Establishment Of Genetic Transformation System In Marchantia Polymorpha L. And Preliminary Study On The Metabolic Engineering Of Mustard Glucosinolates

Marchantia polymorpha L.belongs to the bryophyte,which distributes all over the world.It is quite common in the north and northwest of China.Because of its low genome redundancy,simple genetic transformation,rapid asexual reproduction and cell suspension culture,it has great potential in theory and technology as a bioreactor.Because of its eukaryotic expression system consistent with that of higher plants,it has great potential in production of secondary metabolites or proteins from higher plants.Glucosinolate is a secondary metabolite containing sulfur.When plants are injured by pathogenic bacteria or mechanical damage,the tissues in vivo are destroyed,which makes glucosinolates and myrosinase fully contacted,then glucosinolates are hydrolyzed.These hydrolysates have many biological activities.For plants,these hydrolysates can be used as signaling molecules to stimulate plant defensive responses against pathogens and pests.For humans,they can be used to prevent and treat cancer and also to produce biological pesticides and fungicides.Indoles glucosinolates are a kind of glucosinolates derived from tryptophan.Its degradation products have the effects of inhibiting tumor activity and treating colon cancer.Although indole glucosinolates and their degradation products are pretty beneficial to humans,they are rarely found in plants.Therefore,the production of glucosinolates by metabolic engineering has important research significance.This study intends to use Marchantia polymorpha L.as a bioreactor to conduct a preliminary study on glucosinolates metabolism.Tissue culture and genetic transformation system were established and optimized,and the metabolic engineering strategy of indole glucosinolates is preliminarily designed.The advantages and feasibility of using Marchantia polymorpha L.as bioreactor were discussed,which provided theoretical and technical reference for developing new efficient bioreactor and the biosynthesis of glucosinolates.The main results of this paper are as follows:Successfully domesticated wild Marchantia polymorpha L.and established and optimized its tissue culture system.To optimize the influencing factors of genetic transformation efficiency,the optimum genetic transformation system was determined as follows:1-day pre-culture of germs in 1/2B5 medium containing 1% sucrose,10 minutes immersion in transformation buffer containing 0.01% Tween 20,3 days co-culture with Agrobacterium in dark environment,2 weeks screening with 50 mg/m L kanamycin to survive.After the surviving thallus grow gemmaes,screening kanamycin at 10 mg/m L,obtain resistant gemmaes.The driving efficiency of endogenous promoter Mp EF1? and 35 S are compared by using reporter gene.Genes in indole glucosinolate metabolic pathway were cloned and plant expression vectors were constructed.A preliminary strategy was designed for the metabolic engineering of glucosinolates in the bioreactor of Marchantia polymorpha L.