| Summary: | 碩士 === 國立中興大學 === 農業生物科技學研究所 === 84 === Photobacterium leiognathi 的螢光調控機制仍不甚瞭解，其螢光表現
顯受調控。本研究嘗試選殖可調控 P. leiognathi lux operon 之基因；
以 in trans com-plementation 之方法建立 P. leiognathi gene
library， 選殖 lux operon 的 regula-tory gene 或可 enhance lux
operon 螢光表現之基因。經選殖得一質體 pHC4 明顯 enhances P.
leiognathi lux operon 之螢光表現；pHC4 具一 2.1 kb P. leiognathi
genomic DNA，經 DNA 序列定序與分析，此段 DNA 序列的基因排列如下
： pkI-ter--- R&R-luxZ其中一完整基因，定名為 luxZ gene 為可調
控 lux operon 之基因。luxZ gene encodes 一 221 amino acid
residues 之蛋白質，其分子量 Mr 25 kDa，經氨基酸分析比對其與
Pseudomonas strain LB400 之 orf0 基因類似，可能為一屬於 GntR
family 之 regulatory protein。序列分析發現在 luxZ gene 上游具有
調控區域 (Regulatory Region, R&R)，以 promoter-proving vector/
terminator-proving vector 及 bioluminoassay in vivo 之方法，可確
認此區域為 luxZ 及其相關基因的 regu-latory region R&R, 具有
upstream activating seguence WUAS, dual promoters, operator。 經
maxicell 方法分析，可知選殖的 luxZ gene 可主導 LuxZ protein 的合
成。調節蛋白 LuxZ 經功能分析後，得知其並非 P. leiognathi lux
operon 之專一性的調節基因 (regulatory gene)。luxZ gene enhances
P. leiognathi lux operon 之螢光表現，其功能可能與 cAMP-CRP
binding site 及 cAMP-CRP complex binding 有關；luxZ gene 應是
positive regulatory gene。Glucose repression 明顯的抑制 LuxZ
enhances P. leiognathi lux operon 的螢光表現的作用。
The specific gene designated as luxZ gene that enhances
bioluminescence of the lux operon from Photobacterium leiognathi
PL741 in E. coli was cloned and identified. Nucleotide sequence
of the regulatory region R&R with the luxZ gene has been
determined, and the amino acid sequence of the protein encoded
by the luxZ gene is deduced. Functional analysis elucidates that
the regulatory region R&R, which inludes upstream activator-
binding site WUAS, dual -10/-35 canonical promoters, putative
operator and Shine-Dalgarno (SD) sequence, indeed functions for
gene expression and regulation of the luxZ and related genes.
The LuxZ protein has a calculated Mr of 25,807 and comprises 221
amino acid residues. The luxZ gene, which apparently enhances
bioluminescence of the lux operon from P. leiognathi in E.
coli, was identified as regulatory gene for the lux operon by in
trans complementation bioluminoassays in vivo. The LuxZ protein
was identified as the putative transcriptional regulator by
significant ssimilarity with the hypothetical transcriptional
regulator encoded by orf0 genefrom Pseudomonas sp. LB400. The
LuxZ protein, which functions as positive regulator for the lux
operon from P. leiognathi to enhance bioluminescence, is likely
to be the DNA-binding protein related to the GntR family of
transcripti-onal regulators. In addition, partial 3'-end
nucleotide sequence of the pkI gene from P. leiognathi PL741 has
been cloned and determined, and the amino acid sequence of
pyruvate kinase I encoded by the pkI gene is deduced. Pyruvate
ki-nase I is the key enzyme, which converts phosphoenol pyruvate
to pyruvate, for glycolysis. Alignment and comparison of
pyruvate kinases I from P. leiogathi and E. coli shows that they
are homologous. Nucleotide sequence reveals that the pkI gene is
linked to the luxZ gene that enhances bioluminescence of the lux
operon from P. leiogathi. The gene order of the pkI and luxZ
genes is pkI ter->-R&R-luxZ-> (R&R: regula-tory region; ter,
terminator), whereas the R&R is the regulatory region for the
luxZ gene, and ter is the transcriptional terminator for the pkI
and related genes. It clearly elicits that pkI gene and luxZ
gene belong to two diffenent operons. Functional ana-lysis
confirms that the potential hair-pin loop WT is the
transcriptional terminator for the pkI and related genes. It
suggests that the pkI and related genes are simply linked to the
luxZ gene in genome.
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