目的 探究温度调控对人参皂苷积累及其合成途径关键酶基因表达量的影响。方法 以培养23 d人参愈伤组织为实验材料，置于5、10、15、20、25、30℃ 6个培养箱中恒温培养，每天取样1次连续取样8 d，测定干、鲜质量和皂苷含量，确定响应敏感温度，以GAPDH为内参基因，real-time PCR检测皂苷合成途径中9个关键酶3-羟基-3-甲基戊二酰辅酶A还原酶（HMGR2）、法尼基焦磷酸合成酶（FPS）、角鲨烯合成酶（SS1）、角鲨烯环氧酶（SE1）、达玛烷二醇合成酶（DS-Ⅱ）、β-香树素合成酶（PNY1）、β-香树素C-28位羟基化酶（CYP716A52v2）、原人参三醇合成酶（CYP716A53v2）、原人参二醇合成酶（CYP716A47）基因表达量。结果 20℃为人参愈伤组织干、鲜质量积累最佳温度，5、10、15℃皂苷含量2~3 d达到最大值，5℃为人参皂苷积累响应敏感温度，与对照组差异显著，人参皂苷Re、Rg₁和总皂苷分别为对照组1.93、11.93和1.54倍。HMGR2、SS1、DS-Ⅱ、SE1和CYP716A52v2基因表达量在低温2~4 d均达最大值，为对照组2.8、1.6、3.5、3.7和3.8倍。相关性分析发现SE1与人参皂苷Re、Rg₁和总皂苷呈显著正相关关系。结论 适度的低温有利于人参皂苷的快速积累，SS1、DS-Ⅱ、SE1、HMGR2、CYP716A52v2为响应低温的关键基因，在人参皂苷生物合成抵御低温的过程中发挥重要作用。

Objective To explore the effect of temperature regulation on the accumulation of ginsenosides and the expression of key enzyme genes in the synthetic pathway. Methods The ginseng callus cultured for 23 d was used as the test material and placed in six incubators at 5, 10, 15, 20, 25, and 30℃. The dry fresh weight and saponin content were measured to determine the response sensitive temperature. GAPDH was used as the internal reference gene. Real-time PCR was used to detect nine key enzymes in the saponin synthesis pathway, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR2), Farnesyl pyrophosphate synthase (FPS), squalene synthetase (SS1), squalene epoxidase (SE1), dammarane diol synthase (DS-II), β-xanthin synthase (PNY1), β-xanthophyll C-28 hydroxylase (CYP716A52v2), protopanaxatriol synthase (CYP716A53v2), and protopanaxadiol synthase (CYP716A47). Results 20℃ was the optimal temperature for the accumulation of dry and fresh weight of ginseng callus. The content of saponin in 5, 10, and 15℃ reached the maximum value for 2-3 d. Re, Rg₁, and total saponins were 1.93, 11.93, and 1.54 times that of the control group, respectively. The expression levels of HMGR2, SS1, DS-II, SE1 and CYP716A52v2 reached their maximum values at 2-4 d of low temperature, which were 2.8, 1.6, 3.5, 3.7, and 3.8 times higher than those of the control group. Correlation analysis found that SE1 was significantly positively correlated with Re, Rg₁ and total saponins. Conclusion Moderate low temperature is conducive to the rapid accumulation of ginsenosides. SS1, DS-II, SE1, HMGR2, and CYP716A52v2 are key genes that respond to low temperatures, which play an important role in the process of ginsenoside biosynthesis against low temperatures.

R282.12

2020年吉林省中医药科技项目（2020042）；长春中医药大学“杏林学者青年科学家”资助项目；2020年长春中医药大学大学生创新创业训练计划支持项目；2020年吉林省中医药科技项目（2020052）