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研究生:黃俊源
研究生(外文):Jun-Yuan Huang
論文名稱:洛德乳酸桿菌質體pK50-2之複製起始區分子特性分析
論文名稱(外文):Molecular Characterization of the Replication origin of Plasmid pK50-2 from Lactobacillus reuteri
指導教授:張登欽張登欽引用關係
指導教授(外文):Tung-Ching Chung
學位類別:碩士
校院名稱:國立中興大學
系所名稱:微生物暨公共衛生學研究所
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:70
中文關鍵詞:洛德乳酸桿菌質體
外文關鍵詞:Lactobacillus reuteriplasmidRCR
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本論文為研究洛德乳酸桿菌K50菌株的內源性質體pK50-2,並評估此質體的複製起始區是否適合構築載體。首先利用限制酵素EcoRI切割pK50-2,並以全長核酸片段轉殖於複製起始區篩選載體pUE80(-),形成重組質體pUE802-E,並將pK50-2全長核酸片段進行定序及分析。pK50-2是由1866個鹼基對組成,G+C百分比為35%,具有一個開讀框,將其轉譯的蛋白進行比對,發現分別與Rep B家族的洛德乳酸桿菌PA-16質體複製蛋白及乳酸球菌pAR141質體複製蛋白具有64%與56%的相似度。此外,屬於dso的高度保留nic區域及具正向重複序列的bind區域也被辨認 (nt1021-1111),而距離複製蛋白基因上游280個鹼基的區域 (nt1597-1732),發現與ssoT具有相似的DNA二級結構。因此認為pK50-2屬於滾輪式複製質體中的pMV158家族。為了測試pK50-2的複製區穩定度,接著構築一重組質體pUE802-HS,並以此質體轉形洛德乳酸桿菌 DSM 20016;於培養36代,72代,100代時,可觀察到重組質體的穩定度百分比分別為98%,77%與75%,證實pK50-2的複製起始區具高度穩定性。利用瓊脂膠體分析pUE802-HS於洛德乳酸桿菌 DSM 20016的質體複製數量為28。综合以上,證實pK50-2之複製起始區具有供洛德乳酸桿菌構築良好選殖載體之潛力。
The aim of this study was to investigate the plasmid pK50-2, an indigenous plasmid of Lactobacillus reuteri K50 strain, and to evaluate the possible use of pK50-2 origin for vector construction. PK50-2 was digested with EcoRI and cloned into the vector pUE80(-), a replicon-probe vector for Lactobacillus. The recombinant plasmid pUE802-E was sequenced and characterized. The pK50-2 consisted of a 1866 bp circular molecule with G+C content of 35%. An open reading frame ( ORF ) was predicted from the full length sequence of pK50-2. Alignment of the ORF protein reveals that this protein shared 64% and 56% identity, respectively, with Rep proteins of L. reuteri PA-16 plasmid and pAR141 of the Rep B superfamily. Furthermore, the highly conserved nic sequence and direct-repeated bind region within the dso was discerned ( nt1021-1111 ). About 280 bp upstream of the rep gene, a putative ssoT region was predicted ( nt1597-1732 ), suggesting the belonging of pK50-2 to the pMV158-RCR family. In order to test the stability of the pK50-2 ori, the recombinant plasmid pUE802-HS was transformed into L. reuteri DSM 20016, in which a high stability index of pUE802-HS, ca. 98%, 77% and 75% after 36, 72 and 100 generations, was observed. The copy number of pUE802-HS was estimated to be 28 copies per cell by agarose gel assay. Based on the data obtained, the origin of pK50-2 seems to be a good element for constructing efficient cloning vectors for use in L. reuteri.
目次
中文摘要………………………………………………………………… i
英文摘要………………………………………………………………… ii
第一章 緒言…………………………………………………………… 1
第二章 文獻探討……………………………………………………… 2
第一節 乳酸菌之介紹…………………………………………….. 2
1-1乳酸菌的定義....…………………………………………. 2
1-2益生菌 ( probiotics ) 的介紹……………………………. 2
1-3益生菌作用機轉與臨床應用……………………………... 3
第二節 洛德乳酸桿菌 ( Lactobacillus reuteri ) 的特性………….. 4
2-1洛德乳酸桿菌之介紹……………………………………… 4
2-2洛德因 ( reuterin ) 的研究……………………................... 4
第三節 洛德乳酸桿菌之質體在分子生物學的研究……………… 5
第四節 洛德乳酸桿菌染色體的全長定序………………………… 8
第五節 質體複製機制……………………………………………… 8
5-1 Theta質體的介紹………………………………………….. 9
5-2 Theta質體的分類…………………………………………... 9
