臺灣博碩士論文加值系統

十字花科黑腐病菌為革蘭氏陰性菌, 其染色體 DNA 之 G+C 比率約 65
%。 在農業方面， 為引起十字花科黑腐病之病原菌； 另一方面，其產生
之胞外黏多醣黃原膠， 因具有特殊黏性， 在工業上有廣泛之利用價值。
本實驗之主要目的為建立 Xc17 之物理圖譜及把基因定位於物理圖譜上
。 以包埋法製備之完整染色體 DNA 分別以 PacI, SpeI 及 SwaI切割，
並用不同條件進行脈衝電泳 (pulsed-field gel electrophoresis 簡稱
PFGE) 分離, 以求得到最佳之解析度。 各片段之大小經與 size marker
(lambda II, lambda ladder and yeast chromosomes) 比較後， 可計算
每一亮帶之大小。 但於 SpeI 切割之情形, 只經一次 PFGE 分離會出現
重疊亮帶。 後來利用第二次脈衝電泳 (two-dimensional PFGE) 把這問
題解決。 同時經過計算後, 估計 Xc17 之基因組大小為 4725 kb。 pUT-
Tn5(pfm)CmKm 一為 Tn5 之衍生質體, 其 Tn5 上帶有多個 rare-cut
restriction enzyme 切位, 包括 PacI, SpeI 和 SwaI 等。對 Xc17 進
行誘導突變後, 篩選到與 Gum-、 色素、 營養需求、 蛋白質分解和胞外
分泌有關之突變株。 因 Tn5 插入染色體後, 突變株染色體中會比 wild
type Xc 多了一些 rare-cut restriction enzyme 切位， 經過 PFGE
和 DNA hybridization 後， 就可知 Tn5插入之位置, 亦即該基因之位置
。綜合 partial digestion, two dimensional PFGE, 及分析 Tn5突變株
之結果, 定出 SwaI 的片段以 WA-WC-WD-WF-WB-WE 之排列,形成環狀物理
圖譜。 同時亦把有關 uracil 合成基因 ,黃原膠合成基因串, 及 dif
site (deletion induced filamentation) 之位置定位於 SwaI 物理圖譜
上。

Xanthomonas campestris pv. campestris is a Gram (-) bacterium
which has a G+C content of about 65%. It is important in
causing black rot in crucifers. In addition, it produces
xanthan gum which has high viscosity and is widely used in
industry. The purpose of this study was to construct a physical
map of strain Xc17 and localize some genes in the physical map.
Intact chromosomal DNA was prepared by embedding method,
subjected to enzyme digestions with PacI, SpeI and SwaI,
respectively, followed by pulsed-field gel electrophoresis
running at different parameters to get optimum resolution. The
sizes of the fragments were calculated by using lambda II,
lambda ladder and yeast chromosomes as markers. During this
study, several multi-bands with similar fragment sizes were
found in the SpeI-digested samples. These problems are solved
by two dimensional PFGE. By calculating the sum of the length
of the fragments released by enzyme digestions, the Xc17 genome
size was estimated to be 4725 kb. A Tn5 derivative, pUT-Tn5(pfm)
CmKm, which carries sites for rare-cut restriction enzymes,
including PacI, SpeI and SwaI, was used to mutagenize the Xc17
genes. Different type of mutants were thus obtained, which
included Gum-, pigmentation, auxotrophic, protease and
secretion mutants. The chromosomal DNAs of these mutant have
one more rare cut restriction sites than that of the wild type
at sites of Tn5 insertion. After PFGE and hybridization with
appropriate probes, the site of Tn5 insertion in a mutant was
determined. This in turn represents the location of that gene
in the physical map. A combination of partial digestion, two
dimensional PFGE, and Tn5 mutant analysis were the strategies
employed for SwaI physical mapping. The results thus showed a
fragment order WA-WC-WD-WF-WB-WE in the SwaI circular map. In
addition, locations for the genes involved in uracil synthesis,
a gene cluster required for the synthesis of xanthan gum and
the dif site were assigned in physical map of SwaI.