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研究生:黃紹昌
研究生(外文):Shau-Chang Huang
論文名稱:十字花科Xanthomonas屬hrpF(+)及hrpF(–)菌株之研究
論文名稱(外文):The studies on hrpF(+) and hrpF(–) strains of Xanthomonas spp. from cruciferous plants
指導教授:李永安李永安引用關係
指導教授(外文):Yung-An Lee
口試委員:張雅君蘇睿智
口試委員(外文):Ya-Chun ChangRuey-Chih Su
口試日期:2013-07-25
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:2013
畢業學年度:101
語文別:中文
論文頁數:109
外文關鍵詞:Xanthomonascruciferous
相關次數:
  • 被引用被引用:3
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以Xan-D培養基檢測十字花科種子,有些種子可分離出水亮、上凸且具黃綠色之Xanthomonas spp.。將這些菌株接種至甘藍菜葉片,結果發現:有些菌株可引發黑腐病病徵(black rot),應為十字花科黑腐病菌(X. campestris pv. campestris;XCC),有些菌株則引起葉斑病徵 (leaf spot),應為十字花科葉斑病菌(X. campestris pv. raphani;XCR),但有些菌株則無法引起病徵,應為腐生性Xanthomonas spp.。可引起病徵之菌株,不論是XCC或XCR菌株,以hrpF設計之PCR引子組,進行PCR反應,均可擴增出特定的DNA片段 [稱為XCC-hrpF(+)或XCR-hrpF(+)菌株];而無法引起病徵的菌株,則無法擴增出特定的DNA片段 [稱為Xan-hrpF(–)菌株]。將自種子分離出的Xan-hrpF(–)菌株進行鑑定,發現Xan-hrpF(–)菌株確實為Xanthomonas屬細菌。自具有斑點病的蘿蔔的病組織中,可分離到XCR菌株,進行hrpF-PCR,發現有些菌株為hrpF(+),而有些則為hrpF(–),因此認為XCR-hrpF(+)及Xan-hrpF(–)菌株共存於植物上。以脈衝式電泳對XCR-hrpF(+)與Xan-hrpF(–)菌株進行分析,發現兩菌株之間的分子分型並不相同。為了解hrpF對XCC及XCR致病能力的影響,將EZ-Tn5 跳躍子送入hrpF基因,再以marker exchange方法,將XCC-hrpF(+)或XCR-hrpF(+)菌株的hrpF 基因剔除(knockout),得到XCC hrpF knockout 及XCR hrpF knockout突變株經接種後,發現XCC hrpF knockout突變株仍具病原性,但病徵明顯減輕,但XCR hrpF knockout突變株則喪失致病能力。因此單一hrpF的缺乏,不能使XCC喪失病原性。為了解Xan-hrpF(–)菌株除hrpF之外,是否還有其他基因的缺失,進一步以PCR及Southern hybridization進行測試:(a) XCC及XCR-hrpF(+)菌株均具有pathogenicity island上相關的hrc基因,而Xan-hrpF(–)菌株不僅不含hrpF,也不具有整段pathogenicity island;(b) XCC及XCR-hrpF(+)菌株所具有的effector protein基因數量並不相同,而Xan-hrpF(–)則可能具有部份的effector protein基因。本實驗已對兩株Xan-hrpF(–)菌株進行whole genome定序,結果發現Xan-hrpF(–)菌株不具有pathogenicity island,故Xan-hrpF(–)菌株應不具有第三型分泌系統。將Xan-hrpF(–)菌株接種至煙草(Nicotiana benthamiana)葉片中,發現不會引發過敏性反應。將Xan-hrpF(–)菌株接種至甘藍葉片中,發現至少可在葉片中存活七天以上。利用不具致病能力的Xan-hrpF(–)菌株及XCR hrpF knockout突變株,與XCC-hrpF(+)菌株混合接種,發現Xan-hrpF(–)菌株及XCR hrpF knockout突變株可明顯抑制XCC-hrpF(+)的致病能力。
The Xan-D medium was used for the isolation of xanthomonads from cruciferous seeds. The wet-shining, convex and yellow-green bacterial colonies on the Xan-D media were supposed to be xanthomonads and chosen for further experiments. The cabbage leaves were inoculated with the isolated xanthomonad strains. Some strains that could cause black rot symptoms were X. campestris pv. campestris (XCC), and some strains causing leaf spot symptoms were X. campestris pv. raphani (XCR). Some other strains that did not produce any disease were non-pathogenic xanthomonads. The isolated XCC and XCR strains could amplify a specific DNA fragment in a PCR using hrpF primers (called XCC or XCR-hrpF(+) strains), but nonpathogenic xanthomonads did not produce any fragments (called Xan-hrpF(–) strains). The Xan-hrpF(–) strains were indeed identified as Xanthomonas spp. by 16S rRNA gene sequence analysis, Biolog and PCR tests. In addition to seeds, XCR-hrpF(+) and Xan-hrpF(–) strains were isolated from radish tissues with bacterial leaf spot symptoms. The molecular typing patterns of isolated XCR-hrpF(+) and Xan-hrpF(–) strains were determined by pulsed-field gel electrophoresis (PFGE), and the results showed that XCR-hrpF(+) and Xan-hrpF(–) strains are heterogeneous. To understand the effect of hrpF on the pathogenicity of XCC-hrpF(+) and XCR-hrpF(+) strains, XCC and XCR hrpF-knockout mutant strains were constructed by EZ-Tn5 transposon mutagenesis and marker exchange methods. The XCC hrpF-knockout strain only reduced the virulence on the inoculated cabbage leaves, but the XCR hrpF-knockout strain did not cause any symptoms and totally lost the pathogenicity. Therefore, the lack of hrpF might not cause pathogenic xanthomonads to lose their pathogenicity. The Xan-hrpF(–) strains were further found that the strains not only lost hrpF but also lost the entire pathogenicity island and some effector protein genes determined by PCR, Southern hybridization and whole genome sequence draft analyses. Xan-hrpF(–) strains did not induce the hypersensitive response (HR) on the tobacco leaves (Nicotiana benthamiana). Although Xan-hrpF(–) strains did not cause diseases, they could survive at least seven days inside the cabbage leaves. Xan-hrpF(–) and XCR hrpF-knockout strains could reduce the symptom development of XCC-hrpF(+) when co-inoculated on cabbage leaves with XCC-hrpF(+), indicating that both Xan-hrpF(–) and XCR hrpF knockout strains could suppress the virulence of XCC-hrpF(+).
