Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/49653
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dc.contributor.author莊敦堯zh_TW
dc.contributor.other行政院農業委員會zh_TW
dc.contributor.other中興大學化學系zh_TW
dc.date2011zh_TW
dc.date.accessioned2014-06-06T08:35:30Z-
dc.date.available2014-06-06T08:35:30Z-
dc.identifier99農科-1.1.1-檢-B1(5)zh_TW
dc.identifier.urihttp://hdl.handle.net/11455/49653-
dc.description.abstract軟腐病菌可生產兩種不同型態的細菌素,一種是類似病毒結構的高分子量細菌素,另一種是以小段蛋白質形式存在的低分子量細菌素。我們利用transposon Tn5插入軟腐病菌菌株WF7的染色體中,並且個別分離出了約2000株插入突變株,分析了其低分子量細菌素表現活性,在經過了2次的以上的活性測試交叉分析後,我們確認了WF7相關的插入突變株15株完全喪失了低分子量細菌素表現能力。我們進一步的進行TAIL-PCR分析其插入位置周圍的基因序列,而插入突變株TW07的Tn-5是插入與可能是E. coli所生產的colicin E3有同質性DNA序列的位置,我們將此命名為Carocin S4。我們進一步的分析Carocin S4的基因,從親株WF7的染色體DNA的選殖與定序後,我們從所解出DNA序列中發現到2個完整的ORF,第1個ORF與E. coli所生產的colicin E3等有約24%的同質性,其有可能為DNA水解酵素低分子量細菌素,我們將第1個ORF命名為caroS4K gene;而第2個ORF與NCBI基因資料庫中無任何同質性蛋白資料可比對,但由於該基因是緊接於第一個ORF之後,有可能為其相對應的免疫蛋白,我們將其命名為caroS4I gene,這兩個基因我們稱之為carocin S4。我們這兩個基因導入pET32a載體中,製成pET32aS4-42表現載體中,並將其分別導入至宿主細胞大腸桿菌XL1-blue中,其結果顯示帶有pET32aS4-42質體的菌株均能表現出低分子量細菌素carocin S4活性,從此結果顯示我們成功的將carocin S4選質並表現於無致病性菌株中。 本年度計畫中我們將進一步的將Carocin S4蛋白大量純化出來,並確認其殺菌機制。除此之外,我們也將同時尋找能長期保存且且具有高度抗軟腐菌活性的方法,以利將來在田間實驗使用。此外,我們也計畫在今年度中大量蒐集中部地區軟腐菌株的種類,以期能獲得具有更多更廣泛的抗軟腐菌生長的細菌素高度表現能力菌株,以期本研究將來能有極高度的實用價值。 我們將在本年度內將carocin S4基因導入無病源性Erwinia carotovora及E. coli菌株中,以利用其基因轉錄、修飾、及分泌機制來將低分子量細菌素基因輸送至胞外,以期能做出高抗軟腐菌生產能力且無病源性的軟腐菌,使其能於生物防治上應用,以達成抑制軟腐病病害發生的可能性。zh_TW
dc.description.abstractWe inserted the transposon Tn5 into the pectobacterium carotovorum subsp. carotovorum WF7 chromosomal, which can express the low-molecular-weight bacteriocins, then we obtained about 2000 insertional mutagensis strains. We test the Bacteriocin acticity, and there are 15 strains lose the production ability for Bacteriocin. We analyze the surrounding sequence from these strains by TAIL-PCR, and confirmed the sequence data by genebank database. The TW07 strain's data showed that the DNA sequence data is similar with colicin E3, which is a low-molecular-weight bacteriocin and produced by E. coli, and has 24% homologous. We continue the sequence from the parent strain, WF7, and obtain about 2800 bp. After analyzed the DNA sequence, Two complete ORFs were obtained. The first ORF is similar with colicin E3 about 24% homoglous, but the second ORF has bit any homologous with protein database. Both of two bacteriocins are nuclease activity. We named the first ORF gene is carocin S4K, and the second ORF gene is carocin S4I. After subclone the carocin S4 gene and transfer it into DH5 or BL21 cell, we found the carocin S4 gene can be expressed and Carocin activity can be detected in XL1-blue cell by bacteriocin activity assay. This year we will isolate the Carocin S4 protein, and confirm its antibiotic's activity. In addition, we also plan to collect a large number of Erwinia carotovora strains, and make a strain-bank of Erwinia carotovora for middle-Taiwan.en_US
dc.language.isozh_TWzh_TW
dc.relation.urihttp://grbsearch.stpi.narl.org.tw/GRB/result.jsp?id=2087907&plan_no=99%E8%BE%B2%E7%A7%91-1.1.1-%E6%AA%A2-B1%285%29&plan_year=99&projkey=PW9905-1211&target=plan&highStr=*&check=0&pnchDesc=%E8%BB%9F%E8%85%90%E7%97%85%E8%8F%8C%E7%B4%B0%E8%8F%8C%E7%B4%A0%E5%9F%BA%E5%9B%A0%E9%81%B8%E6%AE%96%E5%9C%A8%E8%BB%9F%E8%85%90%E7%97%85%E9%98%B2%E6%B2%BB%E6%B3%95%E4%B8%8A%E4%B9%8B%E6%87%89%E7%94%A8en_US
dc.subject生物技術, 植物保護類zh_TW
dc.subject技術發展zh_TW
dc.subjectgene cloningen_US
dc.subject基因選殖zh_TW
dc.subject細菌素zh_TW
dc.subject軟腐病zh_TW
dc.subject軟腐病菌zh_TW
dc.subjectbacteriocinen_US
dc.subjectsoft-rot diseaseen_US
dc.subjectErwinia carotovoraen_US
dc.title軟腐病菌細菌素基因選殖在軟腐病防治法上之應用zh_TW
dc.titleApplication of the Bacteriocin Gene Cloning from Erwinia carotovora and Soft-rot Disease Controlleden_US
dc.typeResearch Reportszh_TW
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