Résumé
Protein complexes are involved in the synthesis of multiple secondary metabolites in plants, and their separation is essential to elucidate plant secondary metabolism and improve in vitro catalytic efficiency. In this study, the transgenic hairy roots of CYP76AH1, a key enzyme of tanshinone synthesis pathway, was constructed and the transgenic hairy roots of Danshen overexpressing CYP76AH1 protein were screened by Western blotting and used as a tissue culture material for the subsequent extraction of protein complex in tanshinone synthesis pathway. By optimizing the type and concentration of the detergent in the protein extraction buffer, the buffer containing 0.5% Triton X-100 was selected as the best extraction buffer, and a relatively large amount of soluble CYP76AH1 protein was isolated. This study lays the foundation for the further separation and purification of protein complexes interacting with CYP76AH1, and provides the idea for deep analysis of tanshinone metabolic pathway.
Résumé
Establishment of a hairy root culture system from the medicine plants transformed by the Agrobacterium rhizogenes has unique advantages in large scale production of secondary metabolites and provides an effective solution to the shortage of resources. Meanwhile, root-induced(Ri) plasmid is also an ideal vector for plant genetic engineering. Insect-resistant, disease-resistant genes or genes encoding key enzymes involved in biosynthesis of plant secondary metabolites can be harbored by Ri plasmid and integrated into the host plant genome for expression, thus improving the plant traits and enhancing the secondary metabolite content. Establishment of plant transgenic hairy root culture system has laid a solid foundation for regulating plant secondary metabolites content and for industrial production of pharmaceutical active ingredients by using of genetic metabolic engineering strategies. This paper reviews the latest research advances in this field and the related applications.