The Influence Of Temperature On The Mortierella Isabellina M6-22 Protein Expression Profile

Protein extraction is the first step of proteomics research.Its quality directly affects the protein expression analysis studies and the accuracy of the results.In order to extract total cellular protein from Mortierella isabellina strain M6-22 for further proteomics and some other related research,we tried five protein extraction methods.Protein concentration and SDS-PAGE analysis of protein abundancy were used to evaluate the effects of these methods.One of them was selected and furhter improved,by which a total protein concentration of 2.99 mg/ml was obtained.The improved method had advantages of simpler procedures and obtaining higher protein abundancy than the original one.It was further used to extract the total protein of five other fungi,and the results showed that similar extraction effect could be obtained,indicating that this method can be potentially used as an efficient total protein extraction method for fungi.Using the improved method,the total protein of the Mortierella isabellina M6-22 cultivated at 28 ? and 15 ? respectively was extracted.Desalting through dialysis to reduce the salt effect on the pH gradient in isoelectric focusing,the proteins were then used to analyze the difference of the protein expression patterns by 2-dimensional electrophoresis technology.The results showed that 111 protein spots including 69 up-regulation spots and 42 down-regulation spots at 15 ? were found.7 of these up-regulation proteins were specifically expressed at 15 ?,but no protein was specifically expressed at 28 ?.61 spots with low molecular weight and more than two-fold difference were selected for Mass Spectrometry analysis,and 41 proteins were identified.The results showed those proteins belonged to hydrolase,transferase,ligase,ATP binding protein,GTP binding protein,transportor and hypothetical protein etc,locating in cell membrane,organelle,cytoplasm,nuclei and cytoskeleton etc.Gene Ontology(GO)analysis indicated that their functions were involved in cell component synthesis,glycometabolism,metabolism of nucleic acids,protein metabolism,organic acid metabolism,protein translation and modification,transcription,transportation and autophagy etc.All the results indicated that the response of Mortierellaisabellina M6-22 to low temperature was a comprehensive network involved with multiple molecular and physiological mechanisms.This study provides a theoretical basis for understanding the adaptation mechanism of Mortierella isabella and filamentous fungi to low temperature at the molecular level,especially at the proteome level.