Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/24031
標題: LuxR[T] 與 cAMP-CRP 調控 Photobacterium leiognathi lux operon 轉錄表現之探討
Regulatory roles of LuxR[T] and cAMP-CRP on the lux operon of Photobacterium leiognathi
作者: 羅怡婷
Luo, Yi-Ting
關鍵字: Photobacterium leiognathi;海生螢光菌;LuxR[T]
出版社: 生物化學研究所
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摘要: 
Photobacterium leiognathi ATCC 25521 lux operon 之調節區域含有四個對稱序列 ∑IV、∑III、∑II、∑I,一組 cAMP-CRP 結合位,以及兩組啟動子 P[DII]、P[DI]。其相關位置排列為:-∑IV-∑III-∑II-∑I-∑cAMP-CRP-P[DII]-P[DI]-SD-lux operon。利用 5’RACE 分析得知 lux operon 的轉錄起始點座落在 start codon 上游 -68 nt 的“C”核苷酸位置。利用胞內互補性螢光分析方式進行 glucose repression 試驗,證實此調控區含有 cAMP-CRP 之結合位,此位置座落在轉錄起始點上游 -71 nt 之位置,屬於 Class I cAMP-CRP dependent promoter。並於 E. coli中分析一系列 lux operon 調節區域中對稱序列刪除之重組質體,得知 ∑II 為一負向調控區,∑I 為一正向調控區。將 ∑I 與前之研究篩選獲得的可強化螢光表現之 LuxR[T] 進行一系列胞內反應,推測 LuxR[T] 會結合 ∑I,因而提升螢光之表現。凝膠遲滯分析結果亦顯示 (His)6-LuxR[T] 蛋白可與 ∑I 結合。因此,確認 LuxR[T] 可正調節 lux operon 螢光表現。此外,將包含 lux operon 調節區域質體的對稱序列 ∑II 進行部分鹼基突變,再以螢光分析該質體於 E. coli 之螢光表現,證實 ∑II 的確存在負調控機制,可能與未知蛋白結合,影響螢光表現。進一步以 EZ-Tn5TM TransposomeTM Kit 建構一含有任意基因被突變之 E. coil JM103y 族群,再送入含有 lux operon 調節區域之 plasmid,篩選螢光表現增強之轉殖株。目前,尚未篩選到能負調節 lux operon 表現之基因。最後,將可增強 lux operon 螢光之 luxR[T] 及 crp 基因,一起轉形至含有 lux operon 調節區域質體之 E. coil 中,分析螢光值。結果發現,同時存在 LuxR[T] 及 CRP 時,螢光表現提升,但提升程度與 CRP 單獨存在時之結果相似。因此,推測此二蛋白間存在排它效應 (exclusive effect)。本研究得知,lux operon 確實受到 cAMP-CRP 及 LuxR[T] 的正向調節作用,其作用機制仍待進一步研究分析。

The regulatory region R&R of the lux operon from Photobacterium leiognathi ATCC 25521 was cloned and identified. The R&R includes four symmetrical sequence, a CRP binding site, and two putative promoters, arranging in the order of ∑IV-∑III-∑II-∑I-∑cAMP-CRP-P[DII]-P[DI]-SD-lux operon. The functional roles of these sequence in R&R are not clean. In this study, the 5' RACE assay results confirmed that the transcriptional initiation site of lux operon is the C nucleotide -68 nt upstream of the start codon, suggesting that the P[DII]-promoter is the one used for gene expression under this experimental condition. Glucose repression assays indicated that cAMP-CRP is involved in the regulation of lux operon. Belonging to Class I cAMP-CRP dependent promoter, the cAMP-CRP binding site is localized -71 nt upstream of the transcriptional start site. Serial deletion assays elicit that ∑II is a critical negative element and ∑I is a critical positive element in the R&R region. Previous studies have shown that LuxR[T] could enhance bioluminescence of P. leiognathi ATCC 25521 lux operon cloned in E. coli. In trans complementation Lux-bioassays showed that ∑I is closely related to LuxR[T]. Additionally, EMSA results revealed that LuxR[T] protein is able to bind ∑I of the lux operon R&R. On the other hand, the negative regulation effect of ∑II was demonstrated by base mutation. Furthermore, the EZ-Tn5TM TransposomeTM Kit was used to construct a pool of E. coil JM103y random mutant strains, and then, the plasmid carring lux operon R&R was transformed into this mutant pool and screened for transformants with bioluminescence. However, no positive result was obtained. Our bioassay result showed that the lux operon was also enhanced by crp gene. However, no additive effect was observed when luxR[T] gene coupled with the crp gene were cloned for enhancement of lux operon expression. These results clearly demonstrate that P. leiognathi lux operon is regulated by cAMP-CRP and LuxR[T], the response regulation of a two component system.
URI: http://hdl.handle.net/11455/24031
其他識別: U0005-0908201017283000
Appears in Collections:生物化學研究所

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