The Research Of Plant Regeneration System And Genetic Transformation Of G10h,dat Gene In Catharanthus Roseus

Catharanthus roseus contains a group of about 130 terpenoid indole alkaloids (TIAs). Many TIAs are true lead compounds for drug development, including two commercially important cytotoxic dimeric alkaloids; vinblastine and vincristine are utilized in a number of different cancer chemotherapies. Most of these TIAs are produced at low levels in the natural plants and are difficult to be chemically synthesized due to their complex structures. Hairy root culture system and cell culture system offer promising alternatives for the production of these valuable alkaloids. Although transgenic hairy roots and cell cultures can produce a number of TIAs, they seldom produce the most valuable dimeric alkaloids, vinblastine and vincristine,only monomeric alkaloid can be produced in cell and tissue cultures, but at levels too low for commercialization. Metabolic engineering offers one of the most promising approaches for improving the product composition and increasing alkaloids yield in plants.In this study, an efficient and rapid somatic embryogenesis based plant regeneration systerm was standardized for large-scale genetic transformation in Catharanthus roseus. To increase TIAs production, key genes involved in the TIAs biosynthetic pathway, g10h and dat were overexpressed in transgenic Catharanthus roseus plants by using genetic engineering method. The results are as following.(1) An efficient and rapid somatic embryogenesis based plant regeneration systerm from Catharanthus roseus hypocotyls was establishment.(2) Monovalent or bivalent expression vectors (pCAMBIA2300-g10h, pCAMBIA2300-g10h-dat) were constructed which contained the rate-limiting enzyme genes, g10h, and/or dat.(3) A series of transgenic Catharanthus roseus were obtained using agrobacterium-mediated transformation. 19 independent transgenic plants containing g10h alone, 25 independent transgenic plants containing g10h and dat together were obtained.(4) Some transgenic plants were analyzed by real-time PCR. Results showed that foreign DNA fragments were randomly inserted into Catharanthus roseus genome.