Study An Proteomics Of Aspergillus Oryzae Conidia With Different Karyotypes

In this study,the differences of protein expression in different karyotypes conidia of Aspergillus oryzae FS1125 were analyzed by two-dimensional electrophoresis(2-DE)and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry(MALDI-TOF MS),which laid a foundation for the study of genetic regulation mechanism of spore karyotype changes during the growth and development of Aspergillus oryzae.The main contents and results were as follows:1)In order to obtain a large number of conidia,the solid culture conditions of Aspergillus oryzae FS1125 strain were optimized.By using solid Czapek-Dox(CD)medium,humidity and light conditions testing,the optimum solid culture time of FS1125 spore production was determined to be 9-10 days.The optimum sporulation conditions were natural light,humidity 60%and temperature 30C.2)The separation and cell wall breaking method of different karyotype conidia of Aspergillus oryzae FS1125 were established.Membrane filtration was used to separate and enrich mononuclear conidia of Aspergillus orvzae on the basis of previous work in our laboratory.The feasibility of membrane filtration separation was verified by DAPI staining.and it was found that FS1125 conidial spores had nuclear degradation phenomenon during preservation at 4℃.Five methods of conidia wall breaking were compared,and the MP Fast-prep homogenization method for conidia of Aspergillus oryzae FS1125 was established by combining the method of breaking the wall of conidia with the extractions of intracellular proteins suitable for 2-DE.3)The whole proteins from different karyotype conidial spores of Aspergillus oryzae FS1125 were analyzed by two-dimensional electrophoresis.2-DE conditions suitable for protein extractions from conidiospore of Aspergillus oryzae FS1125 were optimized on both of sample quantity and isoelectric focusing program.The differences of protein expression in conidia of different karyotypes of Aspergillus oryzae were analyzed by 2-DE and 41 difference points were screened out.Six unknown proteins and 45 known proteins were identified by mass spectrometry,including heat shock proteins,enzymes involved in many metabolic pathways,and intracellular substance transport related proteins.The 45 proteins were annotated to 21 GO terms by GO classification.KEGG enrichment analysis showed that 45 proteins were annotated to many different metabolic pathways,indicating that there were significant differences in metabolic pathways or metabolic activities between mononuclear conidia and polynuclear conidia.The interaction of 45 proteins identified by String was mainly related to the transport proteins such as Actin.It is speculated that the difference in the nucleation number of conidia of Aspergillus oryzae may be related to the difference in the content or distribution of the proteins related to the transport of these substances.