Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/35990
標題: 楊桃細菌性斑點病原菌的可交換因子區域的選殖及序列分析
Cloning and sequence analysis of exchangeable effector loci (EEL) of the Pseudomonas syringae from carambola
作者: 胡育銘
Hu, Yu-Min
關鍵字: Hrp Pais;楊桃細菌性斑點病原菌;EEL;hrmA;carambola;P. syringae
出版社: 農業生物科技學研究所
摘要: 
楊桃細菌性斑點病為台灣發生之新病害,其最顯著之病徵為葉片上形成紫紅色斑點,周圍有明顯黃色暈環,且葉片易黃化脫落,經生理生化特性測試,顯示此病原菌為 Pseudomonas syringae 之菌株。Alfano等人研究,分析植物病原菌P. syringae pv. syringae (Pss) 61、P. syringae pv. syringae (Pss) B728a 和P. syringae pv. tomato (Pto) DC3000不同菌株的基因序列,顯示有一 Hrp pathogenicity island (Pai) 的基因體,為一個三組不同基因群組成的鑲嵌結構。hrp/ hrc 基因群存在於 Hrp Pais中是一具有保留性的基因群在其兩側有一可交換因子區 (exchangeable effector locus,簡稱EEL) 和一個保留性因子區(conserved effector locus,簡稱CEL)。EEL區域連接於 hrp/ hrc 基因群的左邊位置,常存在一些 effector 蛋白和易變動的遺傳序列如轉移子等。本研究目的主要選殖楊桃細菌性斑點病原菌的 EEL 區域的基因序列,利用菌株 Pss 61 具有保留性基因 queA 和 hrpK 的序列,合成一組引子對EEL1/EEL2,以不同來源的楊桃細菌性斑點病原菌染色體 DNA 當作模板,進行 PCR 反應,則不同來源的楊桃細菌性斑點病原菌皆可增幅出一段約4.3 kb 的EEL 區域DNA片段,再利用限制酵素進行次選殖及設計適當的引子等策略,將楊桃細菌性斑點病原菌HL1的 EEL區域 DNA 片段作完整定序及分析,可得到全長為4291 bp的核酸序列,此核酸及衍生的胺基酸序列經比對後,顯示此 EEL 區域的基因序列,除了兩端有 queA, tRNA-Leu 及hrpK 基因外,主要含有orf1 基因以及被一轉移子 (tnp1’) 插入的hrmA-like 基因,與菌株 Pss 61 的 EEL 區域有較高的相似性,不同之處在於 HL1 的 hrmA-like 基因被轉移子插入,而且 HL1 中的 hrmA-like 及 orf1的轉錄方向與菌株 Pss 61 相反。進一步分析楊桃細菌性斑點病原菌 HL1 與菌株 Pss 61 的 hrmA 及 orf1 基因的功能,顯示 Pss 61的 hrmA 及 orf1能促進楊桃細菌性斑點病原菌 PA5 在菸草中誘發過敏性反應,而 HL1 和 Pss 61 的 hrmA及 orf1 皆能降低楊桃細菌性斑點病原菌對楊桃植株的致病力;另外楊桃細菌性斑點病原菌攜帶分別來自HL1 或 Pss 61 的 hrmA 及 orf1 基因,於楊桃葉片內生長測定試驗中,顯示帶有菌株 Pss 61 的 hrmA 及 orf1 基因的楊桃細菌性斑點病原菌,在楊桃植株中的生長會受到抑制。進一步利用 RFLP 分析方法,來了解楊桃細菌性斑點病原菌 HL1 與其他不同 P. syringae 病原型菌株之間的親源關係,結果顯示楊桃細菌性斑點病原菌 HL1與菌株P. s. pv. phaseolicola 343、P. s. pv. glycinea Race 4、P. s. pv. angulata Pa45R、P. s. pv. tabaci 11528 及 P. s. pv. lachrymans 859 的親源關係較接近。最後依據楊桃細菌性斑點病原菌 HL1 的 hrmA-like 基因序列設計引子對HL1-hrmA01/ HL1-hrmA02,針對楊桃細菌性斑點病原菌和其他不同病原型菌株進行 PCR 反應,結果顯示只有楊桃細菌性斑點病原菌才能增幅一約0.5 kb的DNA片段,所以引子對 HL1-hrmA01/ HL1-hrmA02 可被開發作為快速偵測及鑑定楊桃細菌性病原菌的基礎。

Bacterial spot is a new disease of carambola in Taiwan. The typical symptoms on leaves are purple spots surrounded by yellow haloes, and the infected leaves turn yellow and fall easily. Based on physiological characteristics, the pathogen was identified as a strain of Pseudomonas syringae. According to the report of Alfano et al., DNA sequence analysis of the hrp/hrc regions in P. syringae pv. syringae (Pss) 61. P. syringae pv. syringae (Pss) B728a, and P. syringae pv. tomato (Pto) DC3000 has revealed a Hrp pathogenicity island (Pai) with a tripartite mosaic structure. The hrp/ hrc gene cluster is conserved in Hrp Pais and is flanked by a unique exchangeable effector locus (EEL) and a conserved effector locus (CEL). The EEL region is linkage to the left of hrp/hrc gene cluster and contains some effectors and mobile genetic elements. The aim of this study is cloning the EEL sequences of carambola strains. First, with the sequences of the conserved region of queA and hrpK in Pss 61 to design primer pair EEL1/ EEL2 and the chromosome DNA of carambola strains as a template, polymerase chain reaction (PCR) was performed to clone a 4.3 kb EEL fragment in pGEM-T easy vector. Based on the DNA sequencing and derived amino acid sequence by NCBI BLAST analysis of this insert, it revealed that the region of EEL contains orf1 and a transposase (tnp1') gene inserted hrmA-like, beside that are flanked by the sequences of queA, tRNA-Leu and hrpK genes. The sequences and gene organization are most similar to the region of EEL in Pss 61. But, the transcription direction of the hrmA-like and orf1 genes from P. syringae HL1 is opposite to them from Pss 61. With analysis of biological function of HrmA and ORF1 from P. syringae HL1 and Pss 61, it revealed the HrmA and ORF1 from Pss 61 could modulate P. syringae from carambola strains to elicit HR in tobacco. Both the HrmA and ORF1 from P. syringae HL1 and Pss 61 could reduce the virulence of P. syringae strains from carambola on carambola leaves. Besides, based on population changes of the strains of P. syringae HL1 carrying the hrmA and orf1 from P. syringae HL1 and Pss 61, respectively, it revealed the growth of P. syringae HL1 carrying the hrmA and orf1 gene from Pss 61 would be inhibited in the leaves of carambola. Further, restriction fragment length polymorphism (RFLP) is used to understand the relationship among the strains of P. syringae from carambola and other P. syringae pathovars. It revealed the relationship of P. syringae HL1 and Pss 61 is quite different. Finally, according to the sequences of hrmA-like gene from P. syringae HL1 primer pair HL1-hrmA01/ HL1-hrmA02 was designed to use for PCR based identification . Only the strains of P. syringae from carambola could be amplified a ca. 0.5 kb DNA fragment. The results indicated that the pair HL1-hrmA01/ HL1-hrmA02 could be a useful tool for rapid detection and identification of P. syringae strains from carambola.
URI: http://hdl.handle.net/11455/35990
Appears in Collections:生物科技學研究所

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