金银花细胞悬浮培养生物积累咖啡酰奎宁酸的诱导调控及其分子机制研究

Caffeoylquinic acid is commonly used in the food, healthy products and medicine because of possessing bacteriostatic effects, antioxidant, cancer prevention and so on. It was also suggested caffeoylquinic acid is the most responsible for the bacteriostatic activity of Lonicera japonica leaves by the completed National Science Foundation of China of the applicant. This project aim at elicitation and induction mechanism of caffeoylquinic acid biosynthesis in L. japonica cell suspension cultures using suspension culture plant cell and secondary metabolic elicitation technology to overcome production lack. The suspension cell culture system will be established by culture conditions optimization applying response surface methodology. The elicitor for enhancement accumulation of caffeoylquinic acid will be screened by comparing treatment effect of single and interaction factors among biological, physical and chemical factors. Mechanisms of inducing signal generation and conduction will be explored by comprehensive analysis the relevance between gene expression, enzyme activity and product accumulation. The chromatogram fingerprint will be established based on HPLC-MS method and the kinetic equations of product accumulation will be got through continuous measuring and calculation. Elicitor, induction mechanism and accumulation kinetic equations of caffeoylquinic acid biosynthesis in L. japonica cell suspension cultures will be clarified in this project, which can provide value theoretical basis on elicitation other plant cell suspension cultures for efficient biosynthesis functional secondary metabolites. The launch and completion of this project will widen ideas to break resources bottleneck for further development and utilization caffeoylquinic acid.

咖啡酰奎宁酸具有较强的抑菌、抗氧化、预防肿瘤等功能，在食品、保健品、医药中广泛应用。前个国家基金研究表明咖啡酰奎宁酸是金银花叶抑菌的主要物质，本项目针对其产量匮乏现状，拟利用细胞悬浮培养和次生代谢调控技术，研究金银花细胞悬浮培养生物积累咖啡酰奎宁酸的诱导调控及分子机制。采用响应面法优化培养条件建立悬浮细胞培养体系，通过比较生物、物理、化学单因素及交互因素作用效果系统筛选高产咖啡酰奎宁酸的诱导子，综合分析基因表达、酶活性与产物积累的相关性探索诱导信号产生、传导分子机制，建立基于HPLC-MS法咖啡酰奎宁酸指纹图谱，并通过连续测量计算产物积累动力学方程。通过本项目研究筛选出金银花细胞悬浮培养体系诱导子、阐明诱导作用机制和产物积累规律，对诱导调控其他植物细胞悬浮培养体系高效积累次生代谢产物提供理论依据。项目的开展和完成为突破咖啡酰奎宁酸深度开发的资源瓶颈拓宽思路。

背景：咖啡酰奎宁酸具有较强的抑菌、抗病毒等功能，在食品、保健品、医药中广泛应用。前个国基研究表明咖啡酰奎宁酸是金银花叶抑菌的主要物质，本项目针对其产量匮乏现状，拟利用细胞悬浮培养和次生代谢调控技术，研究金银花细胞悬浮培养高效生物积累咖啡酰奎宁酸的诱导调控及分子机制。.主要内容：建立悬浮细胞培养体系，筛选高产咖啡酰奎宁酸的诱导子，探索悬浮细胞咖啡酰奎宁酸高效积累的分子机制和积累规律，总结悬浮细胞中营养素消耗规律。.重要结果、关键数据等及其科学意义：.建立金银花细胞悬浮培养体系：金银花芽消毒处理后接种在激素配比为NAA 1.5 mg/L + 2,4-D 1.0 mg/L + KT 0.75 mg/L +6-BA 0.15 mg/L的MS培养基中诱导出愈伤组织，经激素组合为6-BA 1.5 mg/L +NAA 0.2 mg/L + 2,4-D 0.1 mg/L MS培养基继代培养后，将夹碎的愈伤组织按接种量为0.2g/mL接种到蔗糖浓度为30 g/L、激素配比为6-BA 1.4 mg/L+ NAA 0.5 mg/L+2,4-D 0.7 mg/L、初始pH为5.8的MS液态培养基中建立金银花细胞悬浮培养体系。.筛选出金银花细胞悬浮培养体系诱导子：茉莉酸甲酯（MeJA）、水杨酸（SA）、紫外线（UV）为高效诱导子，诱导条件为MeJA 200μmol/L、SA 50μmol/L以及UV照射2h。.阐明诱导作用机制：通过转录组测序分析，TRINITY_DN15699_c4_g2(C4H)、TRINITY_DN15990_c3_g3(PAL)、TRINITY_DN14333_c0_g1(HCT)、TRINITY_DN14013_c1_g9(C3’H)、TRINITY_DN13411_c1_g1(CAD)、TRINITY_DN14179_c1_g1(4CL)上调表达，可能为CGA生物合成的关键酶基因，悬浮细胞中绿原酸合成途径可能为由C3'H羟基化生成CGA。.明晰产物积累规律：绿原酸类物质总含量在干细胞中高达9.68%，产物中异绿原酸A含量最高。.探明悬浮细胞营养素消耗规律：碳、氮和磷都是先快速下降，后缓慢减少，诱导组的消耗速度更慢。总糖的消耗速度慢于还原糖；铵态氮则快于硝态氮，氮源的量到后期均有所回升。.科学意义：项目为咖啡酰奎宁酸的生物积累提供了一个方便、快速的工厂化生产方式。