5-3 Theta質體在分子生物學的研究…………………………… 10
5-4 RCR ( rolling-circle replication ) 質體的介紹…………….. 11
5-5 RCR質體的元件介紹與分類………………………………. 12
5-6 RCR質體在分子生物學的研究……………………………. 13
第三章 材料與方法………………………………………………………. 15
第一節 菌株與質體…………………………………………………. 15
1-1 菌株的培養…………………………………………………. 15
1-2 菌株的保存…………………………………………………. 15
第二節 洛德乳酸桿菌K50菌株內源性質體的製備與純化………… 16
2-1 大量質體之製備……………………………………………… 16
2-2 小量質體之製備……………………………………………… 17
2-3 .L. reuteri K50質體pK50-2 DNA的純化…………………… 18
2-4 質體之限制酶切割及瓊脂膠體電泳分析…………………… 19
第三節 內源性質體pK50-2核酸全長的定序………………………. 19
3-1 限制酶切割、去磷與接合作用…………………………….. 19
3-2 轉形用勝任細胞 ( competent cell ) 之製備………………… 21
3-3 以重組質體pUE802-E轉形E. coli TG1……………………… 21
3-4 重組質體pUE802-E的抽取與確認…………………………… 21
3-5 pK50-2核酸全長的自動定序………………………………… 22
第四節 利用pK50-2複製區構築E. coli-L. reuteri穿梭質體
pUE802-HS……………………………………………………. 22
4-1 L. reuteri電孔轉形勝任細胞 ( competent cell ) 之製備…….. 23
4-2電孔轉形……………………………………………………….. 23
第五節 洛德乳酸桿菌在MRS broth中的生長曲線試驗…………….. 24
5-1以傾注平板法 ( pour-plating ) 計數菌落,建立生長曲線….. 24
5-2 L. reuteri DSM 20016 generation time的計算………………… 24
第六節 pUE802-HS質體穩定度分析………………………………….. 25
第七節 pUE802-HS質體拷貝數 ( plasmid copy number )之測定…… 25
7-1 L. reuteri DSM 20016-802HS總DNA的製備……………….. 25
7-2以瓊脂膠體分析測定質體拷貝數……………………………. 26
第四章 結果…………………………………………………………………. 28
第一節 L. reuteri K50內源性質體pK50-2的萃取…………………… 28
第二節 L. reuteri K50內源性質體pK50-2全長序列分析…………… 28
2-1 Rep蛋白胺基酸序列分析……………………………………… 28
2-2 dso核酸序列分析…………………………………………….. 29
2-3 sso核酸序列分析……………………………………………. 30
第三節 以E. coli-L. reuteri穿梭質體pUE802-HS 轉形
L. reuteri DSM 20016………………………………………….. 30
第四節 L. reuteri在MRS broth的生長曲線…………………………. 30
第五節 pUE802-HS質體穩定度………………………………………. 31
第六節 pUE802-HS質體拷貝數測定…………………………………. 31
第五章 討論…………………………………………………………………. 52
參考文獻……………………………………………………………………… 54
附錄…………………………………………………………………………… 61
表目次
表1 本論文所使用之菌株與質體…………………………………………… 32
表2 pK50-2全長定序所使用的引子……………………………………........ 34
表3 .RCR質體pMV158 family的Rep蛋白序列比對……………………... 35
圖目次
圖1 洛德乳酸桿菌之PFGE電泳圖與分離自洛德乳酸桿菌K50
之內源性質體 pK50-2電泳圖............................................................... 37
圖2 ..pUE80(-)限制酶圖譜與經限制酶切割電泳圖....................................... 38
圖3 pK50-2限制酶圖譜與經限制酶切割電泳圖........................................ 39
圖4 重組質體pUE802-E之構築圖................................................................. 40
圖5 分離自E. coli TG1-802E之pUE802-E,及其經EcoRI切割之電泳圖. 41
圖6 ..pK50-2全長核酸序列與胺基酸序列分析…………………………....... 42
圖7 .pK50-2複製區之預測ssoT-like區域與Bacillus質體pBAA1之
ssoT核酸序列及二級結構比對................................................................ 45
圖8 重組質體pUE802-HS之構築圖................................................................ 46
圖9 由E. coli TG1-802HS分離pUE802-HS,經EcoRI切割
以證實構築成功......................................................................................... 47
圖10 分離自轉形菌株L. reuteri 20016-802HS之重組穿梭質體pUE802-HS
,經EcoRI切割電泳圖........................................................................... 48
圖11 .L. reuteri DSM 20016與L. reuteri DSM 20016-802HS在MRS broth
的生長曲線……………………………………………........................... 49
圖12 pUE802-HS質體穩定度分析.................................................................... 50
圖13 pUE802-HS質體拷貝數分析.................................................................... 51
參考文獻

