臺灣博碩士論文加值系統

自Xanthomonas campestris pv. campestris (Xcc) strain 1-1基因組中選殖出了三段帶有IS1404a、IS1404c與IS1404d的不同DNA片段，分析這些IS1404座落位置兩側的核酸序列發現，IS1404a位在一個類似IS476且屬於IS3 family的一個新插入序列ISA2的transposase基因ORFb內；除IS1404a外，ISA2還被另外一個與IS1400近似也屬於IS3 family的新插入序列ISA1所插入。IS1404c的插入位置是在IS5 family的IS1478的transposase基因中。IS1404d則是座落在一個與Rickettsia prowazekii RP293、Agrobacterium tumefaciens virD4、Escherichia coli traG及taxB、Helicobacter pylori病原小島(pathogenicity island)內的cag5等基因相似的新基因內，定名為vag1 (virD4-taxB-cag5-like gene)。將此三段IS1404在基因組內座落位置的兩側序列加上另一來自Xcc14-1的IS1404b兩側序列並列比較分析後，並未發現IS1404在Xcc1-1基因組內的插入位置具有特定的序列存在，不過IS1404有選擇G+C比例較高的目標序列的傾向，並在插入位置兩側的序列中發現有一組5 bp對稱的保守序列。
除了被IS1404d插入破壞的vag1基因外，在Xcc1-1的基因組中還有3.9-kb 及8.3-kb兩段EcoRI DNA片段含有與vag1相似的基因。選殖出3.9-kb EcoRI DNA片段並經核酸定序及分析後發現，此片段中含有由兩個完整的開放讀架(open reading frame; ORF)，命名為vag2A與vag2B，所組成的operon (vag2AB)。vag2A與vag1基因的核酸序列有92.34%的相同度。 Vag2A的N端部分帶有signal peptide及兩段可能為membrane-spanning domain的hydrophobic regions，並且也含有兩個在RP293、VirD4、TraG、TaxB與Cag5間所高度保留的NTP-binding Walker motifs。另外，以polymerase chain reaction (PCR)方法檢測8.3-kb EcoRI DNA片段，發現此片段中含有一段與vag2A相似且完整的ORF，稱為vag3，因此Xcc1-1基因組中vag基因的存在情形可暫表示為vag1::IS1404, vag2AB, vag3。
當以vag2A基因的內部片段作為探針時，對台灣的三個不同的黑腐病菌group, A, B, 及C,菌株的基因組DNA進行南方氏雜合反應，發現三個group間有明顯不同的雜合反應型式(hybridization pattern)，並且均有二或三條不同大小的EcoRI DNA片段可產生雜合反應訊息，顯示黑腐病菌中至少含二個以上的vag基因。進一步以PCR及南方氏雜合反應實驗結果發現(一)所有測試group A 菌株及部份的group B菌株均有一個vag基因被IS1404所插入，並且IS1404在group A 菌株的插入位置與在group B菌株中不同。在group C 菌株中則未發現有插入的情形。(二) group A, B, 及C菌株均至少有一段與vag2A相似的完整的ORF，如屬於group A的Xcc1-1含有的vag2A及vag3。(三) vag2B基因及vag2AB operon只存在於部份的group A菌株中。由以上的結果顯示黑腐病菌所共有而完整的vag 基因應為Xcc1-1的vag3基因，而Xcc1-1所含有的vag2AB operon可能是經由horizontal transfer引入黑腐病菌的某些菌株內。以marker-exchange方法得到Xcc1-1的vag2A-knock out而vag3仍完整的突變菌株(Xcc1-1 vag1::IS1404, vag2A::Ω B, vag3)，將此菌株接種在寄主植物的甘藍及非寄主植物的煙草上，發現其產生病徵及誘發過敏性反應的能力與Xcc1-1 wild type菌株並無顯著差別。
除在黑腐病菌外，vag基因仍廣泛存在於Xanthomonas各種類的病原小種(pathovar)間，但不存在於其它的植物病原細菌如Erwinia、Pseudomonas及Ralstonia solanacearum內， 因此vag基因為一個Xanthomonas-specific的基因。

