Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21279
標題: 嗜鹽甲烷古生菌質體pML序列分析與滾環形複製機制之探討
Sequence analysis and rolling circle replication mechanism of plasmid pML in Methanohalophilus mahii
作者: 余秉弘
Yu, Ping-Hung
關鍵字: 甲烷古生菌
methanogenic archaea
質體
開放閱讀框架
滾環型複製
反轉序列
雙股複製起始
單股複製起始
單股中間產物 去氧核糖核酸
plasmid
open reading frame (ORF)
rolling circle replication (RCR)
inverted repeat
double strand origin (DSO)
single strand origin (SSO)
single strand DNA intermediates (ss DNA)
出版社: 植物學系
摘要: 自嗜鹽甲烷古生菌Methanohalophilus mahii的質體pML (2163 bp)的迷你質體經序列分析顯示具有一與高鹽古生菌Halobacterium spp.的滾環型(rolling circle replication, RCR)質體pGRB1複製蛋白 (Rep) 有34 %序列相同的開放閱讀框架open reading frame (ORF1)。進一步比對後發現ORF1具有滾環型複製蛋白所特有的三個保留區域,分別為motif1, motif2, motif3。依據motif3的tyrosine殘基數目,可將pML歸類在RCR superfamily I。且orf1上游的反轉序列 (inverted repeat),具有RCR質體所特有的複製起始 (double strand origin, DSO) 切割位置TGATA,此反轉序列亦具有stem-loop 二級結構。亦預測出可能為single strand origin (SSO) 的位置。由序列分析結果顯示pML具有RCR質體的特徵,因此推測pML為一滾環型複製質體,亦是第一個發現具有滾環型複製特色的甲烷古生菌。同時利用膠體電泳及南方墨漬法分析M.mahii的全部核酸時亦發現質體單股DNA中間產物存在。此外,含pML質體的大腸桿菌及具有orf1基因在表現載體pET21b的大腸桿菌之全蛋白分析均具有一22 kD的蛋白條帶,此蛋白可能是滾環型複製蛋白。 目錄 頁 中文摘要………………………………………………………………….I 英文摘要…………………………………………………………………II 壹、前言…………………………………………………………………..1 貳、前人研究……………………………………………………………..2 一、太古生物 (古生菌) …………………………………………………2 二、嗜鹽甲烷古生菌…………………………………………………….7 三、古生菌質體………………………………………………………….9 四、質體複製機制………………………………………………………11 五、質體的滾環形複製機制與特性……………………………………14 六、古生菌質體的複製機制……………………………………………16 七、嗜鹽甲烷菌質體……………………………………………………17 參、材料與方法 一、菌種與質體…………………………………………………………20 二、培養基組成…………………………………………………………20 三、藥品…………………………………………………………………22 四、接菌及菌體生長……………………………………………………23 五、質體之抽取與純化…………………………………………………23 六、DNA回收與純化…………………………………………………26 七、核酸純度之鑑定與定量分析……………………………………..26 八、DNA黏合反應……………………………………………………27 九、質體的轉形作用…………………………………………………..27 十、核酸膠體電泳分析與紀錄………………………………………..28 十一、全細胞蛋白質製備……………………………………………..29 十二、蛋白質電泳……………………………………………………..29 十三、質體複製單股核酸中間物質的偵測…………………………..32 1.Toatal DNA之製備………………………………………………..32 2.DNA轉漬………………………………………………………….32 3.探針之製作……………………………………………….……….34 4.DNA雜合………………………………………………………….35 5.螢光偵測法……………………………………………….……….36 6.AP Based Chemilunimescene Substrate Incubation……………….38 十四、Rep 的酵素活性分析………………………………………..38 十五、Rep基因選殖與表現…………………………………………..38 1.引子的設計……………………………………………….………………38 2.聚合 連鎖反應…………………………………………………...39 3.PCR產物純化…………………………………………….………..40 4.表現質體pET21b-orf1 (rep)的構築……………………………….41 5.Rep蛋白的大量表現與製備細胞萃取液…………………………42 6.Ni2+樹脂回收純化Rep蛋白……………………………………….43 十六、核酸序列分析軟體之應用………………………………………43 1.Open reading frames ( orfs ) 之預測與相似蛋白產物之比對……44 2.