芥蓝BaAOP2基因不同拷贝在芥子油苷侧链修饰与代谢中的作用机制研究

Glucosinolates are a group plant secondary metabolites that mainly found in the order of cruciferaes, which was known to have miscellaneous biological function, such as anticancer activity. To date, glucosinolate biosynthetic pathway has been clarified in model plant Arabidopsis. However, significant differences in glucosinolate composition and content were found between Arabidopsis and Brassica plants, which primarily due to the side chain modification of glucosinolate. Therefore, glucosinolate biosynthesis in Brassica should require further complement and expansion. In our previous research, alkenylation of glucosinolate in Chinese kale was extremely high, and the alkenylation reaction was not inhibited after BaAOP2.1 gene silenced, indicating that other copies of BaAOP2 gene probably play important roles in the process. The objectives of this project are to artificially create single, double and triple mutants via the latest CRISPR/Cas9 genome editing technique, elucidate the function and mechanism of BaAOP2 gene copies in glucosinolate side chain modification, and explore to building the CRISPR/Cas9 application system on Brassica plant. This research will enrich our understanding to glucosinolate biosynthesis and metabolism network, and also provide theoretic foundation and technical support for improving the quality of Brassica vegetable using the method of genome editing technique.

植物次生代谢物质芥子油苷广泛存在于十字花科植物中，具有抗癌活性等多种重要的生物学功能。模式植物拟南芥中芥子油苷生物合成的途径已经基本清楚，但拟南芥与芸薹属植物中的芥子油苷组分与含量存在显著差异，侧链修饰是造成这种差异的重要原因。因此，芸薹属植物中芥子油苷的生物合成，特别是侧链修饰还需要进一步地补充和展开。我们前期研究发现芥蓝中芥子油苷烯基化程度极高，但沉默芥蓝BaAOP2.1基因后，烯基化反应并未被抑制，推测BaAOP2基因的其他拷贝极有可能在此过程中发挥重要作用。本项目拟通过最新的CRISPR/Cas9基因组编辑技术创制不同拷贝的单突、双突和三突突变体，进而揭示其在芥子油苷侧链修饰与代谢中的作用机制，并探索建立CRISPR/Cas9技术在芸薹属植物中的应用体系。该项目的实施将丰富对芥子油苷生物合成与代谢网络的认知，同时还为通过基因组编辑技术进行芸薹属蔬菜品质改良奠定理论基础。

芥子油苷是主要存在于十字花科植物中的一类植物次生代谢物质，具有重要的生物学功能。但是芥子油苷在拟南芥与芸薹属植物中组分与含量差异显著，侧链修饰是造成这种差异的重要原因。本项目针对前期研究发现的问题，深入研究了芥蓝中侧链修饰基因BaAOP2的不同拷贝在芥子油苷侧链修饰与代谢中的作用机制。主要结果有：1）克隆了芥蓝BaAOP2基因不同拷贝，揭示了不同拷贝的时空表达模式；2）建立了芥蓝叶肉原生质体分离纯化和瞬时表达体系，揭示了不同拷贝的亚细胞定位情况；3）通过原核表达等手段尝试探索了芥蓝BaAOP2基因不同拷贝的体外活性；4）系统地建立了芥蓝CRISPR/Cas9基因编辑体系，基于该技术，获得了芥蓝BaAOP2基因不同拷贝的CRISPR/Cas9沉默突变株，通过分子检测与表型鉴定等手段，初步确认了BaAOP2.1是芥蓝中控制芥子油苷侧链烯基化的主效基因，揭示了不同拷贝间的相互关系。项目研究结果丰富了对芥子油苷生物合成与代谢网络的认知，同时还为通过基因编辑技术进行芸薹属蔬菜品质改良奠定理论基础。