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標題: Identification and Analysis of the Genes that Ehance Bioluminescence of the lux Operon from Photobacterium leignathi
可調節 Photobacterium leiognathi lux operon 螢光表現之基因
作者: 林碧晶
Lin, Bi-Jing
關鍵字: 螢光;bioluminescence;分子生物學;生物學;lux operon;P. leiognathi;MOLECULAR-BIOLOGY;BIOLOGY
出版社: 分子生物研究所
The lux operon of p. leiognathi PL741 in E. coli shows low
bioluminescence.The fact suggests that the regulatory gene(s) or
factor(s) is required for the lux operon of P.leiognathi to
enhance or induce the gene expression of bioluminescence. P.
leiognathi genome library was constructed to clone the specific
regulatory genes, which enable to regulate or enhance
bioluminescene. pXY2 containing ~6.4-kb P. leiognathi genomic
DNA fragment were clonedby in trans complementation
bioluminescence in vivo. pBJ03 subcloned frompXY2 enhances
bioluminescence as well as pXY2. Sequence analysis reveals that
there are two complete open reading frames ORFs (ufoI, ufoII
genes) and two incomplete ORFs (pefD, sod genes) on pBJ03; there
are 489 bp, 984 bp and 513 bp for the ufoI, ufoII and sod genes,
respectively. The gene order of thesegenes is --pefD-ufoI-
ufoII->-R&R-sod-> (R&R: regulatory region), whereas theR&R is
the regulatory region of the sod operon. It clearly elucidates
that the ufoII gene and sod gene belong to two diffenent
operons. Functional analysisconfirms that the potential hairpin
loops WT-WO are the transcriptional terminator for the sod and
related genes. The maxicell protein assays were usedto identify
the ORFII and CuZn-SOD proteins in the cells. In trans comple-
mentation bioluminoassays elicit that the ORFII and CuZn-SOD are
able to enhance bioluminescence of the lux operon from P.
leiognathi in E. coli.However, the function of the ORFII and
CuZn-SOD proteins enhanced biolumi-nescence is not clealy

以 in trans complementation bioluminoassays in vivo 的方式構
築 P. leiognathi genome library,篩選到一個具有 ~6.4-kb P.
leiognathi genomic DNA 的質體pXY2;pXY2 可使螢光表現增強約二倍。
依鑑識圖譜構築次選殖體 (sub-clones) ,將之轉形至宿主細胞 pL741
in E. coli 進行互補性螢光表現,結果 pBJ03 和 pBJ04 保有增強lux
operon 螢光表現的功能。DNA 序列分析顯示 pBJ03 的 P. leiognathi
genomic DNA 包含二個完整的 open reading frames ORFs (ufoI, ufoII
genes),二個不完整的ORFs (pefD, sod genes) 和一段長 649 bp
(2005th to 2649th bp) 的調節區域 (regulatory region, R&R)。ufoI,
ufoII genes 之序列分析未能確認是為何基因,而 pefD, sod genes分別
與 Salmonella typhi-murium pefD gene, P. leiognathi
bacteriocuprein superoxide dismutase (sod) gene 具同源性。其基
因序列 (gene order) 是 --pefD-ufoI-ufoII->-R&R-sod->;其中
ufoII, sod genes 分別主導 36.6 kDa, 18.6 kDa 之蛋白質。位於
ufoII, sod genes間的調節區域以 promoter/terminator-proving
vector 及 bioluminoassays in vivo 之方法分析,確認 potential
hairpin loops WT-Wo 為 pef operon transcriptional terminator;而
sod operon 之調節區域 R&R 其 promoter 需要 regulatory protein 或
enhancer 的協助方能啟動,以引導基因表現。ufoI, ufoII, sod genes
以 maxicell 蛋白質分析法和螢光表現分析,確定 ufoII, sod genes 主
導 ~36 kDa 之 ORFII 和 ~16 kDa CuZn-SOD 的合成;而 ufoII, sod
genes 在 R&Rlac promoter 的引導下可增強 lux operon 的螢光表現;
推測 CuZn-SOD 分解 superoxide 產生的 O2,可增加細胞內含氧量,間
接促進螢光表現;至於 ORFII 對螢光表現之影響仍不知,有待了解。
Appears in Collections:分子生物學研究所

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