the molecular mechanism of specific quality formation of S. miltiorrhiza. Method: The fingerprints of components in S. miltiorrhiza from eight different habitats and varieties were obtained by high-performance liquid chromatography(HPLC). The full-length cDNA of three functional genes acetyl-CoA C-acetyltransferase(SmAACT),4-diphosphocytidyl-2-Cmethyl-D-erythritol kinase(SmCMK)and isopentenyl diphosphate isomerase(SmIPPI)in tanshinone metabolic pathway were amplified by polymerase chain reaction(PCR),cloned, and sequenced,followed by bioinformatics analysis. Result: The full-length cDNA sequences of three functional genes SmAACT,SmCMK,and SmIPPI in tanshinone metabolic pathway were obtained from 23 strains of S. miltiorrhiza from eight different habitats and varieties. As revealed by the analysis of SNP and amino acid polymorphisms of three functional genes,18,16,and 14 SNP sites were found respectively. HPLC results showed the samples from Beijing,Hubei,Shandong(No. SDB),Shanxi,Henan,and Shandong(No. SDZ)were clustered into one branch,and those from Hebei and Inner Mongolia were clustered into another branch,which suggested that the variation trend of S.miltiorrhiza components had little correlation with geographical distance,but the variety was a critical factor for the quality. Conclusion: There was an obvious genetic differentiation trend in S. miltiorrhiza from different habitats,and different origin-specific genotypes were formed. The molecular mechanism of the formation of the specific quality of S. miltiorrhiza from different habitats was discussed,which laid a foundation for the stability and effectiveness of clinical medication,and guided the breeding of excellent varieties of S. miltiorrhiza.