Functional Analysis Of AMPK/Cnrk1 In The The Transcription Regulation Of Cellulase Genes In Trichoderma Reesei

Filamentous fungi are the dominant microorganisms in nature that exert the ability of degrading cellulose. Trichoderma reesei is one of the most prolific cellulase producers as type strain in industry. The exoglucanase CBH1 account for approximately 60% of the total enzyme system secreted. Research results indicated that cbhl gene was strictly regulated by carbon source. The expression level of CBHl was 1000-fold higher in the presence of cellulose than that from glycerol. Moreover, cbhl gene was completely inhibited when glucose was used as sole carbon source,which is called carbon catabolite repression (CCR).AMP-activated protein kinase (AMPK) is conserved in essentially all eukaryotes as heterotrimeric complexes comprising a catalytic a-subunit and regulatory β- and y-subunits, and any rise in AMP,which signifies falling energy status, causes the increase in protein kinase activity of AMPK. The yeast and Tricoderma reesei ortholog of AMPK has an essential role in CCR in Saccharomyces cerevisiae that includes inactivating the transcription repressor Migl, and its corresponding counterpart in Tricoderma reesei is Crel. But in multicellular filamentous fungi the function of AMPK maybe different from that in yeast:researches show that H. jecorina AMPK readily phosphorylated yeast Migl, but not H. jecorina Cre1; in A. nidulans CreA is not regulated by AMPK.Moreover, AMPK affects glucose signaling via Snfl and Rgt2/Snf3 pathways participated in yeast. It also regulate gene expression at both the transcriptional and post-transcriptional levels:it appears to affect multiple steps in gene regulation including transcription factor binding, RNA polymerase II activity, cytoplasmic mRNA stability, regulation of acetyl-CoA homeostasis and histone acetylation; at post-transcriptional level, AMPK transcriptionally down-regulates expression of genes encoding amino acid biosynthetic enzymes by inhibiting translation of the master regulator Gcn4.So in Trichoderma reesei whether AMPK play a role in regulation of cellulase transcription? To this point, this thesis focused on the function of AMPK in the transcription of cellulase genes. Results can be seen as followed:1. T. reesei Cnrkl was not sufficient to complement the defect of S. cerevisiae A AMPK strain to utilize sucrose as carbon source. But the fused protein of the amino terminal (N-terminal,l-300 aa) of T. reesei Cnrkl and the carboxyl terminal (C-terminal,336-633 aa) of S. cere AMPK could do it; On the other hand, S. cere AMPK could not complement a T. reesei Δcnrkl phenotype as well. Therefore, we believe there is really functional differences between Trichoderma reesei Cnrkl and Saccharomyces cerevisiae AMPK while this difference may be caused by low C-terminal conservative property of the proteins.2.The results proved that in contrast to the parental strain TU6, Trichoderma reesei Cnrkl (homologous protein of Saccharomyces cerevisiae AMPK) played a critical role in the growth on alternative carbon sources, filamentous growth, spores viability and transcriptional level of specific genes including cellulase encoding.3. Trichoderma reesei Cnrkl functioned as a negative regulation factor in cellulase transcription. Complementation mutant strains of T. reesei with different Cnrkl protein kinase activities were constructed. The most significantly effect was that the expectant hyperactive form Cnrk1G18R lost the induction ability to use cellulose and lactose as carbon sources,but the effect to xylan induction pathway was slightly.4.Thirteen kinds of candidate proteins may interact with Trichoderma reesei Cnrkl were screened by yeast two-hybrid technology, including AMPKP-subunit, AMPKy-subunit, heat shock 70 kd protein cognate 1, Glucosidase I, Phosphatidic acid-preferring phospholipase Al, C3H1-type Zn-finger protein, a predicted oxidoreductas, protein O-mannosyl transferase,60S ribosomal protein L38, Nucleotide excision repair protein RAD 16,40S ribosomal protein S2 and two hypothetical proteins. The results will be helpful for further research of Trichoderma reesei Cnrkl function.