目的 克隆刺五加的β-香树酯醇合成酶(β-amyrin synthase,bAS)基因,并分析其表达对皂苷量的影响。方法 利用同源克隆法克隆刺五加的bAS基因。利用Real time PCR技术,分析刺五加bAS基因的表达规律,分光光度法测定总皂苷量。结果 克隆到cDNA长度分别为1 223、1 226 bp的刺五加bAS1、bAS2基因。bAS1和bAS2基因在各时期和器官中均有表达,但表达量差异显著(P < 0.05)。bAS1基因在萌芽期的表达量最高,之后迅速降低,并基本维持恒定。在整个生长期中bAS2表现出低→高→低的表达特点。bAS1基因在各器官中的表达量基本恒定,bAS2基因在叶片中的表达量最高。茉莉酸甲酯(MeJA)处理后,bAS1基因的表达未发生显著变化,bAS2的表达量显著上升。刺五加的皂苷量与bAS2呈极显著的正相关关系(P < 0.01),bAS1基因未达显著水平。结论 bAS2可能是催化刺五加三萜皂苷生物合成的关键酶。

Objective To clone β-amyrin synthase (bAS) gene from Eleutherococcus senticosus and analyze the effect of its expression on saponin contents. Methods The sequence of cDNA of E. senticosus bAS was cloned by homologous cloning strategy. Quantitative real time PCR was developed to analyze the expression pattern of E. senticosus bAS gene. And E. senticosus saponin contents were measured by spectrophotometry method. Results Length 1 223 and 1 226 bp of E. senticosus bAS1 and bAS2 genes were cloned. The results showed that bAS1 and bAS2 were expressed in the each growth period and every organ of E. senticosus, and the expression differed significantly (P < 0.05). bAS1 showed the highest expression when the plants were grown in germination stage, then rapidly depressed, and changed slightly in the end. The expression of bAS2 showed the characteristic of low-high-low. The expression of bAS1 in different organs of E. senticosus was constant, but the highest content of the expression of bAS2 was in the leaves. With the treatment of methyl jasmonate (MeJA), bAS2 expression has been significantly improved and bAS1 without a significant changing. There exists significantly positive correlation (P < 0.01) between the content of E. senticosus saponins and the expression levels of bAS2, and bAS1 without a significant difference. Conclusion bAS2 may be a key enzyme gene in the biosynthesis of triterpenoid saponins.

河北省自然科学基金——石药集团医药联合研究项目(H2012401006);河北省教育厅资助科研项目(QN2014102);华北理工大学培育基金(GP201306)