Regulation Of Flavonoid Biosynthesis By Overexpression Chalcone Isomerase Gene In Saussurea Involucrata

Saussurea involucrata Kar. et Kir. is a traditional medicinal plant that is well known for its richness in flavonoids. Supply of this plant has been from only its wild populations, since trials for field cultivation remain unsuccessful. In the wild, S. involucrata grows on snowy mountains at altitudes of 4,000 to 5,000 m, and now this valuable plant species is under an enormous pressure for survival. An alternative way to produce the bioactive compounds would be by in vitro tissue/cell culture systems, but cell lines tend to accumulate only low levels of secondary metabolites and are genetically unstable. Hairy root cultures are more attractive by the facts of rapid growth, frequent branching and, more importantly, their ability to synthesize the same compounds as the intact plants do. However, in most cases the yields of specific metabolites are not high enough for commercial exploitation. Metabolic engineering can be a useful approach to boost the accumulation of targeted metabolites.In the present study, Saussurea medusa chalcone isomerase (CHI; EC 5.5.1.6) was chosen for metabolic engineering of S. involucrata. The goal has been to establish an in vitro system for S. involucrata with a satisfactory efficiency of flavoniod accumulation in general and apigenin biosynthesis in particular. Research for this thesis was executed in three areas, which are summarized below.First, a functional bioassay for S. medusa CHI (SmCHI) was performed using tobacco (Nicotiana tabacum) as a model system. Tobacco plants were transformed by SmCHI cDNA in either sense or antisense orientation under the regulation of the cauliflower mosaic virus (CaMV) 35S promoter. Transgenic tobacco plants overexpressing sense SmCHI accumulated higher levels of total flavonoids than wild-type control tobacco did, line CS-5, which was the best performer, produced 5-fold higher total flavonoids than wild-type tobacco plants, mainly due to an enhanced accumulation of rutin. Transgenic plants with antisense SmCHI accumulated less flavonoids; this is apparently brought about by suppressed expression of the endogenous chi gene. Further, both SmCHI gene expression and...