碩士 / 國立中興大學 / 化學系所 / 106 / Pectobacterium carotovorum subsp. carotovorum (P.c.c.) is a gram negative bacterium, causing a heavy damage on multiple economic plants and food plants. The soft rot disease results from the plants infected with Pectobacterium carotovorum subsp. carotovorum, interfering with the plant growing. Up to now there are no effective way to control P.c.c. infection. We just take a physical precaution against the infection and want to find the low harm therapy, that is, the biological control to environments, humans, plants possibly infected with P.c.c..
In gram negative bacterium, bacterium can not make it survive in the tough environment, so they may secrete bacterocins to inhabit growth of the closely related bacteria strains and get more other nutritions from. From the previous studies, a mutant obtained by transposon Tn5 insertional mutagenesis using P.c.c., it can’t secrete bacteriocin and observe inhibition zone from bacteriocin assay. Then, the inserted sequence showed high homology with the genes ysaT and flhA. In addition, the genes ysaT and flhA knockout mutant can’t deliver Carocin to the extracellular medium, it confirm the carocin are delivered though the needle T3SS as well as flagellar T3SS. the flagellur T3SS not only swim but also secrete the effector (bacterocin) in P.c.c.. Due to the previus study, the study purpose is to suggest if the protein YsaT and FlhA have the interaction to bind together. It will be determined the needle T3SS and the flagellar T3SS may not assemble respectively but assemble a new secretion complex system with both the protein from the needle T3SS and the flagellar T3SS.
The studies show the new way for western blot to support the purpose. The new way is to remove the step about transferring proteins to the nitrocellular membrane, then add directly primary antibody, secondary antibody in order into the buffer. For centrifuge completed, take protein precipitates and transfer it to the nitrocellular membrane, exposed to the UV light for 10 minute. It make both proteins and nitrocellular membrane have interaction with covalent bond. The last, add ECL, and visualize the luminescence to check if there are protein YsaT and FlhA . The result show the expression of protein YsaT and FlhA are translated successfully by the new construction.
| Identifer | oai:union.ndltd.org:TW/106NCHU5065024 |
| Date | January 2018 |
| Creators | Mei-Chen Kuo, 郭玫辰 |
| Contributors | Duen-Yau Chuang, 莊敦堯 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 60 |
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