中文摘要
英文摘要
前言
材料與方法
01.菌種取得來源、培養條件及儲存方法
02.十字花科種子檢測流程
03.十字花科作物(蘿蔔)病徵檢測流程
04.Xanthomonas屬細菌之病原性測試
4-1 X. campestris pv. campestris接種方法
4-2 X. campestris pv. raphani接種方法
05.分離之Xanthomonas屬細菌之hrpF基因檢測
5-1 hrpF-PCR
5-2 hrpF-Southern hybridization
06..Xanthomonas hrpF(–)菌株之鑑定
6-1 16S rDNA gene選殖、定序及比對
6-2 利用Biolog GN2 MicroPlateTM作檢測
6-3 利用Biolog GNIII MicroPlateTM作檢測
6-4 利用cel3535-PCR作檢測
07.利用脈衝式電泳(Pulsed-field gel electrophoresis, PFGE)對X. campestris pv. raphani與Xan-hrpF(–)菌株作分子分型(molecular typing)
08. X. campestris pv. raphani XCR-R1-2-3 hrpF knock out菌株製作
8-1 利用hrpF-PCR檢測X. campestris pv. raphani XCR-R1-2-3 hrpF knockout菌株
8-2 利用hrpF-Southern hybridization檢測X. campestris pv. raphani XCR-R1-2-3 hrpF knockout菌株
8-3 檢測X. campestris pv. campestris與X. campestris pv. raphani hrpF knock out菌株之致病能力
09.分離之Xanthomonas屬細菌之pathogenicity island基因檢測
9-1 hrcN、hrcU、hrcR-PCR
9-2 hrcN、hrcU、hrcR-Southern hybridization
10.分離之Xanthomonas屬細菌之effector protein genes檢測
10-1 xopD、xopF1、xopP、xopR-PCR
10-2 xopD、xopF1、xopP、xopR- Southern hybridization
11..Xan-hrpF(–)菌株whole genome定序與比對
12.分離之Xanthomonas屬細菌於煙草上之過敏性反應測試
13.檢測Xan-hrpF(–)菌株於甘藍葉片的生長情形
14.Xan-hrpF(–)、X. campestris pv. raphani hrpF knockout菌株分別與X. campestris pv. campestris等比例菌量混合接種甘藍葉片試驗
15.基礎分生技術
實驗結果
01.自十字花科種子與蘿蔔病徵分離之Xanthomonas屬細菌之病原性測試
02.分離之Xanthomonas屬細菌之hrpF基因檢測結果
03. Xan-hrpF(–)菌株之鑑定結果
04. X. campestris pv. raphani hrpF(+)與Xan-hrpF(–)菌株之脈衝式電泳之類型(pattern)
05. X. campestris pv. raphani hrpF knockout菌株之構築結果
06. X. campestris pv. campestris與X. campestris pv. raphani hrpF knockout菌株接種結果
07.分離之Xanthomonas屬細菌之pathogenicity island基因之檢測結果
08.分離之Xanthomonas屬細菌之effector protein genes之檢測結果
09. Xan-hrpF(–)菌株whole genome定序草圖(draft)之比對分析結果
10.分離之X. campestris pv. campestris、X. campestris pv. raphani與Xan-hrpF(–)菌株於煙草上之過敏性反應測試結果
11..Xan-hrpF(–)菌株於甘藍葉片的生長情形
12. Xan-hrpF(–)菌株、X. campestris pv. raphani hrpF knock out分別與X. campestris pv. campestris等比例菌量混合接種甘藍葉片試驗
討論
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