劉雅芬, 2001. 以脈衝電泳法估算洛德乳酸桿菌的染色體大小並分析台灣雞隻腸道內此菌的分佈. 國立中興大學碩士論文。台中市。

Alpert, C. A., et al., 2003. Characterization of a theta-type plasmid from Lactobacillus sakei: a potential basis for low-copy-number vectors in lactobacilli. Appl Environ Microbiol. 69, 5574-84.

Alvarez-Olmos, M. I., Oberhelman, R. A., 2001. Probiotic agents and infectious diseases: a modern perspective on a traditional therapy. Clin Infect Dis. 32, 1567-76.

Anderson, D. G., McKay, L. L., 1983. Simple and rapid method for isolating large plasmid DNA from lactic streptococci. Appl Environ Microbiol. 46, 549-52.

Arvola, T., et al., 1999. Prophylactic Lactobacillus GG reduces antibiotic-associated diarrhea in children with respiratory infections: a randomized study. Pediatrics. 104, e64.

Axelsson, L. T., et al., 1988. Identification and cloning of a plasmid-encoded erythromycin resistance determinant from Lactobacillus reuteri. Plasmid. 20, 171-4.

Benachour, A., et al., 1995. Characterization and replication mode determination of the minimal replicon of Tetragenococcus halophila ATCC33315 plasmid pUCL287. Biochimie. 77, 868-74.

Bruand, C., et al., 1993. A fourth class of theta-replicating plasmids: the pAM beta 1 family from gram-positive bacteria. Proc Natl Acad Sci U S A. 90, 11668-72.

Copeland, A., et al., 2009. Complete sequence of chromosome of Lactobacillus reuteri DSM 20016. Unpublished.

del Solar, G., et al., 1998. Replication and control of circular bacterial plasmids. Microbiol Mol Biol Rev. 62, 434-64.

Duan, K., et al., 1998. Involvement of antisense RNA in replication control of the lactococcal plasmid pND324. FEMS Microbiol Lett. 164, 419-26.

Egervarn, M., et al., 2009. Identification and characterization of antibiotic resistance genes in Lactobacillus reuteri and Lactobacillus plantarum. J Appl Microbiol. 107, 1658-68.

Ennahar, S., et al., 2003. Phylogenetic diversity of lactic acid bacteria associated with paddy rice silage as determined by 16S ribosomal DNA analysis. Appl Environ Microbiol. 69, 444-51.

Fang, F., et al., 2008. Characterization of endogenous plasmids from Lactobacillus salivarius UCC118. Appl Environ Microbiol. 74, 3216-28.

Feld, L., et al., 2009. Characterization of a small erythromycin resistance plasmid pLFE1 from the food-isolate Lactobacillus plantarum M345. Plasmid. 61, 159-70.

Gibson, G. R., Roberfroid, M. B., 1995. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J Nutr. 125, 1401-12.

Gruss, A. D., et al., 1987. Functional analysis of a palindromic sequence required for normal replication of several staphylococcal plasmids. Proc Natl Acad Sci U S A. 84, 2165-9.

Harmsen, H. J., et al., 2000. Analysis of intestinal flora development in breast-fed and formula-fed infants by using molecular identification and detection methods. J Pediatr Gastroenterol Nutr. 30, 61-7.

Hefford, M. A., et al., 1993. The complete nucleotide sequence of a small cryptic plasmid from a rumen bacterium of the genus Butyrivibrio. Plasmid. 29, 63-9.

