基于枇杷悬浮细胞系的熊果酸合成机制研究

Ursane-type and oleanane-type triterpene were two main triterpenoid skeletons. Amyrin Synthase(AS) was the first and the most critical enzyme to determine these two branches in the metabolic pathway, and its function are still unstated clearly, which became the critical node in ursolic acid (UA) synthesis mechanism. Applicants have established stable cell suspension lines from loquat, and obtained high levels of UA by adding NAA 0.5 mg/L. The suspension cultures can be used as an ideal material for AS function research, on the other hand, the regulation of loquat cell suspension to extract UA was a different technology way from the UA extraction of loquat leaves in production, which can overcome low productivity and high cost limitations. This research on UA synthesis mechanism will help improve the efficiency of further regulation.The project include the following several main contents: 1) Isolation several mRNA sequences of AS in loquat suspension cell lines by screening transcriptome database combined with RT-PCR and RACE, and cloning DNA sequences by PCR , and then analyzing those sequences by bioinformatics software; 2) Differential expression analysis of AS genes in different regulation conditions in loquat cell suspension cultures using real-time PCR method; 3)Construction of eukaryotic expression vector, and expression AS in yeast expression system and transient expression AS in loquat suspension cell; 4) Qualitative and quantitative detection of the catalytic products and target products by using GC-MS method and HPLC method; 5) Construction model by mathematical methods to describe UA synthesis mechanism in loquat suspension cells based on differences gene sequences of AS, differences expression of AS and differences function of AS.

熊果烷型和齐墩果烷型是植物五环三萜化合物最主要的骨架形式，香树素合酶（AS）是决定代谢途径这两分枝的首个关键酶，其功能未阐明成为熊果酸（UA）合成机制关键节点。申请者已建立稳定枇杷悬浮细胞系并通过添加NAA0.5mg/L获得富含UA的调控，可作为研究AS功能的理想材料，并且调控枇杷细胞悬浮系提取UA不同于生产上枇杷叶提取UA的技术路线，可克服后者产率低、成本高，UA合成机制研究有利于提高进一步的调控效率。本项目拟采用转录组测序结合RT-PCR和RACE在枇杷悬浮细胞中明确几个AS的核酸序列，用生物信息学进行序列分析；采用实时荧光定量PCR分析不同悬浮细胞调控体系中AS的表达差异；构建AS真核表达载体，转酵母表达系统和枇杷悬浮细胞瞬时表达系统，用GC-MS对催化产物及用HPLC对目标产物进行定性定量检测；用数学方法对AS的序列、基因表达、功能差异建立联系，阐述枇杷悬浮细胞UA合成的某种机制。

熊果烷型和齐墩果烷型是植物五环三萜化合物最主要的骨架形式，香树素合酶（AS）是决定代谢途径这两分枝的首个关键酶，阐明其功能成为熊果酸（UA）合成机制关键节点。调控枇杷悬浮细胞提取UA不同于传统生产上枇杷叶提取的技术路线，以富含UA的枇杷悬浮细胞的不同调控和枇杷叶为材料，用转录组测序结合RACE获取ASs成员的核酸序列；用实时荧光定量PCR分析ASs的表达差异；构建AS真核表达载体转化枇杷悬浮细胞瞬时表达，对催化产物定性定量检测；对AS的序列、基因表达、功能进行关联分析。此外，拓展：提纯萎陵菜酸（含量最高的一种五环三萜，干重>6%）进行体外抗肝癌细胞活性研究; 提取总三萜研发1个洗护用品。结果：1.枇杷悬浮细胞中存在两个AS， AS1的ORF与本研究者获得的枇杷叶AS1序列一致，其表达量与UA的变化趋势相似。AS2表达丰度极低且与UA的变化无相关性, 3端序列可能存在复杂的可变剪接，尝试多种方法仍未获得5端序列，推测与其是假基因有关（近缘物种苹果中AS2亦推测为假基因）。2.构建AS1过表达载体，通过农杆菌侵染瞬时转化枇杷悬浮细胞，AS1的过表达可以提高α-香树素含量2.52倍，提高β-香树素含量3.13倍。AS1属于α-/β-混合型（且α- amyrin ：β- amyrin=1:1），在枇杷UA代谢中起决定作用。3.拓展：萎陵菜酸具有体外抗肝癌细胞HepG2、Bel-7405、Sk-hep-1的潜能，并对正常细胞伤害很小；五种天然五环三萜以天然配比方式存在，共同构成 “美黑、抗菌消炎头皮按摩膏” 研发产品功能性的核心。意义：1.两个AS基因序列信息及表达特征，转录组数据及差异基因，为完善植物五环三萜类化合物合成机制和人工调控产物研究提供资料。2.首次构建枇杷悬浮细胞瞬时转化体系，可为基因功能研究奠定基础。3. 拓展:萎陵菜酸和总三萜可分别用于药品和化妆品的开发应用。