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學校名稱輔仁大學
系所名稱生命科學系
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學號492546135
研究生(中)李京倫
研究生(英)Ching-Lun Lee
論文名稱(中)Xanthomonas屬植物病原細菌之兩段基因間序列之分析及應用
論文名稱(英)Sequence analysis and application of two intergenic regions of Xanthomonas spp.
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指導教授(中)李永安
指導教授(英)Yung-An Li
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關鍵字(中)黃原桿菌屬 核酸陣列 分子分型
關鍵字(英)Xanthomonas DNA array molecular typing
摘要(中)本實驗室在研究十字花科黑腐病菌( Xanthomonas campestris pv. campestris 1-1;XCC 1-1 )內插入序列IS1404座落位置時,發現其中一個IS1404座落於NTPase基因內,與已公佈的X. campestris pv. campestris ATCC33913 ( XCC ATCC33913 )、X. axonopodis pv. citri 306 ( XAC 306 )及X. oryzae pv. oryzae KACC10331 ( XOO KACC10331 )三個基因體序列分析比對,發現在XCC ATCC33913中,NTPase基因內亦有一個IS1404插入,但XAC306卻無NTPase基因,NTPase在XCC ATCC33913中兩端基因存在的情形為mrdB-NTPase-chp-hp-peh-1,XAC 306則為mrdB-peh-1,缺少NTPase-chp-hp約8-kb的DNA片段(簡稱NTPase片段),而XOO KACC10331則為mrdB-ucp1-hp-ucp2-ucp3-peh-1 ( 簡稱ucp片段 )。為了確認NTPase片段是否只存在於XCC,分別以PCR 及Southern hybridization檢測,結果發現NTPase片段只存在於XCC,而其他Xanthomonas屬細菌則缺乏此片段,進一步測試XCC不同菌株,除均含有NTPase片段外,此片段也均位於mrdB- peh-1兩基因之間,顯示此NTPase片段可能經由水平基因轉移方式,移入XCC之共同祖先,而成為XCC菌株所共有。針對NTPase片段內的chp基因,設計PCR引子對XCCchp-F/ XCCchp-R1,發現所測試的XCC菌株皆可擴增出443-bp大小的DNA片段,而其他Xanthomonas屬或非Xanthomonas屬植物病原細菌,除X. campestris pv. aberrans及X. bromi外,均無法擴增出任何的DNA片段,因此XCCchp-F/ XCCchp-R1之PCR引子對,可用於對十字花科黑腐病菌進行專一性的檢測之用。此外,將XCC細菌與甘藍種子混合培養48小時後,可利用XCC專一引子自混合培養液中擴增出目標片段。同樣地,針對XOO獨有的ucp片段中之ucp2基因設計PCR引子對Xo-ucp-F/ Xo-ucp-R,僅XOO菌株可擴增出643-bp大小的DNA片段,因此可用於對XOO的專一性檢測。利用mrdB與peh-1二基因專一性引子探針及跨基因引子對Xanthomonas屬不同菌株進行測試,得知它們皆具有此二基因,但其間序列長短情形有差異。以mrdB-peh-1跨基因引子mrdB-F2L/ Xpeh-R分別將Xanthomonas屬內11個種的菌株mrdB-peh-1間序列選殖出並定序比對,依結果可分為A~H八種類型,其中除了X. campestris pv. syngonii有一IS插入,其餘菌株在此部份並沒有任何功能基因存在。利用此處的序列差異,分別將A~G七種類型菌株所特有的核酸序列以PCR方式擴增出並製成探針,發現各探針對相對應類型的菌株具有專一性,因此可利用此mrdB-peh-1間序列對Xanthomonas屬細菌進行初步鑑定。除了mrdB-peh-1為可能的水平轉移區域外,本實驗室也在另外一段區域發現了類似的情形。先前在研究Xanthomonas代謝quinate的能力時,發現僅X. arboricola擁有此能力,其後進一步自X. arboricola pv. juglandis XJC5菌株中將相關基因qumA-qumB選殖出,經定序分析結果得知,qumA-qumB基因兩側尚有具相同轉錄方向的unknown與pep基因,基因排列順序由5’端開始依序為unknown-qumA-qumB-pep。比對已知的Xanthomonas基因體序列XCC ATCC33913、XAC 306以及XOO KACC 10331,發現此處基因排列順序皆為unknown-pep,缺少qumA-qumB約4.4-kb的片段,因此推測qumA-qumB也是經由水平基因轉移的方式移入X. arboricola的共同祖先。以unknown-pep跨基因引子UN-F6/ pep-R7分別將Xanthomonas屬內12個種的菌株unknown-pep間序列選殖出並定序比對,依結果可分為七種類型,其中X. campestris pv. zantedeschiae與X. arboricola同樣具有qumA-qumB片段,而X. oryzae pv. oryzicola、X. hortorum pv. pelargonii有IS插入,其餘菌株在此部份沒有任何功能基因存在。將此分類結果與mrdB-peh-1基因間序列的分類結果進行比較,推測XCC的NTPase片段、XOO的ucp片段以及X. arboricola的qumA-qumB片段可能分別經由水平基因轉移移入Xanthomonas中。
摘要(英)The study on insertion sequence IS1404 of Xanthomonas campestris pv. campestris XCC1-1 led to a discovery of NTPase gene which is interrupted by IS1404. Based on whole genome sequences of X. campestris pv. campestris ATCC33913 ( XCC ATCC33913 ), X. axonopodis pv. citri 306 ( XAC 306 ), and X. oryzae pv. oryzae KACC10331 ( XOO KACC10331 ), it was found that an IS1404 is located inside the NTPase of XCC ATCC33913, but XAC 306 and XOO KACC10331 do not contain NTPase gene. In addition, the three Xanthomonas spp. have different gene content in this region. In XCC ATCC33913, the order of genes is