Global And Functional Analysis Of Differential Phosphorylation Proteomics In Trichoderma Reesei

Trichoderma reesei(teleomorph Hypocrea jecorina) is widely used in industry as a source of cellulase and hemicellulase. T. reesei has excellent ability of protein synthesis and secretion. In the post-genomic era, proteomics is becoming one of the most popular research directions. Protein phosphorylation is one of the most important reversible modifications in nature. It is involved in many cellular processes such as proliferation, differentiation, cellular signaling, transcriptional and translational regulation, interaction with other proteins, subcellular localization and the degradation of protein. So phosphorylation is one of the most common and best characterized post-translational modifications(PTM) of cellular proteins.To find the phosphoproteins participating in regulation of cellulase expression in T. reesei, in the first part of this work, LC-MS and Label-free quantification were used to find the key protein in the regulation of cellulase expression, and to analyze the features of phosphorylation proteomics in regulation of cellulase expression. T. reesei was cultivated in inducing condition(A, with avicel as the carbon source), repressor condition(G, with glucose as the carbon source) and mediate condition(Y, as glycerol asn the carbon source), respectively, and the total intracellular protein of T. reesei was extracted and analyzed quantitatively using label-free technology. By comparing the protein expression pattern of different cultivating conditions, we found 90 differentially expressed proteins between group A and group G, 61 differentially expressed proteins between group A and group Y, and 90 differentially expressed proteins between group G and group Y. Most of the differentially expressed proteins were located in the following functional group: 1) zinc finger binding protein activity; 2) ATP binding protein activity; 3) N-acetyltransferase activity; 4) oxidoreductase function. Most of proteins involved in glycolysis, protein synthesis, DNA repair and other processes. Understanding of the function of the phosphorylation proteins and the pathways in which they are involved might provide new approaches to improve the ability of cellulase expression and secretion in T. reesei.Although the cellulase expression of T. reesei can be improved through gene overexpression, gene knockout and so on, our understanding on regulation of cellulase expression in T. reesei is still insufficient. Comprehensive understanding of cellulase gene expression regulation of T. reesei could further explore the potential of its excellent biological functions and improve cellulase production. In the second part of this work, the stable isotope dimethyl labeling technology was used to quantify the phosphoprotein of T. reesei that was resepectively cultivitated in inducive condition and repressive condition, and 32 differentially expressed phosphoproteins were found. After analyzing the functions of the phosphoproteins that were expressed with significant difference in the two conditions, we finally selected the GTP domain protein for functional characterization.A GTP protein overexpression vector was constructed and transformed into T. reesei. The cellulase production efficiency of the recombinant T. reesei strain G9 was analyzed. The results showed that GTP protein can improve cellulase activity. Comparison of the filter paper activities between the GTP protein over-expression strain and the wild type strain, the cellulase activity of over-expression strain improved 82%, while the CMC activity of the GTP over-expression strain improved 1.73 times. The results of this work demonstrated that analysis of the phosphorylation proteomics of T. reesei can provide a new approach to study the metabolism and gene expression regulation and to improve the cellulase productivity in this organism.