滾環形複製蛋白胺基酸序列之全長比對………………………...44 3.Motifs alignment……………………………………………………44 4.Double strand origin ( DSO ) 預測與二級結構之建立…………..44 5.Singlestrand origin (SSO) 的預測…………………………………45 肆、結果與討論………………………………………………………..46 一、質體pML核酸序列分析………………………………………….46 二、質體pML的orf1可能轉錄RCR複製的蛋白…………………...47 三、質體pML具有滾環形複製的double strand origin ( DSO )……...49 四、質體pML具有滾環形複製的single strand origin ( SSO )……….50 五、帶抗藥基因之pML衍生質體的構築……………………………..50 六、在載體pGEM7Zf-的pML之基因表現……………………………51 七、orf1 (rep)基因之選殖大量表現與活性分析………………………52 伍、表與圖………………………………………………………………56 陸、參考文獻……………………………………………………………76 摘要 質體pML (2163 bp) 是自嗜鹽甲烷古生菌Methanohalophilus mahii中發現的迷你質體,經過序列分析顯示一open reading frame (ORF1) 的氨基酸序列,與高鹽古生菌Halobacterium spp.的滾環型(rolling circle replication, RCR)質體pGRB1複製蛋白 (Rep) 有34 %序列相同。進一步比對後發現ORF1具有滾環型複製蛋白所特有的三個保留區域,分別為motif1, motif2, motif3。依據motif3的tyrosine殘基數目,可將pML歸類在RCR superfamily I。在orf1的上游發現到一個反轉序列 (inverted repeat),此序列包含有RCR質體所特有的複製起始 (double strand origin, DSO) 切割位置TGATA,利用PC / GENE模擬出此反轉序列之二級結構 (stem-loop structure),亦預測出single strand origin (SSO)。經由序列分析所得之結果符合RCR質體所具備之特徵,因此,推測pML為一滾環形複製質體,此亦是甲烷古生菌首次發現具此特性的質體。同時以膠體電泳分析質體pML的total DNA發現到可能有單股DNA中間產物存在,更進一步利用南方墨漬法發現在超螺旋的下方有一可能為單股DNA中間產物的訊號。另外,更進一步將orf1基因接入表現載體pET21b進行大量表現後,利用Ni2+親和性管柱純化蛋白進行in vitro活性測試,以確認ORF1蛋白是否具有滾環形複製蛋白 (Rep) 的功能與活性。
A small cryptic plasmid (2.16-kb), designated pML, was isolated from the moderately halophilic methanogen Methanohalophilus mahii SLP. Sequence analysis indicated the possibility of the two open reading frames (ORF 1, ORF 2). The predicted protein of ORF1 showed 34 % sequence identity to a putative replication protein (Rep) of pGRB1 from Halobacterium spp. Both putative proteins contained three sequence motifs (motif1, motif 2 and motif 3) that conserved in Rep proteins of rolling circle replication (RCR) mechanism. Based on the numbers of Tyr residues in the motif 3, pML was belonged to superfamily I of RCR. Moreover, two inverted repeated sequences around the nick site (TGATA) within the double strand origin (DSO) of plasmids and phages that replicate via a RCR mechanism.And the nicking site of pML was situated at loop region of IRI stem-loop structure. we also find single strand origin (SSO) near orf1. ss DNA intermediates were confirmed by Southern analysis. Based on above analyses, plasmid pML from the halophilic methanogen was proposed to replicate via a RCR mechanism and is the first RC plasmid discussed in methanogenic archaea. Predicted ORF1 encoded a putative 20.8 kDa protein consists of 176 amino acids. Further step, the orf1 was amplified with PCR technique with the additional restriction enzyme cut sites of Nde I and Xho I. The orf1 was further subcloned into the expression vector pET21b. After IPTG induction of the T7 lac promoter of pET21b, a 22 kDa polypeptide was heavily accumulated only at strain with orf1 gene. Next, study the activities of its encoded polypeptide in vitro will help us to understand RCR more.
URI: http://hdl.handle.net/11455/21279
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