Heng, N. C., et al., 1999. Influence of different functional elements of plasmid pGT232 on maintenance of recombinant plasmids in Lactobacillus reuteri populations in vitro and in vivo. Appl Environ Microbiol. 65, 5378-85.

Holzapfel, W. H., et al., 2001. Taxonomy and important features of probiotic microorganisms in food and nutrition. Am J Clin Nutr. 73, 365S-373S.

Ingledew, W. J., et al., 1995. Studies on a stabilisation of ubisemiquinone by Escherichia coli quinol oxidase, cytochrome bo. Eur J Biochem. 227, 903-8.

Isolauri, E., et al., 1994. Oral bacteriotherapy for viral gastroenteritis. Dig Dis Sci. 39, 2595-600.

Jin, L. Z., et al., 2000. A strain of Enterococcus faecium (18C23) inhibits adhesion of enterotoxigenic Escherichia coli K88 to porcine small intestine mucus. Appl Environ Microbiol. 66, 4200-4.

Kandler., O., et al., 1980. Lactobacillus reuteri sp. nov., a new species of heterofermentative lactobacilli. Zentbl Bakteriol Mikrobiol Hyg I Abt Orig C1 264-269

Khan, S. A., 1997. Rolling-circle replication of bacterial plasmids. Microbiol Mol Biol Rev. 61, 442-55.

Khan, S. A., 2005. Plasmid rolling-circle replication: highlights of two decades of research. Plasmid. 53, 126-36.

Kiewiet, R., et al., 1993. Theta replication of the lactococcal plasmid pWVO2. Mol Microbiol. 10, 319-27.

Klein, G., et al., 1998. Taxonomy and physiology of probiotic lactic acid bacteria. Int J Food Microbiol. 41, 103-25.

Le Chatelier, E., et al., 1994. The pAM beta 1 CopF repressor regulates plasmid copy number by controlling transcription of the repE gene. Mol Microbiol. 14, 463-71.

Lee, J. H., et al., 2007. Comparative sequence analysis of plasmids from Lactobacillus delbrueckii and construction of a shuttle cloning vector. Appl Environ Microbiol. 73, 4417-24.

Lee, J. H., O''Sullivan, D. J., 2006. Sequence analysis of two cryptic plasmids from Bifidobacterium longum DJO10A and construction of a shuttle cloning vector. Appl Environ Microbiol. 72, 527-35.

Li, R., et al., 2009. Characterization of a rolling-circle replication plasmid pLR1 from Lactobacillus plantarum LR1. Curr Microbiol. 58, 106-10.

Lin, C. F., 1999. Molecular characterization of antibiotic resistance genes and replication regions from indigenous plasmids of Lactobacillus reuteri and the construction of cloning vectors. Ph. D. thesis, Graduate Institute of National Chung-Hsing University, Taiwan, ROC.

Lin, C. F., Chung, T. C., 1999. Cloning of erythromycin-resistance determinants and replication origins from indigenous plasmids of Lactobacillus reuteri for potential use in construction of cloning vectors. Plasmid. 42, 31-41.

Lin, C. F., et al., 1996. Molecular characterization of a plasmid-borne (pTC82) chloramphenicol resistance determinant (cat-TC) from Lactobacillus reuteri G4. Plasmid. 36, 116-24.

Lin, C. F., et al., 2001. Characterization of the replication region of the Lactobacillus reuteri plasmid pTC82 potentially used in the construction of cloning vector. Biosci Biotechnol Biochem. 65, 1495-503.

Luchansky, J. B., et al., 1988. Application of electroporation for transfer of plasmid DNA to Lactobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Bacillus, Staphylococcus, Enterococcus and Propionibacterium. Mol Microbiol. 2, 637-46.

Luthi-Peng, Q., et al., 2002. Effect of glucose on glycerol bioconversion by Lactobacillus reuteri. Appl Microbiol Biotechnol. 59, 289-96.

Macfarlane, G. T., Cummings, J. H., 1999. Probiotics and prebiotics: can regulating the activities of intestinal bacteria benefit health? BMJ. 318, 999-1003.

Mack, D. R., et al., 1999. Probiotics inhibit enteropathogenic E. coli adherence in vitro by inducing intestinal mucin gene expression. Am J Physiol. 276, G941-50.

Mayer, M. P., 1995. A new set of useful cloning and expression vectors derived from pBlueScript. Gene. 163, 41-6.

