基于蛋白质组和转录组的紫外诱导银杏叶次生代谢产物生物合成研究

Ginkgo biloba extract and its preparations are important natural medicine for the treatment of cardiovascular and cerebrovascular diseases. However, influenced by producing area and harvest season, flavonoids content in Ginkgo biloba was unstable and low while phenolic acid content was relatively high, which has been the main factor to constrain the improvement of Ginkgo biloba extract. Our preliminary research demonstrated that total flavonoids content was significantly increased and phenolic acid content was dramatically decreased in Ginkgo biloba after induced by UV. Comparative proteomics study showed that the expression of the major rate-limiting enzyme, chalcone synthase (CHS) and phenylalanine ammonia lyase (PAL) , were significantly increased in the biosynthetic pathway of flavonoids in UV-induced Ginkgo biloba. HPLC analysis of the changes of flavonoids in chloroplast before and after UV radiation indicated that the chloroplast might be one of the flavonoid biosynthesis places. .This project intends to further develop the comparative proteomics study of Ginkgo biloba chloroplast and determine the function of chloroplast to participate in the biosynthesis of flavonoids. Combined with transcriptome studies, through fitting analysis between different expressed proteins and different expressing genes and validated by RT-PCR, We can explore the biosynthetic mechanism of the secondary metabolites of UV-induced Ginkgo biloba from protein level and transcriptional level and provide new methods and technologies to optimize medicinal quality and inrich medicinal resources.

银杏叶提取物及其制剂是治疗心脑血管疾病重要的天然药物，但受产地和采收季节等因素的影响，银杏叶黄酮含量偏低以及银杏酚酸含量偏高已成为制约银杏叶提取物质量达标的主要因素。我们前期研究表明，银杏叶经紫外诱导后总黄酮含量显著提高，银杏酚酸的含量降低明显；差异蛋白质组学研究发现，银杏叶中类黄酮成分生物合成途径中主要的限速酶-查尔酮合酶（CHS）、苯丙氨酸解氨酶（PAL）经紫外诱导后表达均显著上调；HPLC分析诱导前后叶绿体类黄酮成分的变化，显示叶绿体可能为银杏叶类黄酮成分生物合成场所之一。.本课题拟进一步开展银杏叶叶绿体的差异蛋白质组学研究，定位叶绿体在银杏叶类黄酮成分代谢网络中的功能；结合转录组学研究，通过差异蛋白与转录组差异表达基因间的相互匹配分析，并辅以RT-PCR验证，从蛋白质水平和基因水平诠释银杏叶相关次生代谢产物响应紫外诱导的生物合成机理，为优化药材质量、丰富药材资源提供新的技术手段。

本课题按照计划书安排，在充分调研文献及设计论证实验技术路线的基础上，建立了UV-B诱导离体银杏叶以提高其中活性产物含量的方法。通过对诱导前后银杏叶总黄酮和酚酸含量的分析测定以及诱导前后银杏叶抗氧化活性的对比，证明UV-B诱导能显著提高银杏叶活性产物含量并降低有毒成分酚酸的含量，该方法的建立为提高银杏叶药材的质量提供了一条新的潜在的技术手段。. 本课题还对诱导前后银杏叶及其叶绿体蛋白质组的差异表达进行研究。银杏叶片总蛋白和叶绿体蛋白分别鉴定到30和43个功能注解差异蛋白，这些蛋白质分别与光合作用、糖代谢、能量代谢、次生代谢、氨基酸合成、防御和抗氧化、翻译后修饰、信号转导和调控相关。其中，果糖-1,6-二磷酸醛缩酶、烯醇酶、苯丙氨酸解氨酶、查尔酮合酶等与植物黄酮类成分合成密切相关的蛋白质表达量发生改变，可能与酚酸含量的降低相关的 S-腺苷甲硫氨酸脱羧酶的表达量发生改变，上述数据从蛋白水平结果初步揭示了UV-B诱导下银杏叶活性成分变化的分子机理。本课题还选取了黄酮生物合成途径中的6个关键基因进行UV-B辐射下的表达趋势分析，与对照组相比，6个关键基因在UV-B照射阶段整体表现为下调表达，暗培养阶段表达上调，这些上调表达的基因从转录水平证实了银杏叶黄酮类成分积累的作用机理。