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研究生:陳宥溱
研究生(外文):You-Jen Chen
論文名稱:台灣茄科細菌性斑點病菌avrBs2的基因功能分析
論文名稱(外文):Functional analysis of avrBs2 from Taiwan-isolated Xanthomonas vesicatoria Xvt147
指導教授:鄧文玲鄧文玲引用關係
學位類別:碩士
校院名稱:國立中興大學
系所名稱:植物病理學系所
學門:農業科學學門
學類:植物保護學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:52
中文關鍵詞:第三型分泌系統
外文關鍵詞:Xanthomonas vesicatoriaavrBs2Type III secretion system
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在高溫高濕的環境中 Xanthomonas campestris pv. vesicatoria (Xcv) 會在茄
科作物番茄與甜椒上造成細菌性斑點病,其主要的病徵為會在葉片、莖部、花器
及果實處造成斑點型外圍帶有黃暈的壞疽病斑。Xanthomonas spp. 的致病能力由
第三型蛋白質分泌系統與一系列藉由第三型分泌系統分泌出去的 effectors 決
定,本研究探討在 Xanthomonas spp. 中普遍存在的 AvrBs2 在細菌感染植物的
過程中可能具有的功能。AvrBs2 可被抗性寄主植物內的 Bs2 蛋白辨識,另外,
在感染寄主植物中則可藉由未知的機制促進病原性。由 AvrBs2 胺基酸序列分析
後發現其 271-422 aa 與Agrobacterium tumefaciens 的 agrocinopine synthase (acs)
以及 E. coli 的 glycerophosphoryl diester phosphodiesterase (GDPD, encoded by
ugpQ) 酵素活性位置具有高度相似性,除此之外由不同的 Xanthomonas spp. 所
選殖而來的 avrBs2 alleles 在同樣的位置也具有高度保留性,故推測此片段對於
AvrBs2 在感染植物時可能具有重要的功能。另外利用 gain-of-function 與
lost-of-function 的方式分析台灣所分離來的 X. vesicatoria (Xvt147) 與 X.
campestris pv. raphani (cxsp3),以 marker exchange 的方式致變 Xvt147 之
avrBs2,得到突變株後以番茄進行接種測試,發現 Xvt147 avrBs2 接種番茄後
的病徵量與在番茄內的族群數與野生型菌株無明顯差異;但在限制植物水分供應
的情況下,過量表現 AvrBs2 則會使 Xvt147 感染的植物出現嚴重萎凋病徵。而
在原本即不帶有 avrBs2 之 cxsp3 內表現 Xvt147 來源的 avrBs2,並對甘藍及
番茄進行接種測試,發現帶有 AvrBs2 之 cxsp3 菌株會使得病徵出現時間提
早,另外,轉型菌株在植物內初期的生長速率高於野生型的 cxsp3,顯示 Xvt147
AvrBs2 可影響植物病徵的呈現。有趣的是在培養 cxsp3 的過程中,發現avrBs2
可避免 cxsp3 於培養晚期中產生細胞凝聚的情形,推測 AvrBs2 可能具有提供
細菌適應培養基與寄主內環境的功能。
Bacterial spot disease of pepper and tomato caused by Xanthomonas campestris
pv. vesicatoria (Xcv) is a major problem where high temperatures and high humidity
occur simultaneously. The pathogenicity of Xanthomonas spp. depends on a
functional Type III secretion system (T3SS) and an array of effectors translocating
through the T3SS. One effector of interest found in nearly all examined Xanthomonas
spp. is AvrBs2. AvrBs2 is readily recognized by the R protein of Bs2 in resistant
plants and is involved in promoting virulence in susceptible plants by unknown
mechanisms. Sequence analysis reveals the amino acid residues 271-422 of Xv
AvrBs2 are homologous to the catalytic domain residing in agrocinopine synthase
(encoded by acs) of Agrobacterium tumefaciens and glycerophosphoryl diester
phosphodiesterase (GDPD, encoded by ugpQ) of E. coli. In addition, diverse avrBs2
alleles identified in other Xanthomonas spp. are highly conserved in the catalytic
domain, suggesting it is important for AvrBs2 activities in planta. Genetic studies of
AvrBs2 function are done using gain-of-function and loss-of-function strategies in X.