mrdB-NTPase-chp-hp-peh-1 ( NTPase fragment ), whereas XAC 306 is mrdB-peh-1, and XOO KACC10331 is mrdB-ucp1-hp-ucp2-ucp3-peh-1 ( ucp fragment ). To determine weather NTPase fragment exists only in XCC, PCR and Southern hybridization are used to test different Xanthomonas species and XCC strains. The results indicated that NTPase fragment only existed in XCC strains and was located between mrdB and peh-1 in all XCC strains tested. However, two flanking genes, mrdB and peh-1 were highly conserved and widely distributed in Xanthomonas spp., Indicating that NTPase fragment was acquired by XCC through horizontal gene transfer. A specific primer set XCCchp-F/ XCCchp-R1 for chp gene of NTPase fragment was designed and could amplify a 443-bp DNA fragment from all XCC strains, but not from non-XCC strains ( except X. campestris pv. aberrans and X. bromi ) or non-Xanthomonas plant pathogenic bacteria in PCR tests. The primer set was used to detect the presence of XCC in artificially inoculated cabbage seeds. The same strategy was applied to XOO, a specific primer Xo-ucp-F/ Xo-ucp-R for ucp2 gene of ucp fragment was designed and could only amplify a 643-bp DNA fragment from all XOO strains tested, but not from other Xanthomonas spp. The mrdB-peh-1 intergenic region of other species of Xanthomonas were amplified using primer mrdB-F2L/ Xpeh-R and sequenced. Based on sequence analysis, the regions could be seperated into eight different types. The nucleotide sequence of each type was used as a DNA microarray probe to identify different species of Xanthomonas. In addition to the region of mrdB-peh-1, we also found another DNA region which was probably acquired by horizontal gene transfer. In previous study, X. arboricola has qumA and qumB involved in quinate metabolism, which were absent in other Xanthomonas spp. The flanking genes of qumA and qumB in X. arboricola pv. juglandis XJC5 is unknown-qumA-qumB-pep. Based on whole genome sequences of XCC ATCC33913, XAC 306, and XOOKACC 10331, these Xanthomonas spp. have unknown and pep, but do not contain 4-kb qumA-qumB DNA frament. Amplification and sequencing of twelve different species of Xanthomonas showed variable nucleotide sequences in unknown-pep intergenic region. The regions could be separated into seven different types. The studies on mrdB-peh-1 and qumA-qumB allowed us to find specific DNA fragments of XCC and XOO, respectively, and to group Xanthomonas spp. by these two intergenic nucleotide sequences.
論文目次中文摘要 英文摘要 前言 材料與方法 菌種取得及培養條件 自Xanthomonas中選殖mrdB-peh-1及unknown-pep intergenic region 甘藍菜種子內XCC的增殖與偵測 序列比對分析 以Dot Blot區分六種類型的Xanthomonas 探針的製作 標的物的製作 Dot Blot操作流程 雜合反應 清洗 呈色反應偵測 利用NJ(neighbor-joining)建立親源關係樹 基礎分生技術 結果 mrdB-peh-1 cluster: 在Xanthomonas中之mrdB-peh-1 cluster內基因種類與坐落位置的分析 mrdB與peh-1二基因的序列分析 XCC之chp基因在Xanthomonas或其它屬植物病原細菌的存在情形 利用XCC的chp基因專一性引子檢測甘藍菜種子內之XCC XOO之ucp2基因在Xanthomonas中的存在情形 mrdB-peh-1 cluster在非XCC、XOO的Xanthomonas中排列情形 mrdB-peh-1間序列分析與分子類型區分 以Dot blot的方式區分不同類型的Xanthomonas 利用mrdB-peh-1cluster建立演化關係樹 unknown-pep cluster: 在Xanthomonas中之unknown-pep cluster內基因種類與坐落位置的分析 unknown與pep二基因在Xanthomonas中的存在情形與序列分析 unknown-pep間序列分析與分子類型區分 利用unknown-pep cluster建立演化關係樹 討論 參考文獻 表 圖
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