Xanthomonas 在基因表現與調控方面之分子生物學資料非常缺乏.為了增
加這方面之了解, 需要篩選較多 Xc 菌體之啟動子以進行分析,因此首先
構築了泛寄主啟動子選殖載體 pFY5 與 pFY7.此二質體均具有已刪除啟動
子區域之螢光脢基因 luxA-luxB 作為報導基因泛寄主質體 RK2 之複製起
始點 oriV 及複製所需之 trfA 基因, 在報導基因上游有多個選殖位和一
個 threonine 轉錄終止子. Xc 染色體經鑑識脢切割後將小於 1000 bp
之 DNA 片段選殖到 pFY5 及 pFY7, 構成啟動子庫, 這些啟動子庫分別被
送入 Xc 及 E. coli, 選取有螢光表現之菌落.從 E.coli 菌落中挑選到
六個有螢光表現之轉形株, 抽取其重組質體再送入 Xc 中, 發現皆可表現
螢光. 從 Xc 菌落中挑選到三十個有螢光表現之轉形株, 抽取其重組質體
送入 E.coli 中, 結果只有三個可表現螢光. 從 Xc 挑選到之三十個重組
質體之插入 DNA 片段大小從小於 300 bp 到 1500 bp 不等. 挑選七個
在 Xc 內螢光表現較強之重組質體, 定序其插入 DNA片段之核甘酸序列.
有四個片段於報導基因上游有 50 個胺基酸以上之 open reading
frame. 以 E.coli 啟動子序列為依據, 分析這七個片段,發現在 Xc 內具
啟動子功能之片段不一定有 -10, -35 區域. 實驗結果顯示 E.coli RNA
polymerase 能辨認的序列, Xc RNA polymerase 均能辨認 . 而 Xc RNA
polymerase 能辨認的序列, E.coli RNA polymerase 大部分不能辨認.
又已定序之 Xc 啟動子片段不一定有 E.coli 啟動子之 -10, -35 區域.
顯示二者之啟動子序列有差異. 至於 RNA polymerase 所辨認之 Xc 啟動
子序列為何? 除了有待選殖並定序更多啟動子, 定出轉錄起始點, 縮小分
析泛圍, 再作進一步比對外, 亦須配合 RNA polymerase 與啟動子辨認作
用機制之探討, 才能有更深入之了解.
To initiate transdription, the RNA polymerase recognizes and
binds to specific promoter sites on DNA. Promoter for E.coli
RNA polymerase have been shown to contain two regions of
consensus sequence, about 10 and 35 base pairs upstream of the
transcriptional start site. Little is known about the promoter
sequence in Xanthomonas campestris pv. campestris (Xc), a Gram-
negtive bacterium that causes black rot in crucifers.
Therefore, when a new Xc gene is cloned and sequenced its
promoter region is hard to be determined merely by comparing
structural features of nucleotide sequence. To study Xc
promoters, the first step of this study was to construct a
promoter-proving vector for selection of promoter sequences.
pFY5 and pFY7 containing promoterless luxA-luxB genes as
repoter and replication origin (oriV) from the broad-host-
range plasmid RK2 were constructed. These two vectors contained
a threonine terminator snd a set of cloning sites at upstream
of the reporter. Fragments ( about 500 bp ) of Xc chromosomal
DNA were cloned, for construction of "promoter banks", into
these two promoter-proving vectors. The banks were then
transformed into Xc17 and E.coli, scoring for bioluminescence.
All recombinant plasmids ( six clones ) isolated from the
luminescent E.coli showed promoter functions in Xc. Thirty
different clones with inserts ranging from smaller than 300 bp
to 1000 bp in size were obtained from luminecent Xc
transformants , however, only three of them were found to
express luciferase activity in E.coli. Seven of the clones
including those directed strong luminescence in Xc only or in
both E.coli and Xc were sequenced, and four were found to carry
an open reading frame containing more than 50 amino acids at
upstream of the reporter. None of these seven inserts possessed
sequences consensus to E.coli promoter. These results suggest
that significant differences exist between the promoter of Xc
and E.coli.