Three DNA fragments containing iso-IS1404s, named IS1404a, IS1404c and IS1404d, from Xanthomonas campestris pv. campestris Xcc1-1 genomic DNA were cloned and sequenced. Based on nucleotide sequence analysis of the flanking sequences of these iso-IS1404s, IS1404a is located in the ORFb of transposase gene of a new IS3 family member and IS476-like insertion sequence, ISA2. In addition to IS1404a, another new IS3 family member and IS1400-like insertion sequence, ISA1, also integrate the same ISA2. IS1404c is located in the transposase gene of an IS5 family insertion sequence, IS1478. IS1404d is transposed in an open reading frame (ORF) whose deduced amino acid sequence shows homology to virD4 of Agrobacterium tumefaciens, traG and taxB of Escherichia coli, and cag5 pathogenicity island gene of Helicobacter pylori. The ORF was named vag1 for virD4-taxB--traG-like gene. Further analysis of the flanking sequences of four iso-IS1404s, including IS1404b from Xcc14-1, revealed that there is not any conserved sequence or hot spot for IS1404 transposition, but the sequences are GC-rich and contain a 5-bp inverted-repeat consensus sequence
In addition to vag1, another two DNA fragments of Xcc1-1 genome, 3.9-kb and 8.3-kb EcoRI DNA fragments, contained genes homologous to vag1 determined by Southern hybridization. The 3.9-kb EcoRI DNA fragment were cloned and sequenced. Sequence analysis revealed that the fragment contains an operon (named vag2AB) with two open reading frames, named vag2A and vag2B, respectively. There is 92.34% identity between vag1 and vag2A nucleotide sequence. The amino-terminus region of Vag2A has a signal peptide and two hydrophobic regions, which may function for membrane-spanning. Vag2A also contains two NTP-binding Walker motifs, which are highly conserved in VirD4, TraG, TaxB, and Cag5. In 8.3-kb EcoRI DNA fragment, there is another complete and vag2A-like gene, named vag3, determined by polymerase chain reaction (PCR). The results indicated that vag genes in the genome of Xcc1-1 exist in three different forms and can be expressed as vag1::IS1404, vag2AB, vag3.
When an internal fragment of vag2A was used as a probe, three heterogeneous groups (A, B, and C) of X. campestris pv. campestris can be easily distinguished by hybridization patterns. Strains in each group have two or three EcoRI fragments hybridized to vag2A, indicating that strains contain at least two vag genes. Further PCR and Southern hybridization analyses revealed (1) that all tested strains in group A and some strains in group B have a vag gene integrated by IS1404, but no IS1404-integrated vag gene was found in group C strains; (2) that strains in group A, B, and C contain at least one complete and vag2A-like gene. For example, Xcc1-1 of group A contains vag2A and vag3; (3) that vag2B and vag2AB operon only exist in strains of group A, but not in group B and C. The results suggested that vag3 of Xcc1-1 might be a common vag gene existing in X. campestris pv. campestris, and vag2AB was acquired by horizontal gene transfer. A Xcc1-1 vag2A knock-out mutant strain (vag1::IS1404, vag2A::WB, vag3) was obtained by a marker-exchange method. No difference was found between vag2A knock-out mutant and wild type of Xcc1-1 in the capacities to cause disease in cabbage (host plants) and to induce hypersensitive reaction in tobacco (non-host plants).
The vag gene is widely distributed in pathovars of Xanthomonas spp. but do not exist in other phytopathogenic bacteria such as Erwinia, Pseudomonas, and Ralstonia solanacearum. According, vag gene is a Xanthomonas-specific gene.

中文摘要--------------------------------------------------------------------------------------- I
英文摘要------------------------------------------------------------------------------------- III
前言-------------------------------------------------------------------------------------------- 1
材料與方法--------------------------------------------------------------------------------- 5
菌種及培養條件------------------------------------------------------------------------ 5
自十字花科黑腐病菌Xcc1-1中選殖含有IS1404之核酸片段--------------- 5
序列分析--------------------------------------------------------------------------------- 6
vag2基因的選殖----------------------------------------------------------------------- 8
vag基因分布範圍的測定-------------------------------------------------------------- 8
vag基因knock out菌株的取得------------------------------------------------------ 9
植株與栽培------------------------------------------------------------------------------ 10
植物接種及過敏性反應測試-------------------------------------------------------- 10
基礎分生技術--------------------------------------------------------------------------- 11
實驗結果-------------------------------------------------------------------------------------- 24
選殖Xcc1-1中帶有IS1404之DNA片段---------------------------------------- 24
含有IS1404a之2.7 kb HindIII片段核酸序列分析------------------------------- 24
含有IS1404c之3.8 kb EcoRI核酸序列片段分析------------------------------- 27
含有IS1404d之4.1 kb EcoRI片段核酸序列分析--------------------------------- 27
IS1404插入位置兩側序列並列分析-------------------------------------------------- 28
確認Xcc1-1基因組中是否存在其它完整vag基因------------------------------- 29
vag2的基因結構與DNA序列分析--------------------------------------------------- 30
Vag2A胺基酸序列分析----------------------------------------------------------------- 31
Vag2A與其它TraG family protein的比較---------------------------------------- 32
Vag2B胺基酸序列分析--------------------------------------------------------------- 32
vag3在Xcc1-1基因組中的存在情形----------------------------------------------- 32
vag基因在Xcc不同菌株間的存在情形-------------------------------------------- 33
Xcc不同菌株間被IS1404插入與完整vag基因存在情形---------------------- 34
Xcc1-1 vag2A-knock out菌株的篩選與確認---------------------------------------- 35
Xcc1-1 vag2A-knock out菌株的接種------------------------------------------------- 36
vag基因在Xanthomonas各種不同病原小種間的分布-------------------------- 37
vag基因在各種植物病原細菌中的分布-------------------------------------------- 37
討論--------------------------------------------------------------------------------------------- 38
參考文獻---------------------------------------------------------------------------------------- 53
圖表---------------------------------------------------------------------------------------------- 61

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