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研究生:楊川賢
研究生(外文):Yang Chuen-Hsien
論文名稱:十字花科蔬菜種子上之Xanthomonas屬細菌病原性分析及種類之鑑別
論文名稱(外文):The pathogenicity and differentiation of Xanthomonas spp. from cruciferous vegetable seeds
指導教授:李永安李永安引用關係
指導教授(外文):Lee Yung-An
口試委員:張雅君朱宇敏
口試日期:2012-07-30
學位類別:碩士
校院名稱:輔仁大學
系所名稱:生命科學系碩士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:93
中文關鍵詞:十字花科種子細菌種類鑑別
外文關鍵詞:Xanthomonas
相關次數:
  • 被引用被引用:2
  • 點閱點閱:347
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  • 下載下載:12
  • 收藏至我的研究室書目清單書目收藏:0
中文摘要

利用 Xan-D 鑑別性培養基檢測十字花科種子,可分離出呈現黃綠色,水亮上突的Xanthomonas屬細菌,將這些菌株接種於甘藍菜葉片上,另以hrpF基因設計的 PCR 引子組,進行 PCR 反應。接種結果發現有些菌株可引起黑腐病病徵 ( black rot ),為十字花科黑腐病菌 ( Xanthomonas campestris pv. campestris; XCC ),有些菌株可引發葉斑病徵( leaf spot)為十字花科葉斑病菌 ( Xanthomonas campestris pv. raphani; XCR ),這些可引發病徵的 XCC 或 XCR 菌株,以hrpF-PCR 都可擴增出 424 bp DNA 片段 (簡稱hrpF (+) 菌株);另有些菌株則不會使甘藍菜葉片發病,這些菌株在hrpF-PCR 下,均無法擴增出 424 bp 的 DNA 片段 (簡稱hrpF (-) 菌株)。進一步以 pathogenicity island 上的多個hrc基因,對hrpF (+)及hrpF (-) 菌株做 PCR 和Southern hybridization 研究,結果發現hrpF(+)菌株均具有pathogenicity island基因,而hrpF ( - ) 菌株則無。因此hrpF基因的存在與否,可成為用於區分Xanthomonas屬細菌是否具有病原性以及是否含有pathogenicity island的重要指標。hrpF (+) 菌株中包含有 XCC及XCR兩種病原細菌,而兩者的 16S rRNA序列以及生理生化特性十分相似,必須經植物接種後,依病徵的不同來區分。本實驗擬依基因演化機制,尋找 XCC 及 XCR病原細菌兩者基因序列差異,利用 PCR將XCC及XCR病原細菌區分出來。由於植物病原細菌具有分解植物細胞壁的能力,本實驗首先分析纖維水解酵素基因 (cellulase),經序列比對結果發現Xanthomonas屬細菌均具有engXCA這個cellulase基因,依此基因繪製的親緣關係圖和目前以16S rRNA 及 23S rRNA 序列所繪製的親緣關係圖的分類一致,且以engXCA比對所繪製的樹狀圖的 bootstrap 值較16S rRNA 比對所繪製的樹狀圖高,表示以纖維水解酵素engXCA所繪製的親緣關係樹狀圖可信度較高。因此認為engXCA cellulase 基因為一直系同源基因 (ortholog)。在基因比對的過程中發現基因engXCA在一些Xanthomonas屬細菌中發生基因複製 (duplication),形成的旁系同源基因( paralog )。在 XCC 病原細菌中,此旁系同源基因為另一個纖維水解酵素基因 XCC3535,在XCR 病原細菌中,則為另一個纖維水解酵素基因 XCR_3876,利用旁系同源基因 XCC3535及 XCR_3876累積的序列差異,可分別設計出對 XCC及 XCR病原細菌具專一性的 PCR 引子組,Xan-cel3535-F/ Xan-cel3535-R2引子組可對 XCC 病原細菌擴增出 1,429 bp 的DNA 片段,而XCR-cel3535-Fr/ Xan-cel3535-R2引子組可對 XCR 病原細菌擴增出 733 bp 的 DNA 片段,並可將兩組 PCR 引子對混合,進行 multiplex PCR,可在一次的 PCR 反應中區分出 XCC 和 XCR 。由於XCR也可以感染茄科植物,因此也希望利用纖維水解酵素基因engXCA的旁系同源基因對其他感染茄科植物的Xanthomonas屬細菌加以區分。透過基因 XCC3535 的 BlastP 比對,發現在感染茄科植物的Xanthomonas屬細菌X. gardneri和X. vesicatoria也有纖維水解酵素基因engXCA的旁系同源基因,利用這些序列間的差異可設計出對 X. gardneri和 X. vesicatoria有專一性的引子對,(XG-cel3535-F/ Xan-cel3535-R2)對X. gardneri有專一性,產生 423 bp 的 DNA 產物;而另一引子組 (XV-cel3535-F/ Xan-cel3535-R2)對X. vesicatoria有專一性,產生 1642 bp 的 DNA 產物。

