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Please use this identifier to cite or link to this item: http://ir.hwai.edu.tw:8080/ir/handle/310996100Q/1628

Title: Pectobacterium chrysanthemi 病原性相關基因之分析
The analysis of the pathogenicity relative genes in Pectobacterium chrysanthemi
Authors: 蔡孟庭
Tsai, Meng-Ting
Keywords: 軟腐病菌
病原性相關基因
Pectobacterium chrysanthemi
Date: 2013
Issue Date: 2013-10-18 11:37:26 (UTC+8)
Abstract: 蝴蝶蘭是全球最受歡迎的觀賞植物也是為台灣重要外銷觀賞植物之一,近年來蝴蝶蘭已成為台灣重要的外銷花卉,其經濟栽培面積大幅擴增,但因溫室栽培環境高溫多濕,若種植過程管理不當,則易助長蝴蝶蘭病害之發生與蔓延,造成蘭園重大之損失。而蝴蝶蘭細菌性病害的主要的病原菌有三種,分別為褐斑病菌(Acidovorax avenae subsp. cattleyae)、葉斑病菌(Burkholderia gladioli)及軟腐病菌(Pectobacterium chrysanthemi),而其中以軟腐病菌最為嚴重。本研究擬利用生物資訊學比對及分析軟腐病菌Pectobacterium chrysanthemi中第一型及第三型分泌系統中病原性相關之基因,希望能從這些篩選出可鑑別PCH及PCC差異之序列,並開發專一性引子組。本研究從NCBI Genomes資料庫中匯整Pectobacterium chrysanthemi 第一型分泌系統外膜蛋白基因與第三型分泌系統之hrp基因群序列,作為鑑別PCH及PCC分析之依據。利用生物資訊學已篩選出第一型分泌系統之eprF基因及第三型分泌系統之hrcJ、hrcT、hrcU、hrpL、hrpN及hrpX基因,都可用於開發PCR鑑定技術之用途。研究中利用分生技術已成功選殖上述hrp基因群,並將選殖株之基因序列以BLAST軟體進行比對,研究發現選殖株之序列對NCBI資料庫中PCH之hrcJ、hrcT、hrcU、hrpL、hrpN及hrpX基因的相似度分別介於88-96%、83-93%、83-92%、89-96%、85-88%及80-91%。而選殖株之序列對NCBI資料庫中PCC的相似度則較低,依序分別為73%、73%、72%、71%、74%及70%。序列間之異同可用於專一性引子組之開發,本研究已成功利用多序列比對結果設計出專一性引子組,初步測試可用於PCH及PCC的鑑別。此外對於致病相關基因有系統性的分析,發現在許多基因序列對不同來源之PCH仍有程度上的差異,是否與寄主專一性有關仍有待進一步了解。本研究中已成功選殖及表現出eprF基因,此基因的DNA長度為1389bp、分子量為52kDa,相似度為96%,確定為研究中選殖之基因,未來可大量表現蛋白質並純化,期可在抗體製作上加以進行,進一步利用免疫磁珠PCR及ELISA-PCR以增加檢測之敏感度。
Phalaenopsis became the most important products in the agriculture, the cultivation area of orchids increased rapidly. Growing the orchid in the greenhouse caused by the increasing of the temperature and humidity would yield the phytobacteria diseases, which caused enormous losses. The important phytobacteria were Acidovorax avenae subsp. cattleyae, Burkholderia gladioli and Pectobacterium chrysanthemi which caused infected diseases on orchids. One of the most serious bacterial disease agents is Pectobacterium chrysanthemi in Taiwan. This study plans to understand the pathogenicity relative genes in the type I and type III secretion system from Pectobacterium chrysanthemi. In this study, we attend to analysis the diversity between PCH and PCC, and to develop the specific primer-pairs for PCR detection. We collect the sequences of type I secretion system gene and type III secretion system genes to make the database respectively. We use the software of multiple sequences alignment to analysis the similarity among different database to understand the diversity to design specific primers. These specific primer-pairs from different genes would be used to identify PCH. Furthermore we have cloned the genes included hrcJ, hrcT, hrcU, hrpL, hrpN and hrpX genes into E. coli. The sequences of hrcJ, hrcT, hrcU, hrpL, hrpN and hrpX were used to BLAST analysis, that revealed the similarity are 88-96% ,83-93%, 83-92%,89-96%,85-88% and 80-91% to PCH,respectively. The sequences BLAST analysis to PCC revealed the similarity are 73%,73%, 72%, 71%, 74% and 70%, respectively. This study has been used the diversity of multiple sequence alignment to design specific primers. The PCR tests reveal the accuracy to PCH. This study has successfully demonstrated eprF gene, the DNA length is 1389bp, the molecular weight is 52kDa and the similarity is 96%, determine the gene for the study, overexpression of the protein and purification of antibody production Future study immuno-magnetic PCR and ELISA-PCR in order to increase the detection sensitivity.
Appears in Collections:[生物科技系暨生物醫學研究所] 博碩士論文

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