木豆毛状根中黄酮类活性成分诱导增量及其生物合成调控机制初步研究

The use of plant in vitro tissue culture technology to produce bioactive forest ingredients and elucidate the related biosynthesis regulation mechanism, is an important research hotspot in the modern forestry biological agent and forest chemistry engineering fields. Cajanus cajan (L.) Millsp is a perennial woody plant that contains flavonoids as the main active ingredients with multiple medical and health benefits, which have become potential sources of new drugs and new chemical entities. In this project, the stable and high-productive C. cajan hairy root culture system was established via Agrobacterium rhizogenes-mediated transformation, which was used as the model platform for the production of bioactive flavonoids as well as the study of the related biosynthesis regulation mechanism. C. cajan hairy root cultures were treated by exogenous biotic and physicochemical elicitors to explore the relationship between the specific effect, concentration effect and time effect of elicitors and the biosynthesis of target metabolites, which aimed at promoting the increment of bioactive flavonoids. Through the differential transcriptome sequencing of elicitor-treated samples combined with bioinformatics and metabolomics, the key enzyme genes that regulate the biosynthesis of bioactive flavonoids were elucidated. This project is in line with the emerging development trend of the modern forest chemistry combined with the biotechnology and molecular biology, and can provide a technical system of C. cajan hairy root culture with high productivity of bioactive flavonoids for the pharmaceutical and chemical fields. This can get rid of the traditional way to obtain bioactive ingredients from natural forest resources, and promote the development of green emerging industries in forestry fields.

利用植物组织培养技术生产林源活性成分并阐明其生物合成调控机制，是现代林业生物制剂和林产化工领域研究的一项重要内容。木豆是多年生灌木植物，黄酮类化合物是其主要活性成分，具有多种医疗和保健功效，已成为众多新药和新化学实体的潜在来源。本项目通过发根农杆菌介导转化建立木豆毛状根培养体系，作为黄酮类活性成分生产及其生物合成调控机制研究的模式平台；通过外源理化和生物诱导子胁迫处理木豆毛状根，探析诱导子特异效应、浓度效应和时间效应与黄酮类化合物合成之间关系，促使目标活性成分进一步增量；通过诱导样品的差异转录组测序并结合生物信息学和代谢组学分析，初步阐明调控黄酮类活性成分生物合成的关键酶基因。本项目符合现代林产化学与生物技术和分子生物学相结合的新兴发展趋势，可为医药和化工领域提供体外生产黄酮类活性成分的木豆毛状根培养技术体系，摆脱从自然资源中获取高价值林源活性成分的传统方式，推动我国林业绿色新兴产业发展。

利用植物体外培养技术生产林源活性成分并阐明其生物合成调控机制，是现代林业生物制剂和林产化工领域研究的一项重要内容。木豆是多年生灌木植物，其含有的黄酮和芪类化合物具有多种医疗和保健功效。本项目系统优化并建立了发根农杆菌介导的木豆毛状根体外转化及高效液体培养体系，用以生产目标黄酮和芪类活性成分以及作为研究其生物合成调控机制的模式平台；系统开展了系列外源理化和生物因子对木豆毛状根的胁迫诱导研究，明晰了不同诱导子浓度/剂量/处理时间与目标活性成分积累之间关系，发现400 μM水杨酸诱导12 h时总目标黄酮类化合物增量最高为1.98倍，72 h时木豆芪酸增量最高为3.91倍，同时发现UV-B对黄酮和芪类成分的合成诱导出现了此消彼长的调控效果；据此开展了UV-B对木豆毛状根中黄酮和芪类活性成分生物合成调控机制的研究，发现UV-B可通过ROS及伤害/防御信号路径介导苯丙素途径相关生物合成酶基因的转录表达来调控目标活性成分的合成，且以CHS和STS基因主导的代谢流竞争极有可能是导致木豆毛状根响应UV-B辐射下黄酮和芪类活性成分含量变化差异的原因，同时通过分析诱导过程中目标活性成分含量变化和生物合成候选酶基因表达水平变化之间的相关性，初步阐明了一些与目标活性成分生物合成关系密切的关键酶基因。以上研究工作完成了本项目计划的目标和任务，研究结果为未来利用木豆毛状根培养技术工业化生产黄酮和芪类活性成分奠定了基础，同时也为后续深入研究木豆黄酮和芪类活性成分生物合成调控机制提供了理论参考。