Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36143
標題: 楊桃細菌性斑點病原菌hopAJ1及hopAJ2基因的特性分析與藉由轉殖harpin基因對延長矮牽牛花期之可行性
Characterization of hopAJ1 and hopAJ2 genes from Pseudomonas syringae pv. averrhoi and testing on feasibility of extending flower longevity of Petunia plants transformed with a harpin gene
作者: 郭政憲
Kuo, Cheng-Hsien
關鍵字: Pseudomonas syringae pv. averrhoi
楊桃細菌性斑點菌
type III secretion system (T3SS)
transgenic plant
第三型分泌系統
轉基因植物
出版社: 生物科技學研究所
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摘要: 楊桃細菌性斑點病為台灣發生之重要病害,是由楊桃細菌性斑點菌Pseudomonas syringae pv. averrhoi (Pav)HL1所造成,其致病機制為利用第三型蛋白分泌系統 (Type III secretion system, T3SS)將effector蛋白分泌出去,使其能在寄主植物中繁殖。由前人的研究顯示,楊桃細菌性斑點病菌的病原島嶼基因序列類似已被解序完成的植物病原菌P. syringae pv. phaseolicola (Pph)1448A。在本研究中,依據Pph 1448A的hop基因序列,設計出專一性的引子,由楊桃細菌性斑點菌基因庫 (library)及利用PCR方式選殖到hopAJ1及hopAJ2基因。依據胺基酸序列及結構分析,顯示此二蛋白與來自Pph 1448A並無明顯的差異且其都具有MltB (murein lytic transglycosylase B)的保留區域。進一步利用同源性重組的方式構築了hopAJ1、hopAJ2、hopAJ1/hopAJ2與hopAJ1/hopAJ2/orf1的突變株,orf1位於CEL區域,Orf1也是lytic transglycosylase-like蛋白質,但屬於MltD群。植物接種試驗結果顯示hopAJ1、hopAJ2與hopAJ1/hopAJ2的突變株對於在非寄主植物菸草葉片引起過敏性反應並沒有影響,但在寄主植物楊桃的致病力有些微的降低,而hopAJ1/hopAJ2/orf1的突變株在菸草葉片引起過敏性反應與orf1的突變株並無明顯差異,皆有減弱現象,但卻影響在寄主植物楊桃的致病力,顯示HopAJ1及HopAJ2都不是參與過敏性反應唯一必須的蛋白,但在寄主植物楊桃的致病力有其重要性。進一步以CyaA為報導基因,測試HopAJ1或HopAJ2與CyaA的融合蛋白的被分泌情形,結果顯示HopAJ1Pav–CyaA融合蛋白可被分泌到細菌胞外,但無法進入到植物細胞內,而HopAJ2Pav–CyaA融合蛋白則是無法分泌到細菌胞外。至於此兩個effector蛋白在楊桃細菌性斑點菌所扮演的角色仍需進一步探討。 植物病原菌中的harpin蛋白是利用第三型分泌系統被分泌至植物細胞間隙,會在菸草上引發過敏性反應,在P. syringae是由hrpZ基因所編譯的蛋白。由前人的研究結果認為將harpin基因轉殖入植物中,表現Harpin蛋白於植物中可能對植物的開花期會有延長的效果,因此本研究中選用楊桃細菌性斑點菌的hrpZ基因,以探討hrpZ基因表現於矮牽牛中是否對花期有延長的效果。將hrpZ藉由農桿菌 (Agrobacterium tumefaciens)轉入矮牽牛 (Petunia Ultra Rose, Petunia Ultra Blue)中,分別以花椰菜嵌紋病毒 (CaMV)35S連續性 (constitutive)啟動子與hsr515 (hypersensitivity-related gene)誘導性 (inducible)啟動子,調控harpin蛋白於矮牽牛細胞質中的表現,另也構築以PR1 (pathogen-related)基因的訊息胜肽 (signal peptide)來調控harpin蛋白分泌至植物的細胞間隙 (apoplast);以PCR方法與RT-PCR方法確認hrpZ基因已轉入矮牽牛中並表現,並且於所得到的轉基因矮牽牛株系 (lines)中,發現由35S啟動子與35S啟動子帶有PR1訊息蛋白表現的harpin株系中,其開花期相較於未轉殖植株及其他轉基因矮牽牛株系有明顯的延長,且其叢生化現象也較不明顯,至於harpin蛋白與延長植物花期的關係仍需進一步探討。
Bacterial spot of carambola caused by Pseudomonas syringae pv. averrhoi (Pav)HL1 is an important disease in Taiwan. This phytopathogen secretes a repertoire of effector proteins via the type III secretion system (T3SS) to cause disease in the host plant. According to previous studies, the Pai (Pathogenicity island) sequence of Pav HL1 is highly homologous to P. syringae pv. phaseolicola (Pph)1448A whose genome has been completely sequenced. In this study, specific primers were designed from couple of hop genes based on the sequence of Pph 1448A and two genes, hopAJ1 and hopAJ2 were cloned. HopAJ1 and HopAJ2 of Pav HL1 are highly homologous to the ones of Pph 1448A and have the conserved domain of MltB (murein lytic transglycosylase B) by amino acid sequence and structure analysis. Moreover, the strategy of homologous recombination was used to construct the mutants, ΔhopAJ1Pav, ΔhopAJ2Pav, ΔhopAJ1Pav/ΔhopAJ2Pav, and ΔhopAJ1Pav/ΔhopAJ2Pav/Δorf1Pav. The orf1 gene was located in Pai CEL and its product belongs to the MltD group of lytic transglycosylase family. The mutants of ΔhopAJ1Pav, ΔhopAJ2Pav and ΔhopAJ1Pav/ΔhopAJ2Pav elicit typical hypersensitive response (HR) in non-host tobacco, and show slightly reduced virulence in host carambola. The ΔhopAJ1Pav/ΔhopAJ2Pav/Δorf1Pav triple-gene mutant, compared to Δorf1Pav, elicits reduced HR and virulence in tobacco and host carambola, respectively, suggesting that HopAJ1Pav and HopAJ2Pav are not necessary for the elicitation of HR in Pav HL1 but play important roles in virulence. To test whether HopAJ1Pav or HopAJ2Pav is secreted via T3SS, both proteins are fused with the reporter gene CyaA. The HopAJ1Pav-CyaA fusion protein was secreted via T3SS but not translocated into plant cell, while HopAJ2Pav-CyaA fusion protein was not secreted. It is necessary to further investigate the roles of these two proteins in pathogenicity. Harpin is known to be secreted into plant apoplast via T3SS, and it is able to elicit HR in tobacco. In P. syringae, harpin is encoded by hrpZ gene. It has been suggested that transgenes expressing HrpZ delay flower senescence and extend flower longevity. In this study, the hrpZ gene of Pav HL1 was transformed into Petunia using Agrobacterium tumefaciens. The harpin expression is either under the control of a constitutive promoter CaMV 35S or under an inducible promoter hsr515 (hypersensitive response-related gene). In addition, the PR1b signal peptide was used in one of the constructs to secrete HrpZ into apoplast. The transgenic lines were confirmed by PCR and RT-PCR. The extended flower longevity was shown in transgenic plants transformed with 35S-derived hrpZ or 35S-derived hrpZ with PR1b signal peptide constructs, and the rosette of those transgenic plants was not apparent. It is necessary to further investigate the mechanism by which harpin protein can extend flower longevity.
URI: http://hdl.handle.net/11455/36143
其他識別: U0005-0808200718594000
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-0808200718594000
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