Establishing Transgenic Tobacco Plants Expressing L-Amino Acid Oxidases of Trichoderma harziana and Testing Their Antimicrobial Effects
| Autor: | Hsin-Chiao Yu, 游心喬 |
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| Rok vydání: | 2013 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 101 The L-amino acid oxidases (LAAO) are flavoenzymes, which catalyze the stereospecific oxidative deamination of L-amino acid substrates. The LAAO have been demonstrated to exhibit antimicrobial activity. Dr. Peng purified a LAAO (ThLAAO) from Trichoderma harzianum ETS323. They showed that ThLAAO interact with bacteria to cause membrane permeabilization, increase exogenous H2O2 production, and cause bacterial damage. The purpose of this study is to genetically transform the ThLAAO gene into tobacco to elucidate whether ThLAAO alone can contribute antimicrobial effects and play an important role in biocontrol of Trichoderma. Transgene was introduced into Nicotiana tabacum cv. Xanthi by Agrobacterium-mediated transformation. We found that ThLAAO contain endoplamic reticulum (ER) retention signal, KEDEL. We constructed two clones. One is to directly clone the ThLAAO gene into Ti plasmid, pCAMBIA1302, resulting in the expression of ThLAAO in cytosol. The other one is to add a signal peptide of the pathogenesis-related protein 1b (PR1b) on the N-terminus of ThLAAO which allows ThLAAO to be secreted into ER. After antibiotic selection and regeneration, ten transgenic tobacco plants of each clone were obtained (L1 – L10 and P1 – P10). Expression of the transgenes in transgenic tobacco was examined by PCR analysis, Semi-quantification RT-PCR analysis, and western blot analysis. Of these transgenic tobacco plants, only L2, L7, L9, L10, and P2, P4, P7, P10 expressed the ThLAAO transgene. We chose L2, L7, L10, P2, P4, and P10 transgenic tobacco plants for further analysis of antifungal activity. The result showed that all the L2, L7, L10, P2, P4, and P10 showed significant antimicrobial activity against pathogenic fungi, Sclerotinia sclerotiorum and Botrytis cinerea. Futhermore, all the transgenic plants expressing the ThLAAO genes produced H2O2, suggesting that ThLAAO confers resistance against pathogens through the activation of defense systems by H2O2. All the above results indicated that ThLAAO plays an important role in biocontrol of Trichoderma. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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