中文摘要
长春新碱等萜类吲哚生物碱（TIAs）只在长春花中生物合成，但含量甚微。人们曾试图通过组织培养等方法来提高其含量，但由于TIAs生物合成涉及复杂的组织与空间分布，且受到严格的分子调控，采用普通的培养体系而未成功。因此，寻找一种可对TIAs合成进行调控的新型培养体系并探究其调控机制已成为提高TIAs含量的瓶颈科学问题。我们的前期研究提示：长春花干细胞具有TIAs种类多、酶系丰富、易于受到诱导子调控等特质，且转录因子ORCA3可调控多个TIAs合成途径关键酶基因的表达。故，本项目拟利用该新型培养体系，通过基因技术明确ORCA3的调控网络；筛选最佳ORCA3诱导子；阐明诱导子-ORCA3-基因表达-TIAs含量的关系；过表达G10H和DAT基因；实现TIAs合成途径的整体调控。本项目可对长春花干细胞TIAs生物合成途径关键步骤的调控机制给予科学阐述，研究成果可为生物技术法生产TIAs提供科学依据。
英文摘要
Terpenoid indole alkaloids (TIAs, such as vincristine) are only obtained from Catharanthus roseus (L.) G. Don. And the concentration of TIAs in plants is very low. Scientists have been doing a lot to enhance the content of TIAs by cell cultures. However, the results are not satisfactory by using common culture system up to now. Complex tissue and spatial distribution might be the key problem in the biosynthesis of TIAs. Furthermore, the biosynthesis of TIAs is under strict control in the plant. Therefore, regulating the biosynthesis of TIAs with a new almighty cultured cells' system is the bottleneck problem in the procedures of increasing the concentration of TIAs by cell cultures. Our previous research has given us a light that stem cultured cells of C. roseus contain many kinds of TIAs and abundant enzymes, easy to be regulated by elicitors. Transcription factor ORCA3 regulates many genes of the key enzymes related to the biosynthetic pathway of TIAs. Therefore, the stem cell culture system of C. roseus, which retains all the genetic information of parental plant cells, might be used in the current project. In this project, firstly, genes controlled by ORCA3 would be elucidated by the methods of gene overexpression and gene silencing. Secondly, the best ORCA3 promoter would be screened. Based on this foundation, the relationship among elicitors- ORCA3-genes expression-content of TIAs, over expressing G10H and DAT genes in stem cultured cells of C. roseus would be evaluated. Thus, the whole biosynthesis pathway of TIAs could be regulated. The regulatory mechanism of the key step in biosynthesis pathway of TIAs of C. roseus stem cells will be elucidated with the successful completion of this project. And the scientific information about biosynthesis of TIAs in large scale by biotechnology method would be provided.
