Construction Of Rhizopus Genetic Transformation System

ABSTRACT:Rhizopus nigricans belongs to zygomycetes Amon Rhizopus. Its mycelium is not separated and the cell wall composition is complex. The preparation of its protoplast is more difficult and research about it is lack, which hindered its conversion transformation. This experiment tried different enzymes and a variety of enzymatic buffer portfolio to optimize the preparation of Rhizopus nigricans protoplast. We adopted electroporation method to transfer plasmid of pEGFP-C1 and used the expression of green fluorescent protein to screen them. Electroporation method is complicated, in order to establish a quick and easy transformation system of Rhizopus nigricans, we designed the use of liposomes embedding to transform it. The experimental results were as follows:1.After repeated experiment, we got the best conditions of preparing protoplast. That was:0.1 g fresh mycelium, lml 2% dissolved wall enzyme dissolved in 0.6M mannitol stabilizer enzyme Hydrolysate,30℃ water bath for four hours.2. We adopted electroporation to transform the pEGFP-Clinto the protoplast. After the protoplast regenerated, we used fluorescence microscopy and molecular methods to screen them. The results showed that the GFP gene was expressed and the transformation efficiency was 5.91/μg. When reaching the fourth generation, the GFP gene was lost. The reason is still not clear, further research is needed.3. We adopted liposomes to transform the pEGFP-Clinto the protoplast. After the protoplast regenerated, we used fluorescence microscopy and molecular methods to screen them. The results showed that the GFP gene was expressed.The passage was stability and not a gene was lost.Liposomes transformation is a new technology to transform Rhizopus nigricans and this method is fast and stable, which provides a basis for future research. GFP can efficiently screen the transformants, then build practical Rhizopus nigricans engineered bacteria.