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研究生:陳駿義
研究生(外文):Chen, Chun-Yi
論文名稱:Xanthomonascampestrispv.campestrisβ-Iactamase基因之選殖與定序
論文名稱(外文):Isolation and Characterization of β-Iactamase Genes from Xanthomonas campestris pv. campestris
指導教授:翁淑芬翁淑芬引用關係
指導教授(外文):Weng, Shu-Fen
學位類別:碩士
校院名稱:國立中興大學
系所名稱:分子生物學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:1998
畢業學年度:86
語文別:中文
論文頁數:85
中文關鍵詞:十字花科蔬菜黑腐病菌工業微生物
外文關鍵詞:Xanthomonas campestris pv. campestris
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  • 被引用被引用:8
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  十字花科蔬菜黑腐病菌(Xanthomonas campestris pv. campestris),簡稱Xc,是革蘭氏陰性的植物致病菌,亦是重要工業微生物,具有抗ampicillin之抗藥性。經由β-lactamase活性測試,得知此酵素存在於胞質週緣區,分子量大小約為30kDa。為了選殖β-lactamase基因,將Xc11染色體DNA經sau3AI不完全切割後構築成之Xc11基因庫,送入DH5α勝任細胞中,篩選得到一株對ampicillin有抗性之選殖株。經次選殖後,得到仍然能抗ampicillin的最小轉殖株帶有一段約1.7kb之DNA插入片段。經定序分析,發現該片段全長1,758bp,可轉譯出一個完整之ORF:orf295。orf295位於nt 224至1,111,可轉譯出295個胺基酸。其G+C含量為68.2%,pI值為9.1,分子量為31.8kDa。將胺基酸的序列送入GenBank比對後,發現與Stenotrophomonas maltophilia之L2-β-lactamase有達到50.5%的identity。與其它一些革蘭氏性菌之β-lactamase有很高的相似性。這些酵素有一些保守性很高的consensus reigions,包括N端的22個胺基酸的訊號勝□鍵、STXK、SDN、KGT序列,以及距離SDN環狀結構下游34個胺基酸residues的glutamic acid。因此推論Xcll的β-lactamase可分類為Class-A β-lactamase。nt 1至123的反向轉譯序列中,有一不完整之ORF存在,預測可轉譯出41個胺基酸,命名為orf41。此ORF41與Proteus vulgaris、Citrobacfter freundii、Enterobacter cloacae以及Pseudomonas aeruginosa調控β-lactamase之CumR及AmpR調控因子的N端胺基酸序列,分別有63%與51%的identity。這些調控基因都位於β-lactamase基因上游,且轉譯方向相反。利用gene replacement方法,在染色體上的β-lactamase基因中插入一Tcr,構築一Xc的Aps突變株。然後經由complementation test,將包含完整β-lactamase基因的1.7 kb DNA片段送回所構築的Aps突變株,結果回復對ampicillin之抗藥性。上述實驗除了證明Xc11的bla基因只有一個copy外,也說明了篩選到的DNA片段,在E.coli與Xc11中均可表現β-lactamase活性。實驗中已證明bla基因上游有啟動子,而位於bla基因下游亦發現有類似terminator的結構。因此Xc11的bla基因可能是monocistronic。利用Northern hybridization技術無法偵測到bla基因mRNA明顯且單一的雜交訊號。推測Xc11 β-lactamase為一穩定且效能很高的酵素,只需要微量即可將ampicillin的β-lactam ring破壞,使菌體達到抗藥性。因此bla基因的mRNA表現量可能並不高。以Xc11的bla基因作探針,分析14個Xanthomonas菌株,發現其中13株的染色體DNA上皆可偵測到雜配訊號,顯然其間有很高的同質性。又此13株Xanthomonas皆為Apr之菌株。具有β-lactam ring結構之β-lactam抗生素,有許多種類,許多衍生的β-lactam抗生素且不斷地在發展中。利用Kirby-Bauer antibiotic sensitivity testing測試Xc11之β-lactamase對於這些β-lactam抗生素的抗性。結果顯示Xc11對ampicillin、carbenicillin、cefixime、cefotaxime、cephaloridine、cephalothin、oxacillin、penicillin和piperacillin有抗性,對aztreonam和cefoxitin之抗性則顯現為intermediate,對ceftazidime和imipenem則為susceptible。
  The β-lactam antibiotics are among the most often used antimicrobial agents. The most common mechanism of bacterial resistance to β-lactam antibiotics is the synthesis of β-lactamases that cleave the amide bond in the β-lactam ring to generate inactive products. Xanthomonas campestris pv. campestris confers resistance to ampicillin, and the β-lactamase activity was detected with the molecular mass of 30 kDa by in situ assays in sodium dodecyl sulfate-polyacrylamide gels in the periplasmic space between the outer and cytoplasmic membranes. To iso late the bla gene, an Xc11 genomic library, constructed with pBK-CMV as the vector, was transformed into E.coli Dh5α, selecting for ampicillin resistance (Apr) transformants. One of the clones, designated pBK-1, carrying an insert fragment of 4.5 kb was isolated. We subcloned different fragments from this insert into E.coli and found that the bla gene was located on a 1.7-kb fragment. The DNA sequence of this fragment was then determined and analyzed. A complete ORF of 887 bp with a GC content of 68.2%, between nt 224 and 1,111 which could code for a protein of 295 amino acids with a predicted molecular mass of other β-lactamases showed it to be most closely related (50.5%) to the L2 β-lactamase from Stenotrophomonas maltophilia. The predicted amino acid sequence of the bla gene from Xc11 was aligned with those of other β-lactamases, displays very similar regions of consensus with the other class A enzymes, including the consensus sequences STXK, SDN and KTG and the highly conserved glutamic acid residue located 34 residues downstream fro the SDN loop. Upstream of the bla gene, there is a divergently transcribed open reading frame coding for the Nterminal part of a putative protein similar to CumR (63%) and AmpR (51%) required for the regulation of bla genes in Proteus vulgaris and Citrobacter freundii, respectively. The 1.7-kb fragment carring the complete bla gene, cloned in the broad-host-range plasmid pRK415, was able to complement an ampicillin-sensitive mutant constructed by gene replacement. The bla promoter activity was detected and a putative terminator located at downstream of the translational stop codon was found.
  These results suggest that transcription from the bla promoter may produce a monocitronic mRNA. However, no single band was detected after Northern-blot analysis. The distribution of bla sequence away 14 Xanthomonas strains was examined. It seemed that most of the Xanthomonas strains are resistance to ampicillin. In addition, the bla genes in these strains are highly homologous. The Kirby-Bauer antibiotic sensitivity testing for the Xc11 revealed resistance or intermediate susceptibility to the extended penicillins, cephalosporins and carbapenems tested.
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