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根据刺五加鲨烯合酶基因2(squalene synthase,SS2)和鲨烯环氧酶基因1(squalene epoxidase,SE1)的DNA序列,通过Southern杂交和q RT-PCR法确定SS2和SE1的拷贝数。以actin为内参照基因,利用q RT-PCR法和分光光度法确定不同SS2和SE1拷贝数下的基因表达量和皂苷含量。结果表明,刺五加的SS2存在1和2拷贝,SE1存在1、2和3拷贝的类型及其相互组合的6种拷贝数组合基因型。刺五加SS2和SE1的表达量与皂苷含量均随拷贝数的增加而显著提高(p<0.05),两者间存在极显著的正相关关系(P<0.01)。SS2和SE1每增加1个拷贝可使皂苷含量分别提高0.57和0.42倍。研究结果为进一步揭示刺五加皂苷含量差异形成的分子机理奠定了基础。
The copy numbers of SS2 and SE1 were determined by Southern blot and q RT-PCR based on the DNA sequence of squalene synthase 2 (SS2) and squalene epoxidase 1 (SE1) . Using actin as an internal reference gene, q RT-PCR and spectrophotometry were used to determine the gene expression and saponin content under different copy numbers of SS2 and SE1. The results showed that there were 1 and 2 copies of SS2 in Acanthopanax senticosus, 1, 2 and 3 copies of SE1, and 6 copies of the combined genotypes. Acanthopanax senticosus SS2 and SE1 expression levels and saponin content were significantly increased with the copy number increased (p <0.05), there was a significant positive correlation between the two (P <0.01). Each additional copy of SS2 and SE1 increased the saponin content by 0.57 and 0.42 fold, respectively. The results laid the foundation for further revealing the molecular mechanism of the differences in the content of acanthopanax senticosus.