基于长春花干细胞培养体系的萜类吲哚生物碱生物合成及ORCA3 的调控机制研究

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.

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。

长春新碱等萜类吲哚生物碱（TIAs）只在长春花中生物合成，含量甚微，且受到严格的分子调控。长春花干细胞具有TIAs种类多、酶系丰富、易受诱导子调控等特质，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，本项目旨在利用该新型培养体系，通过诱导子和基因技术明确ORCA3的调控网络；阐明诱导子-ORCA3-基因表达-TIAs含量的关系。本项目主要进行了下述研究并取得重要成果：（1）考察了长春花干细胞对多种诱导子的响应情况，筛选出对长春花干细胞TIAs生物合成具有正向调控作用的诱导子：生物诱导子黄曲霉、腐皮镰孢菌和非生物诱导子茉莉酸甲酯、脱落酸、β-环胡精对长春花干细胞TIAs生物合成均有不同程度的促进作用。诱导子处理后，长春花干细胞TIAs的含量提高了1.45-17.5倍。转录组测试分析和qRT-PCR实验表明，生物碱含量的提高与D4H、G10H、GES、IRS、LAMT、SGD、STR、TDC和ORCA3基因表达呈正相关。诱导子处理后，上述基因的表达量增加至对照组的1.6-13.4倍；（2）分别采用CRISPR/Cas9基因编辑法和大麦条纹花叶病毒介导的基因沉默法考察ORCA3对长春花培养体系中生物碱合成的调控作用：沉默转录因子ORCA3后，TIAs生物合成关键酶基因D4H、G10H、GES、IRS、LAMT、SGD、STR和TDC的转录水平下降为空白对照组的0.01-0.23倍；（3）基因过表达法考察ORCA3对长春花培养体系中生物碱合成的调控作用：过表达ORCA3后，相对于对照组，长春花中的TIAs生物合成相关基因表达量均有不同程度的上升，其中D4H，G10H，GES，IRS，LAMT，SGD，STR，TDC等酶基因的表达量分别为对照组的2.22，3.73，2.71，1.12，2.42，1.05，1.99和4.53倍，且ORCA3基因的表达量是对照组的9.28倍；（4）转录因子ORCA3通过调控D4H，G10H，GES，IRS，LAMT，SGD，STR，TDC来调节长春花生物碱的合成。本项目研究阐明了转录因子ORCA3在长春花中的调控网络，明确了诱导子-ORCA3-基因表达-TIAs含量的关系，为生物技术法生产TIAs提供了重要科学依据。