Meijer, W. J., et al., 1995. Characterization of the replication region of the Bacillus subtilis plasmid pLS20: a novel type of replicon. Nucleic Acids Res. 23, 3214-23.

Morelli, L., et al., 1987. Lactobacillus protoplast transformation. Plasmid. 17, 73-5.

O''Sullivan D, J., Klaenhammer, T. R., 1993. Rapid Mini-Prep Isolation of High-Quality Plasmid DNA from Lactococcus and Lactobacillus spp. Appl Environ Microbiol. 59, 2730-2733.

Pothoulakis, C., et al., 1993. Saccharomyces boulardii inhibits Clostridium difficile toxin A binding and enterotoxicity in rat ileum. Gastroenterology. 104, 1108-15.

Pushnova, E. A., et al., 2000. An easy and accurate agarose gel assay for quantitation of bacterial plasmid copy numbers. Anal Biochem. 284, 70-6.

Raha, A. R., et al., 2006. DNA sequence analysis of a small cryptic plasmid from Lactococcus lactis subsp. lactis M14. Plasmid. 56, 53-61.

Reid, G., et al., 2003. Potential uses of probiotics in clinical practice. Clin Microbiol Rev. 16, 658-72.

Talarico, T. L., et al., 1990. Utilization of Glycerol as a Hydrogen Acceptor by Lactobacillus reuteri: Purification of 1,3-Propanediol:NAD Oxidoreductase. Appl Environ Microbiol. 56, 943-948.

Talarico, T. L., et al., 1988. Production and isolation of reuterin, a growth inhibitor produced by Lactobacillus reuteri. Antimicrob Agents Chemother. 32, 1854-8.

Talarico, T. L., Dobrogosz, W. J., 1990. Purification and Characterization of Glycerol Dehydratase from Lactobacillus reuteri. Appl Environ Microbiol. 56, 1195-1197.

Tannock, G. W., 1987. Conjugal transfer of plasmid pAM beta 1 in Lactobacillus reuteri and between lactobacilli and Enterococcus faecalis. Appl Environ Microbiol. 53, 2693-5.

Tannock, G. W., et al., 1994. Molecular characterization of a plasmid-borne (pGT633) erythromycin resistance determinant (ermGT) from Lactobacillus reuteri 100-63. Plasmid. 31, 60-71.

Teresa Alegre, M., et al., 2009. Characterization of pRS5: a theta-type plasmid found in a strain of Pediococcus pentosaceus isolated from wine that can be used to generate cloning vectors for lactic acid bacteria. Plasmid. 61, 130-4.

Vandenbergh, P. A., 1993. Lactic-Acid Bacteria, Their Metabolic Products and Interference with Microbial-Growth. Fems Microbiology Reviews. 12, 221-238

Vollenweider, S., Lacroix, C., 2004. 3-hydroxypropionaldehyde: applications and perspectives of biotechnological production. Appl Microbiol Biotechnol. 64, 16-27.

Wang, T. T., Lee, B. H., 1997. Plasmids in Lactobacillus. Crit Rev Biotechnol. 17, 227-72.

Wu, C. M., Chung, T. C., 2007. Mice protected by oral immunization with Lactobacillus reuteri secreting fusion protein of Escherichia coli enterotoxin subunit protein. FEMS Immunol Med Microbiol. 50, 354-65.

Wu, E., et al., 2007. Characterization of a cryptic plasmid from Bacillus sphaericus strain LP1-G. Plasmid. 57, 296-305.

Yang, E. J., Chang, H. C., 2009. Analysis of pYC2, a cryptic plasmid in Lactobacillus sakei BM5 isolated from kimchi. Biotechnol Lett. 31, 123-30.

Yin, S., et al., 2008. Characterization of a cryptic plasmid pM4 from Lactobacillus plantarum M4. FEMS Microbiol Lett. 285, 183-7.

Yin, S., et al., 2009. Functional analysis of the plasmid pM4 replicon from Lactobacillus plantarum M4: determination of the minimal replicon and functionality identification of the putative sso. Plasmid. 62, 166-71.

Zhai, Z., et al., 2009. Characterization of a novel rolling-circle replication plasmid pYSI8 from Lactobacillus sakei YSI8. Plasmid. 62, 30-4.

Zuker, M., 2003. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406-15.
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