| Background:Penicillium citrinum is a genus of ascomycetous fungi of major importance in industrial settings. Mevastatin is one of the most important secondary metabolite in Penicillium citrinum. It can break the enzyme called HMG-CoA reductase which is involved in the production of various fatty molecules. Blocking this enzyme lowers cholesterol levels. After biotransformation, mevastatin becomes the drug of first choice for treating hyperlipidemia. Studying the biosynthesis mechanism of mevastatin is usful for strain improvement, and an efficient and stable method is needed for research. Nowadays PEG-medium transformation of protoplast, electroporation, Acetic acid lithium method,gene gun bombardment, and restriction enzyme mediated transformation method can be used for genetic transformation of filamentous fungi. As the filamentous fungi is quite different from plant, animal and procaryotic cell, no almighty method has been found until now. The past research shows Agrobactirium tumfacience mediated-transformant is a good method for transformation in filamentous fungi, as it is efficient, simple to operate, widly recepted, single copy inserting and genetic stability.Objective:The experiment tries to establish an efficieny and stable genetic transformation system which can works well in Penicillium citrinum, in order to lay the foundation for studying the biosynthesis mechanism of mevastatin and global regulation. Methods:1optimize the aspects which have an effect on Agrobacterium-Mediated Transformation of Penicillium citrinum as follow:freshness, concentration of spores, concentration, strain of Agrobacterium, concentration of acetosyringone, temperature, time and method for co-culture.2Colone hyg resistence gene to distinguish transformant. Detectes the genetic stability by PCR after subculturing for five generations on no resistance pressure.3Observe and study the phenotype of the steady transformant.4Ferment the transformant and detect the biosynthesis of mevastatin.Results:1The result shows:transformation rate is much more higher by using fresh spores. It is better to use LBA4404instead of EHA105while co-colturing in24℃for36to48hours. The suited concentration of spores is10, the OD600of agrobacterium is about0.8, and the consistence of Acetosyringone should be80u.g/ml. Using liquid co-colture method can be efficient and simplify the operational process.2The resistant marker of hyg being detected in the transformant by PCR confirms the success. After subculture without Hyg for five generations,90%transformant shows genetic stability.3An significant influence on phenotype of Penicillium citrinum has been observed. The change of chromogenesis in some transformant makes the hyphae ture gray.4Biosynthesis of mevastatin increases obviously in the transformant.Conclusion:An efficient and stable method is founded for Agrobacterium-Mediated Transformation of Penicillium citrinum. After condition optimization, this genetic transformation system is used for preliminary research about the function of Fci-laeA. The result lay the foundation for studying the biosynthesis mechanism of mevastatin and global regulation. |
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