Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36062
標題: 楊桃細菌性斑點病菌 harpin(hrpZPsa)與hrpWPsa基因的選殖及特性分析
Cloning and characterization of harpin-coding hrpZPsa and hrpWPsa genes of Pseudomonas syringe isolated from carambola
作者: 蔡東益
Tsai, Dong-Yi
關鍵字: Pseudomonas syringe;楊桃細菌性斑點病菌;harpin;hrpW;harpin;hrpW
出版社: 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,經生理生化特性分析,顯示此病原菌屬於Pseudomonas syringae 的一種新病原型 (pathovar),定名為 Pseudomonas syringae pv. averrhoi (Psa) 。許多植物病原細菌利用第三型蛋白分泌系統將特定的蛋白送入植物體內,其中harpin就經由此途徑被分泌到胞外的蛋白,且已廣泛被証實存在於Pseudomonas (HrpZ)、Erwinia (HrpN)、Xanthomonas (HpaG)、Ralstonia (PopA)等菌屬中,除了造成過敏反應外,在寄主上的生物特性變化很大。因此,本研究目的主要選殖楊桃細菌性斑點病菌的harpinPsa基因,並探討其生物特性。首先利用多個已證實含有harpin基因之P. syringae 菌株在hrpR和hrpD基因取其保留性序列,合成一組引子對,利用PCR方式以楊桃細菌性斑點病菌染色體DNA當作模板,則可增幅出一段約4.5 kb的DNA片段,此核酸及衍生的胺基酸序列經比對後,顯示此片段含有harpinPsa 基因,並編譯出346個胺基酸的蛋白,經由胺基酸序列分析、過敏性反應、西方轉漬法等分析,顯示楊桃細菌性斑點病菌的harpinPsa與已証實的harpin並無明顯的差異,皆具有共同的特性為: (1)含有高量的甘胺酸(glycine),但不含半胱胺酸( cysteine),(2)具有熱穩定性,(3)可在植物的細胞間隙引起過敏性反應,(4) 經由第三型蛋白分泌系統運送。 進一步利用不具有terminator的nptII基因構築 hrpZPsa 基因的非極性突變株,探討harpinPsa在楊桃細菌性斑點病菌中扮演的角色,結果顯示對於在非寄主植物菸草中引起過敏性反應及寄主植物楊桃植株致病能力並没有顯著的影響,只能說明由hrpZPsa基因轉譯的harpinPsa在引起過敏性反應及致病機制中不是唯一必須的蛋白,另外也從楊桃細菌性斑點病菌中選殖類似harpin功能之基因如,hrpWPto基因,經核酸序列的比對與胺基酸序列分析,結果發現楊桃細菌性斑點病菌的hrpWPsa基因序列在331~333 bp處有一終止碼造成無法編譯出一完整的蛋白,且無法在E. coli BL21中被大量表現,以Pto的HrpWPto抗血清進行西方轉漬法分析在病原菌體內也無法偵測到HrpWPsa蛋白。另一值得注意的是以標記致變構築hrpZPsa 基因的非極性突變株過程中,此病菌有27%會發生非預期之基因重組或變異現象,得到另外兩類的突變株,而其基因重組或突變之處則有待進一步分析。這些結果顯示楊桃細菌性斑點病菌相對於P. syringae其它不同病原型的菌株可能存在的差異性,值得日後深入研究。

Bacterial spot is a new disease of carambola in Taiwan. Based on physiological characteristics, the pathogen was identified as a strain of Pseudomonas syringae and named as a new pathovar: P. s. pv. averrhoi (Psa). Many phytopathogenic bacteria use a type III protein secretion system to transfer virulence proteins into the host. Harpins constitute a group of secreted effectors proteins travelling along the type III pathway in plant pathogenic bacteria. Genes encoding such proteins have been identified in Erwinia amylovora (HrpN), P. syringae (HrpZ), Xanthomonas campestris (HpaG) and Ralstonia solanacearum (PopA). Since the biological functions of haprins varied especially on pathogenesis in different pathogens, the aim of this study is to clone harpinPsa gene from Psa strains. The primes corresponding to the conserved regions of hrpR and hrpD in other pathovars were designed, the chromosome DNA of Psa HL1 strain was used as a template, then polymerase chain reation (PCR) was performed to gain a 4.5 kb fragment cloned in pGEM-T easy vector. Based on the DNA sequencing and derived amino acid sequence using NCBI BLAST analysis of this insert, it reveals that this insert contains partial sequences of hrpR,-D genes, and complete hrpS,-A,-Z, and hrcJ genes. hrpZPsa encodes a 346-aa harpin protein, which is also glycine-rich and cysteine-lacking as reported for harpins produced by other pathovars. Moreover, in biological assay, HrpZPsa is secreted in culture when the Hrp (type III) system is expressed and possesses heat-stable HR elicitor activity when infiltrated into the intercellular spaces of tobacco. To further investigate the role of hrpZPsa in host-parasite interactions, a non-polar mutation of PsaHL1 in which hrpZPsa was deleted and replaced with an nptII cassette lacking a rho-independent transcription terminator was constructed by using marker- exchanged mutagenesis. The hrpZPsa mutant M3 retained the wild-type ability to elicit the HR in tobacco leaves and to cause disease in carambola leaves. This observation suggests that HrpZPsa is not the only elicitor secreted by Psa HL1. According to reports that P. syringae pathovars may produce a family of harpin protein, e.g. P. s. pv. tomato DC3000 producing a second harpin HrpW. Here, the corresponding hrpWPto gene was also isolated from PsaHL1 using PCR. The sequence information revealed that this gene carries a point mutation resulted in a stop codon and potentially encodes only 110 N-terminal amino acids. SDS-PAGE and western blot analysis of the expressed HrpWPsa in E. coli BL21 or PsaHL1 reveals that HrpWPsa is not detectable either E. coli BL21 or PsaHL1. So, hrpWPsa may evolve to be a psudogene. Strikingly, PsaHL1 occured unexpected recombination and mutation at around 27% rate during marker-exchanged mutagenesis on hrpZPsa gene. Taken together, those results were obviously different from those of the other pathovars of P. syringae. Therefore, it is very worthy to study further the genetics of P. syringae pv. averrhoi in turn of pathogenesis.
URI: http://hdl.handle.net/11455/36062
Appears in Collections:生物科技學研究所

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