英文摘要

The Xan-D medium was used to isolate and differentiate Xanthomonas spp. from cruciferous seed. Xanthomonas spp. in Xan-D turned yellow-green, wet-shining and surrounded with a smaller milkly zone and a bigger clear zone colonies. The cabbage leaves inoculated with these Xanthomonas strains. The result showed that some leaves received “black rot disease” symptoms caused by Xanthomonas campestris pv. campestris (XCC) , some received “bacterial leaf spot” symptoms caused by Xanthomonas campestris pv. raphani (XCR), the other leaves didn’t get any disease as usual. The PCR amplification using the primer set, hrpF-PCR amplified a 424 bp DNA fragment for these XCC and XCR strains (called hrpF (+) strains) but non-pathogenic Xanthomonas strains (called hrpF(-) strains). Then, these hrpF (+) and hrpF (-) strains’ hrc genes which located on pathogenicity island was detected by PCR and southern hybridization. The result showed the hrpF (+) strains got pathogenicity island but the hrpF (-) strains didn’t. Thus, the hrpF-PCR is a useful method to differentiate pathogenic and non-pathogenic Xanthomonas spp. and detect the presence of pathogenicity island. The 16S rRNA sequence and the result of physiological and biochemical analysis of the hrpF (+) strains, X. campestris pv. campestris and X. campestris pv. raphani, were too similar to differentiate from each other. In this study, we accorded the mechanism of gene evolution to find a gene which can differentiate XCC and XCR by PCR amplification. We first analyzed the cellulase gene due to the ability of degrade plant cell wall is necessary for the plant pathogenic bacteria. Through the genomic DNA alignment, a cellulase gene, engXCA which is presence in most Xanthomonas spp. was found. Compared the phylogenic tree of engXCA to 16S rRNA, two trees showed the similar current, furthermore, the engXCA’s bootstrap value is much higher than 16S rRNA’s. That means the higher credibility of phylogenic tree of engXCA than 16S rRNA. Therefore we though the cellulase gene engXCA is an ortholog of xanthomonads. During the process of gene alignment, a paralog of engXCA formed by engXCA duplicated in XCC and XCR were found. The paralog of engXCA in XCC and XCR became two diverse genes, XCC3535 and XCR_3876. The sequences of XCC3535 and XCR_3876 were used to design specific primer sets, Xan-cel3535-F/ Xan-cel3535-R2 and XCR-cel3535-Fr/ Xan-cel3535-R2. Xan-cel3535-F/ Xan-cel3535-R2 amplified 1429 bp DNA fragments for XCC, and XCR-cel3535-Fr/ Xan-cel3535-R2 amplified 733 bp DNA fragments for XCR in PCR amplification . The multiplex PCR primer set combined two primer sets, differentiated XCC and XCR in once PCR analysis. Because of the pathogenicity of solanaceous of XCR, we wanted to differentiate other solanaceous pathogenic xanthomonads by engXCA’s paralog. To find the paralog of engXCA in the solanaceous xanthomonads, we did XCC3535 BlastP and the engXCA’s paralog in the Solanaceous pathogenic Xanthomonas spp., ,X. gardneri and X. vesicatoria were found. According to those two diverse gene design two primer sets, XG-cel3535-F/ Xan-cel3535-R2 amplifying 423 bp DNA fragment and XV-cel3535-F/ Xan-cel3535-R2 amplifying 1642 bp DNA fragment in PCR amplification, used to classify the solanaceous pathogenic Xanthomonas spp.
目錄