vesicatoria Taiwan strain Xvt147 and Xanthomonas campestris pv. raphani (cxsp3)
Taiwan strain cxsp3, respectively. Xvt147 avrBs2 is mutated by marker exchange and
the resultant mutant phenotypes are assayed in tomato. The degrees of symptom
development and bacterial multiplication of Xvt147 avrBs2 mutant are comparable
to the wild type strain. Nevertheless, overexpression of AvrBs2 in Xvt147 elicits
severe symptoms on tomato when infected plants are subjected to short-term water
deficiency. Heterologous expression of AvrBs2 in cxsp3 lacking the avrBs2 gene
slightly increases bacterial growth at 1 dpi and delays the onset of symptom
development in cabbage, indicating Xvt147 AvrBs2 might affect plant biochemical
activities for symptom production. Interestingly, the expression of AvrBs2 in cxsp3
31
prevents the formation of bacterial aggregates that are usually seen in the stationary
phase-grown bacteria, suggesting the functions of AvrBs2 might be important for
bacterial adaptation in vitro and in planta.
壹、 前言-------------------------------------------------------------------------------- 1
貳、 材料與方法----------------------------------------------------------------------- 6
一、供試菌株及其培養--------------------------------------------------------- 6
二、供試植物---------------------------------------------------------------------- 6
三、Xanthomonas spp. 之 avrBs2 高度保留區域之選殖---------------- 6
四、avrBs2 coding region 之選殖--------------------------------------------- 7
五、突變菌株之製備------------------------------------------------------------- 7
六、染色體 DNA 之抽取與濃度測量--------------------------------------- 8
七、質體 DNA 之抽取與限制酵素分析------------------------------------ 9
八、菌落聚合酶連鎖反應------------------------------------------------------- 10
九、E. coli DH10B 勝任細胞之製備與電轉形作用----------------------- 10
十、南方雜合法 11
(一) 探針之製備----------------------------------------------------------- 11
(二) 核酸之轉漬----------------------------------------------------------- 11
(三) 雜合反應-------------------------------------------------------------- 12
(四) 洗片與壓片----------------------------------------------------------- 12
十一、病原細菌接種試驗----------------------------------------------------- 12
十二、葉片中菌量測試-------------------------------------------------------- 13
參、 結果-------------------------------------------------------------------------------- 14
一、Xanthomonas spp. 菌株間之 avrBs2 的存在情形------------------ 14
二、取得 avrBs2 缺乏之 Xanthomonas vesicatoria 菌株------------ 14
三、avrBs2 影響菌株之生理現象----------------------------- 15
(一) avrBs2 對 Xanthomonas spp. 菌株生長的影響--------------- 15
(二) avrBs2 對 Xanthomonas campestris pv. raphani 菌株生長
的影響----------------------------------------------------------------- 15
(三) DSF 對 Xanthomonas campestris pv. raphani 凝聚現象產生
的影響-----------------------------------------------------------------
16
(四)、不同 pH 的環境對 Xanthomonas campestris pv. raphani
凝聚現象的影響-----------------------------------------------------------
16
四、avrBs2 對植物病原細菌感染寄主時造成之影響-------------------- 17
(一)、Xanthomonas vesicatoria 病原性接種測試-------------------- 17
(二)、Xanthomonas campestris pv. raphani 病原性接種測試---- 18
肆、 討論-------------------------------------------------------------------------------- 20
伍、 參考文獻-------------------------------------------------------------------------- 25
陸、 中文摘要-------------------------------------------------------------------------- 29
柒、 英文摘要-------------------------------------------------------------------------- 30
捌、 圖表-------------------------------------------------------------------------------- 32
玖、 附錄-------------------------------------------------------------------------------- I
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