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標題: 滾環型複製的嗜鹽甲烷古生菌質體pML之rep基因及其表現蛋白質的分析
Analysis of the rep gene and protein in a RCR plasmid pML from the halophilic methanogenic archaeon-Methanohalophilus mahii SLP
作者: 林珀丞
Lin, Po-Chen
關鍵字: Archaea;太古生物;rolling circle replication plasmid;滾環型複製質體
出版社: 生命科學系
中度嗜鹽甲烷古生菌Methanohalophilus mahii SLP的質體pML,經過定序確認全長為2158 bp,其中最長的ORF (orf1)所轉譯出的胺基酸序列 (328 aa),和高鹽古生菌Halobacterium sp.質體pGRB1的滾環型複製 (RCR)蛋白質 (Rep)有34 %的相似性。ORF1序列上也有Rep蛋白質所共有的三個序列保留區 (motif),根據第三個保留區上的tyrosine數目,pML應歸屬在滾環型複製superfamily I。利用南方墨漬法探測到單股的pML,證實pML是以滾環型的方式進行複製。由M. mahii 的全細胞RNA所做的反轉錄聚合酶連鎖反應 (RT-PCR),亦增幅出和orf1一樣大小的DNA片段 (984 bp),將orf1接到表現載體後送到大腸桿菌中也能轉譯出和預測大小相同的胜肽鏈 (38 kDa)。針對表現蛋白質所做的N端胺基酸定序,確認此表現蛋白質的N端胺基酸序列和預測的ORF1 N端胺基酸序列吻合。藉由anti-His (C-term)-HRP抗體對表現蛋白質做西方墨漬法分析的結果顯示, orf1表現蛋白質的C端具有轉譯後額外添加的組胺酸 (Histidine)可被抗體結合。具有Rep蛋白質辨識切位的pMM3和此大量表現的蛋白質作用後,使pMM3被S1核酸水解酶水解,顯示此蛋白質具有Rep蛋白質能辨識核酸序列並造成切口的活性。將已知的古生菌Rep蛋白質胺基酸序列依其相似程度做系統演化分析,嗜鹽甲烷古生菌質體pML上的Rep蛋白質胺基酸序列和在高鹽環境的極端高鹽古生菌質體pGRB1的相似性遠高於其同源的甲烷菌。

Plasmid pML (2158 bp) from the moderately halophilic methanogenic archaeon- Methanohalophilus mahii SLP was sequenced. The deduced amino acid sequence of the largest ORF (328 aa) shows 34 % similarity to the putative replication protein (Rep) of pGRB1 from Halobacterium sp. and contains three sequence motifs that conserved in the Rep proteins of rolling circle replication (RCR) mechanism. Based on the numbers of tyrosine residues in motif 3, pML belongs to the superfamily I of RCR. The single-stranded intermediate form of pML was detected by southern blotting, which confirms that pML replicate through RCR mechanism. RT-PCR with total M. mahii RNA and primers specific for orf1 amplified an RNA species of 984 bp. DNA encoding ORF1 was cloned into Escherichia coli and a polypeptide with expected molecular mass of 38 kDa was expressed. Both the N-terminal amino acid sequence and western blotting with anti-His (C-term)-HRP antibody confirm that this overexpressed protein is the translational product of orf1. This protein binds to pMM3 which contains a putative double-stranded replication region and made this fragment sensitive to S1 nuclease indicating the site-specific nuclease activity of this Rep protein. Phylogenetic analysis based on the amino acid sequences of all known archaeal Rep indicated that Rep of pML is clustered to the plasmids of the aerobic extremely halophilic Euryarchaeota, which they inhabited together at hypersaline environment.
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