中文摘要 ----------------------------------------------------------------------------------------I
英文摘要 ---------------------------------------------------------------------------------------- III
前言 ---------------------------------------------------------------------------------------------- 1
材料與方法 ------------------------------------------------------------------------------------- 9
1 菌種取得、培養條件、儲存方法 ----------------------------------------------------- 9
2 自十字花科種子分離之Xanthomonas屬細菌之病原性測試及引發病徵觀察
2-1 接種用甘藍菜之培養 -------------------------------------------------------------- 9
2-2 接種方法 ----------------------------------------------------------------------------- 10
3 自十字花科種子分離之Xanthomonas屬細菌之hrpF及 Pathogenicity island 上基因存在之測試 --------------------------------------------------------------------- 10
3-1 Xanthomonas屬細菌hrpF PCR 及南方氏雜合反應 --------------------------- 10
3-2 Xanthomonas 屬細菌hrpF (+) 菌株、hrpF (-) 菌株上 pathogenicity island(PAI)上hrc基因之檢測 --------------------------------------------------------- 11
4 利用纖維水解酵素基因 ( cellulase ) 區分十字花科病原細菌X. campestris pv. campestris及X. campestris pv. raphani------------------------------------------------- 11
4-1 Xanthomonas屬細菌纖維水解酵素基因種類檢索及比對 ------------------- 11
4-2 纖維水解酵素基因engXCA基因序列的檢索及比對 ------------------------ 13
4-3 Xanthomonas屬細菌engXCA基因片段之選殖 -------------------------------- 14
4-4 以纖維水解酵素基因engXCA、16S rRNA、23S rRNA 基因序列分析Xanthomonas屬細菌親緣關係圖 ----------------------------------------------- 14
4-5 纖維水解酵素基因engXCA之 旁系同源基因之尋找 ----------------------- 14
4-6 以engXCA旁系同源基因區分Xanthomonas campestris pv. campestris及Xanthomonas campestris pv. raphani-------------------------------------------- 15
5 以纖維水解酵素基因engXCA之旁系同源基因區分感染十字花科及茄科之Xanthomonas屬植物病原細菌 ------------------------------------------------------ 15
5-1 X. gardneri、X. vesicatoria 專一性引子對設計 -------------------------------- 15
5-2 X. gardneri、X. vesicatoria專一性引子對測試 ------------------------------- 16
6 序列比對與分析 ------------------------------------------------------------------------- 16
7 基礎分生技術 ---------------------------------------------------------------------------- 16

結果
1 自十字花科種子分離之Xanthomonas屬細菌之病原性測試及引病徵觀察 -- 23
2 自十字花科種子分離之Xanthomonas屬細菌之hrpF及 Pathogenicity island 上的hrc基因存在之測試結果 ------------------------------------------------------ 23
2-1 Xanthomonas屬細菌hrpF PCR 及南方氏雜合反應結果 ----------------- 23
2-2 Xanthomonas 屬細菌hrpF (+) 菌株、hrpF (-) 菌株上 pathogenicity island(PAI)上hrc基因之檢測結果 -------------------------------------------------- 25
3 利用纖維水解酵素基因 ( cellulase ) 區分十字花科病原細菌X. campestris pv. campestris及X. campestris pv. raphan i------------------------------------------------ 27
3-1 Xanthomonas屬細菌纖維水解酵素基因種類檢索及比對結果 ------------ 28
3-2 纖維水解酵素基因engXCA基因序列的檢索及比對結果 ---------------- 28
3-3 Xanthomonas屬細菌engXCA基因片段之選殖 ----------------------------- 29
3-4 以纖維水解酵素基因engXCA、16S rRNA、23S rRNA 基因序列分析Xanthomonas屬細菌親緣關係圖 ------------------------------------------------ 29
3-5基因engXCA之旁系同源基因之尋找結果 -----------------------------------
3-6以engXCA旁系同源基因區分Xanthomonas campestris pv. campestris及Xanthomonas campestris pv. raphani --------------------------------------------- 30
4 以纖維水解酵素基因engXCA之旁系同源基因區分感染十字花科及茄科之Xanthomonas屬植物病原細菌 ------------------------------------------------------ 31
4-1纖維水解酵素基因 XCC3535 BlastP 比對結果 ------------------------------ 31
4-2 纖維水解酵素基因 XCC3535、XCR_3876、XGA_0271 、XVE_2541比對結果 ------------------------------------------------------------------------------ 31
4-3 X. gardneri、X. vesicatoria專一性引子對測試 -------------------------------- 32

討論---------------------------------------------------------------------------------------- 33
參考文獻 ---------------------------------------------------------------------------------- 40
表 -------------------------------------------------------------------------------------------47
圖 -------------------------------------------------------------------------------------------73


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