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Silencing Mapk and RAC Gene in Trichoderma Harzianum Mediated by Dsrna

Author: ChenYan
Tutor: SongJinZhu
School: Harbin Institute of Technology
Course: Genetics
Keywords: Trichoderma harzianum mapk rac gene silence
CLC: S432.44
Type: Master's thesis
Year: 2009
Downloads: 77
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Abstract


The generation of vegetative organs, reproductive organs and infection structures response to the physical and chemcial signal induction in filamentous fungi, so the signal transduction pathways are important to growth, develpment and infection. Trichoderma harzianum was applied as biological control agent against plant fungal pathogens. The biological mechanisms have been hot spots recent years. MAPKs as signal transcription factor of mitogen-activated protein kinases cascade activate specific expression of proteins by phosphorylation and regulate filamentous growth, sexual reproduction, stress response and virulence producion in filamentous fungi. RAC which belong to small G protein Rho family contribute to celluar morphogenesis and superoxide generation by cycling between active GTP and inactive GDP-bound states. The biological control research of the two genes help interpret the molecule mechanisms and establish the background for the biological control of T. harzianum.Silence vector which developed dsRNA using an RNA silencing approach was transformed into T. harzianum by Agrobacterium tumefaciens-mediated. The silence-transformants which made the mapk and rac gene expressed down were obtained. Enzymatic activity, the growth of osmosis stess and the growth confront with pathogenic between transformants were performed to analysis the regulation of mapk and rac gene to cellulose,β-1,3-glucanase, osmotic stress, and aerial hypha growth.The cDNA sequences of mapk and rac gene were cloned from T. harzianum successfully, and were submited to GenBank, the accession numbers were FJ716099 and FJ595933 respectively. The mapk gene encoded 406 amino acid residues with molecular weight of 46.28 kDa in theory. The rac gene encoded 204 amino acid residues with molecular weight of 22.4 kDa in theory. pCambla1301-mapk/pCambla1301-rac, which made mapk/rac silent, was transformed into T. harzianum by A. tumefaciens-mediated genetic transformation system. pSilent-mapk-T88 and pSilent-rac-T88 were choosed as transformants which made mapk gene express down 27% and rac gene express down 29%. The result revealed that mapk gene participated in the regulation ofβ-1,3-glucanase, cellulose, osmotic stress and aerial hypha growth, and rac gene participated in the regulation ofβ-1,3-glucanase, cellulose and aerial hypha growth.

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CLC: > Agricultural Sciences > Plant Protection > Pest and Disease Control > Plant diseases and their prevention > Transgression ( pass ) an infectious agent harmful > Fungi
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