Establishment Of2-DE Technique System And Studies On Proteome Of The Eisenia Fetida In Pollution Stress By Phenanthrene

In this paper, in order to establish two-dimensional electrophoresis(2-DE) system for Eisenia fetida proteome analysis, the protocols forextracting proteins form Eisenia fetida and appropriate IPG strip,isoelectric focusing parameter and loading quantity of sample wereinvestigated and optimized. The differential expression of proteome inEisenia fetida stressed by phenanthrene(Phe) was analyzed by theestablished2-DE system, several of the differential expression proteinspots were identified by MADIL-TOF-MS. This study laid a foundationfor follow-up research of Eisenia fetida proteome and biomarkersscreening for soil Phe pollution.The results showed that, The improved TCA-A method proved to bemore effective and showed a better ability of impurity removal than thelysis solution method in Eisenia fetida protein extracting.2-DE gels turnout to be high resolution and showed more protein spots with proteinsample prepared by improved TCA-A protin extraction method.While the24cm IPG strip(pH4～7),10h isoelectric focusingparameter and600μg loading sample quantity were considered to be theoptimal condition for two-dimensional electrophoresis analysis of Eisenia fetida proteome, up to450protein spots could be detected on the gel aftercoomassie brilliant blue R-250staining.Extracts of Eisenia fetida collected at days10,20and40days afterexposure by23mg/kg Phe were analyzed by2-DE, up to14,22and35differential expression protein spots were found on the gel compared tothe blank control, these differentially expressed proteins showed asignificantly down-regulated expression. Five differentially expressedprotein spots collected at40days after exposure by Phe were selected formass spectrometry analysis. The identified protein, including ABCtransporter ATP binding protein, DNA repair protein Rad, phosphoserineaminotransferase, band4.1like protein2isoform a and hypotheticalprotein, are involved in immune regulation, energy metabolism, generepair and cell structure, which showed that Phe stress has a great impacton the cellular activities of Eisenia fetida and forced Eisenia fetida tostart stress response to resist Phe stress.