共生培养法促进石杉碱甲增量富集的研究

Study on promoting effect of co-culture system on the increment and enrichment of huperzine A

为了促进蛇足石杉中石杉碱甲增量富集,本文利用内生真菌与蛇足石杉的共生进化关系,以共培养体系(co-culture system,CCS)为基础,提出了增加石杉碱甲产率的新方法。以蛇足石杉植株存活率为指标,确定共培养体系最佳培养条件;以细胞存活率、愈伤组织诱导率和增殖率为指标,确定最佳培养基条件;以石杉碱甲和麦角甾醇为指标性成分,控制植物细胞和内生真菌比例关系,维持培养体系内植物细胞和内生真菌之间的生长平衡,获得最佳的继代时间。结果表明:CCS的最佳培养条件为光照强度100 Lux,湿度95%,MS液体培养基,蔗糖20.0 g/L,2,4-二氯苯氧基乙酸5.0 mg/L和激动素1.0 mg/L;4 d为最佳继代时间,其石杉碱甲的浓度最高,与1 d时相比增加了49.34%,并且石杉碱甲与麦角甾醇的浓度比值控制在1.5~2.2之间时,内生真菌生长对石杉碱甲的富集有良性促进作用。该研究为高效、低成本增加石杉碱甲产量提供新的探索路径,亦可以有效解决药源不足问题,具有很好的应用前景。

In order to promote the efficient enrichment of huperzine A,a new co-culture system(CCS),utilizing the symbiotic relationship between Huperzia serrata and endophytic fungi,was proposed.The optimal culture conditions of the CCS were obtained based on the cell survival rate,callus induction rate and multiplication rate.Huperzine A and ergosterol were applied as indicators to control the proportion of botanical cells and endophytic fungi.The growth balance between botanical cells and endophytic fungi was maintained for the optimal time of subculturing.The results showed that the best culture conditions for CCS was:100 Lux Luminous level,95%humidity,MS liquid medium,20.0 g/L sucrose,5.0 mg/L 2,4-dichlorophenoxyacetic acid and 1.0 mg/L kinetin.The 4 th day was the optimal subculturing time with the highest concentration of huperzine A,which increased by 49.34%compared with the huperzine A level on the 1 st day.The growth of endophytic fungal had a positive effect on the enrichment of huperzine A when the concentration ratio of huperzine A to ergosterol was kept between 1.5 and 2.2.This study provides a new proposal to increase the production of huperzine A with high efficiency and low cost,which can effectively solve the problem of insufficient drug source and has a good application prospect.