碩士 / 國立中興大學 / 植物病理學系 / 92 / Quorum sensing is a system which bacteria monitor their own population densities and regulate genes expression through sensing the levels of signal molecules called autoinducers. Quorum sensing bacteria produce and release chemical signal molecules that increase in concentration as a function of cell density. The most common autoinducers in gram negative bacteria is acylated homoserine lactone (HSL) synthesized by HSL synthase gene. In this study, we cloned and characterized HSL synthase gene from Erwinia carotovora subsp. carotovora ( Ecc ) ZL1 strain and the HSL synthase gene transgenic tobacco were generated. The susceptiblity of the HSL synthase gene transgenic tobacco tissue to soft rot erwinia was examined. Primer pair expI-F / expI-R was designed according to the sequence of expI gene (the HSL synthase gene of Ecc SCC3193) to amplify the HSL synthase gene of Ecc strains from colored calla lily. A 1.2 kb DNA fragment was amplified from each of 20 Ecc strains by polymerase chain reaction (PCR). The 1.2 kb DNA fragment amplified from Ecc ZL1 was further cloned and sequenced. It showed 92% identities with hsl I gene of Ecc 71. HSL synthase gene from Ecc ZL1 was constructed into pT7-7 expression vector and transformed into Escherichia coli BL21 DE3. The total protein profiles of E. coli BLEXP-1 carrying the HSL synthase gene from Ecc ZL1 were assayed by Sodium dodecyl sulfate polyacrylamide gel electrophoroesis (SDS-PAGE). It showed that a 26 kDa protein band was observed. In addition, E. coli ECEXP-1 could accelerate the pit formation by Ecc ZL1 and Ech CAS11 on crystal violet polypectate plate (CVP). Coincubation of high densities of E. coli ECEXP-1 carrying HSL synthase gene from Ecc ZL1 with low densities ( 106 CFU/ml) culture Ecc ZL1 could enhance the production of pectate lyase and polygalacturonase, whereas coincubation of E. coli ECEXP-1 with Ech CAS11 only enhance the production of pectate lyase but not polygalacturonase. A total of 11 lines of HSL synthase gene transgenic tobacco were generated by Agrobacterium-mediated transformation technique. Expression of HSL synthase gene in each of transgenic tobacco lines was confirmed by reverse transcript polymerase chain reaction (RT-PCR) with primer pair 35S-PF2 / hsl I-PR. The susceptibility of transgenic tobacco leaf tissues to soft rot Erwinia was assayed. The results revealed that leaf tissues of all the transgenic tobacco lines were less susceptiblie to Ecc ZL1 and ZL9 than that of wild type or vector transgenic tobacco leaf tissues. Whereas only three tobacco lines were less susceptible to Ech CAS7 and CAS11 than that of wild type or vector transgenic tobacco.
| Identifer | oai:union.ndltd.org:TW/092NCHU0363011 |
| Date | January 2004 |
| Creators | Hau-Ping Chou, 周浩平 |
| Contributors | Kuo-Ching Tzeng, 曾國欽 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 56 |
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