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標題: Xanthomonas細菌細胞凋亡機制之探討與應用
Investigation of the Apoptotic Mechanism in Xanthomonas and Its Application in Biocontrol
作者: 翁淑芬
關鍵字: apoptosis;細胞凋亡;生物技術, 植物保護類;programmed cell death;rapid cell death;Xanthomonas campestris pv;campestris;transcriptome;biocontrol;caspase;細胞計畫性死亡;細胞快速死亡;十字花科蔬菜黑腐病菌;轉錄體;生物防治;基礎研究
細胞凋亡又稱細胞計畫性死亡,而poly ADP-ribose polymerase (PARP)與caspase為凋亡過程中的重要印記。咸認細胞凋亡僅存在於真核生物;近年卻首次發現,在原核的植物病原細菌Xanthomonas campestris pv. glycines (Xcg,引起豆類葉燒病)亦有細胞快速死亡現象(rapid cell death,RCD),且可偵測到caspase 與PARP-like活性;含caspase活性的序列表現之蛋白質產物能被human caspase及PARP抗體辨認。推測參與之基因為pda (主導polysaccharide deacetylase)。近緣的十字花科黑腐病菌X. campestris pv. campestris (Xcc)主導之Pda (901 aa)有三個domains: polysaccharide deacetylase、caspase-like 與tetratricopeptide repeat-like domains。申請人為了探討生物防治,曾將包含pda全長的重組質體送入pda突變株,發現會減緩其細胞生長,導致快速死亡。目前,Pda各domain是否具有活性,其於RCD中所扮演的角色仍不清楚。本計畫之目的與探討策略如下: (1)以生物資訊分析Xcc Pda蛋白之構造,並探討各domain之活性與功能,以了解細菌凋亡機制,以及其與真核生物細胞凋亡在演化上之相關性,(2) 探討pda及其上、下游基因組織(organization),然後選殖啟動子,以報導基因測試不同因子對啟動子活性之影響,以了解調控pda表現之機制,(3)利用微陣列技術探討RCD發生時細菌體內的轉錄體(transcriptome)表現,(4) 以Xcc線狀噬菌體為基礎,發展載體系統,供攜帶plant inducible promoter驅動的pda,以應用於生物防治。本計畫之完成有助於了解細菌凋亡機制,亦能應用於生物防治,具有學術重要性及農業經濟價值。

Apoptosis, also known as programmed cell death (PCD), is a completely normal process in living organisms. It plays an important role in the development and maintenance of the integrity of organisms. Both caspase and poly ADP-ribose polymerase (PARP), one of the important substrates of caspase-3, are important markers in the process of apoptosis. Lacking the complexity of an eukaryotic cell, bacteria were believed to not possess the caspase pathway. Thus, it was surprising to find for the first time that Xanthomonas campestris pv. glycines (Xcg), the causative agent of pustule in soybean, exhibits rapid cell death (RCD) phenotype. Such RCD was found to be associated with the expression of caspase-like and PARP-like activities. Furthermore, antibodies against the human caspase and PARP are able to cross react with the polypeptide over expressed from the Xcg caspase-encoding region. It was suggested that the caspase-like and PARP-like proteins are the gene product of Xcg polysaccharide deacetylase gene, pda. This is the first report showing the presence of a caspase-like activity in bacteria and its involvement in cell death. X. campestris pv. campestris (Xcc) is the phytopathogen causing black rot in crucifers. The protein deduced from Xcc pda gene contains 901 amino acid residues with a molecular mass of 98 kDa. This Pda protein carries three domains: polysaccharide deacetylase, caspase-like, and tetratricopeptide repeat-like domains. A pda mutant of Xcc, with the pda gene mutated by insertion of a Gmr cartridge, was constructed during a study in search of the toxic peptide for biocontrol. This mutant strain exhibited RCD phenotype after transforming with the full length pda gene. It is still unclear if this Pda protein has polysaccharide deacetylase activity and the roles these domains play in RCD remain to be investigated. The goals and strategies of this proposed three-year research are as follows: 1) performing bioinformatic analysis of the Xcc Pda and activity assays of various domains of Pda for understanding the apoptotic mechanism of bacteria and the evolutionary relationships between prokaryotes and eukaryotes in apoptosis, 2) analyzing the regulatory mechanism of the Xcc pda expression, including the gene organization, identification of the promoter region and the effects of various physical and chemical factors on the promoter activity by a reporter gene, 3) performing transcriptome analysis by microarray using the pda-mutated and pda-overexpressing Xcc grown in rich or poor medium, and 4) establishing a platform using a filamentous phage of Xcc as a phagemid to express the cloned pda, which is directed by a plant inducible promoter, for the purpose of biocontrol of black rot disease. The results of this project will be helpful in understanding apoptotic mechanism in bacteria and the evolutionary relationships between eukaryotic and prokaryotic cells in apoptosis. In addition, application of the killing effect of Pda in biocontrol will be of practical values in agriculture.
其他識別: NSC99-2321-B005-010-MY3
Appears in Collections:分子生物學研究所

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