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標題: 極端高鹽太古生物與甲烷太古生物的滾環型複製質體之特性分析
Characterization of rolling-circle plasmids from Haloterrigena sp. H13 and Methanocalculus taiwanensis strain P2F9704a
作者: 朱高鈴
Chu, Kao-Ling
關鍵字: pSN;滾環型複製質體;pYH;rolling circle replication
出版社: 生命科學院碩士在職專班
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極端高鹽太古生物Haloterrigena sp. H13的質體pSN,經定序確認全長為2272 bp,其中最長的open reading frame (orf330)所轉譯出的330個胺基酸序列,經序列比對後發現分別與Halobacterium sp.質體pGRB1、pHSB、pHGN1的滾環型複製蛋白(Rep)有68%、67%、60%的相似性;ORF330序列上也有Rep蛋白質所共有的三個序列保留區(motif),依據Rep的序列同源性來進行滾環型複製質體分類( DPR/DPR_ RCRdata. htm),pSN應歸屬於滾環型複製Group XII。而甲烷古生菌Methanocalculus taiwanensis strain P2F9704a的質體pYH,經定序確認全長為3820 bp,其中最長的open reading frame (orf562)所轉譯出的562個胺基酸序列,經序列比對後發現分別與Sulfolobus islandicus 質體pHEN7的滾環型複製蛋白(Rep)、pRN2的helicase-like 蛋白質有28%的相似性,且與Acidianus ambivalens質體pDL10的primase有27%的相似性,推測pYH的Rep複製起始蛋白可能也具有primase以及helicase的功能,而目前從pYH的Rep序列同源性進行滾環型複製質體分類只能暫時歸類於pRN家族。進一步利用南方墨漬法以及PCR方式探測到單股的pSN以及pYH,顯示pSN與pYH可能以滾環型的方式來進行複製。為了進一步測試Rep複製起始蛋白的切割/接合活性,將pSN的orf330接到表現載體後送到大腸桿菌中也能轉譯出預期的Rep胜肽鏈。藉由anti-His抗體對表現蛋白質做西方墨漬法分析的結果顯示, orf330表現蛋白質的N端具有轉譯後額外添加的組胺酸 (Histidine)可被抗體結合。將已知的古生菌Rep蛋白質胺基酸序列依其相似程度做親源分析,極端高鹽太古生物Haloterrigena sp. H13質體pSN上的RepSN蛋白質胺基酸序列和在高鹽環境的極端高鹽太古生物質體pGRB1、pHSB、pHGN1的相似性最高;甲烷太古生物Methanocalculus taiwanensis strain P2F9704a的質體pYH上的RepYH蛋白質胺基酸序列和在高溫酸性環境的極端高溫太古生物質體pDL10、pRN2、pHEN7有較高的相似性。從Rep胺基酸序列分析所得演化樹圖的結果,可將pSN分群在好氧極端高鹽Euryarchaeota的質體中,而pYH則屬於嗜熱嗜酸的Crenarchaeota質體。

Plasmid pSN (2272 bp) from Haloterrigena sp. H13 was sequenced. The deduced amino acid sequence of the largest ORF (330 a.a.) individually shows 68 %, 67% and 60 % similarity to the putative replication proteins (Rep) of pGRB1, pHSB and pHGN1 from Halobacterium sp. and contains three sequence motifs that conserved in the Rep proteins of rolling circle replication (RCR) mechanism. Based on the homology of double strain origin and Rep protein sequence ( htm), pSN belongs to the Group XII of RCR. Plasmid pYH (3820 bp) from Methanocalculus taiwanensis strain P2F9704a was sequenced. The deduced amino acid sequence of the largest ORF (562 a.a.) individually shows 28 % similarity to the putative replication protein of pHEN7, helicase-like protein of pRN2 from Sulfolobus islandicus and 27 % similarity to primase protein of pDL10 from Acidianus ambivalens. Based on the homology of Rep protein sequence, pYH belongs to the pRN family. The single stranded intermediates form of pSN and pYH were detected by southern blotting and PCR, which confirm that pSN and pYH replicate through RCR mechanism. DNA encoding ORF330 was cloned into Escherichia coli and a expected polypeptide was detected. Western blotting with anti-His antibody confirm that this overexpressed protein is the translational product of orf330. Phylogenetic analysis based on the amino acid sequences of archaeal Rep proteins indicated that RepSN of pSN is clustered to the plasmids of the aerobic extremely halophilic Euryarchaeota. And RepYH of pYH is clustered to the plasmids of the acidophilic and thermophilic Crenarchaeota.
其他識別: U0005-0801201015453400
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