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標題: Identification of rpoH gene and promoter analysis of the rpoD gene from Xanthomonas campestris pv. campestris.
十字花科黑腐病菌rpoH 基因之選殖及 rpoD 基因上游區域之探討
作者: 黃蘭惠
Huang, Lan-Hui
關鍵字: 十字花科黑腐菌;啟動子;sigma 因子;Xanthomonas campestris;sigma factor;promoter
出版社: 分子生物研究所
The aim of this work is to clone the rpoH gene and extend our
understanding of the role of sigma 32 in regulating the
expression of heat shock genes of X. campestris. In addition,
the unique feature in the upstream region of the rpoD gene was
also investigated. Two degenerated oligonucleotides, rpoH22 and
rpoHB, which correspond to the conserved region 2.2 and RpoH box
were designed and used in the polymerase chain reactions. DNA
fragment of 190 bp was produced and sequence analysis indicated
that the amplified DNA fragment has 88.3% identity with the same
region of E. coli sigma 32. By using 190 bp DNA fragment as
probe, two plaques that hybridized with the probe were selected
from the constructed Xc 11 genomic DNA library. In vivo excision
was performed to generate the pBK-CMV derivated phagmids from
the Zap express vectors and designated as pxc57 and pxc65,
respectively. Restriction map and Southern blot analysis
indicated that these two plasmids contain the full length rpoH
gene.The nucleotide sequences on both strands were determined
with the constructed deletion clones and subclones of the 2.8 kb
inserted DNA fragment. Result of DNA sequence analysis showed
that there are two open reading frames (ORF) in the 2833 bp DNA
fragment. The first ORF (ORF241) begins at the nt 231 and ends
at nt 956, from which a 241 amino acids protein could be
translated. The deduced amino acids of ORF241 showed 56%
identity with the E. coli uracil DNA glycosylase. The second ORF
(ORF291) starts at the nt 1157 and contains 876 bp from which a
291 amino acids polypeptide could be translated. Comparison the
deduced animo acids of ORF291 with E. coli sigma 32 showed 59.6%
identity. Results of the DNA sequence analysis have revealed
that (1) there are two putative promoters that can be separately
recognized by (70 and (E at the upstream of ORF 291; (2) the DNA
fragment located at 25 to 43 bp downstream of the ATG is similar
to the downstream box of E. coli and has the potential to
complement with 16S rRNA ; (3) the mRNA in the region between 59
nt upstream and 254 nt downstream of the AUG codon can form a
secondary structure. The rpoH gene was subsequently cloned into
a pET-21b vector and the overexpressed protein was purified
through His-bind resin. The purified sigma 32 protein was used
as an antigen to produce antiserum for Western blot analysis.
Results of 4028 bp DNA sequence analysis revealed that there are
four ORFs in the upstream region of Xc 11 rpoD gene. These four
ORFs were arranged in the order of ORF 377, ORF 211, ORF 306,
and ORF 146. The deduced amino acid sequences of these four ORFs
showed 46.9% identity with ribA, 41% identity with pat, 25%
identity htrB , and 55.9% identity with ORF 0145 of E. coli,
respectively. A transcription start siteof the rpoD gene was
identified by primer extension analysis and a putative -10 and
-35 sequences recognized by E. coli sigma 70 was also observed.
These results demonstrated that rpoD of Xc 11 is monocistronic
and apper not to be a member of MMS operon as that of the E.

本准究的目的主要是對於 X. campestris 的次要 sigma 32 因子進行其
基因 rpoH 的選殖、DNA 定序及菌體內蛋白表現的分析。並對於 rpoD 基
因的上游區域進行 DNA 定序分析以觀察是否具有與 E. coli 類似的大分
子操作組。由於次要 sigma 因子和主要 sigma 因子的相似度並不高,故
首先以位於次要 sigma 因子最具保留性的區域 2.2 及 sigma 32 特有的
保留性區域 RpoH box 分別設計二條退化性寡核酸引子,以 Xc11 染色
體 DNA 為模版,進行聚合連鎖反應增幅出 190 bp 的 PCR 產物,經
DNA 定序後,此 190 bp DNA 片段所推衍出的胺基酸序列與 E. coli 的
sigma 32 有 88.3% 的同質性。以此片段為探針,對所構築的 Xc11 的基
訊息的質體pxc57 及 pxc65,再以鑑識圖譜及膠體內雜交法分析,確定
此二個質體帶有完整的 rpoH 基因。接著利用所構築的部份刪除殖株及次
選殖株的方式進行 DNA 定序工作,總計共完成 2833 bp,經分析後其中
包含有二個 open reading frame (ORF),ORF 241 起始於第 231 bp,可
轉譯出 241 個胺基酸,經分析比對結果與 E. coli 的 Uracil DNA
glycosylase (UNG) 有 56% 的同質性;另一個 ORF 291 則起始於第
1157 bp,具有 876 bp ,可轉譯出 291 個胺基酸,胺基酸比對結果與
E. coli 的 rpoH 基因有 59.6% 的同質性,此外,DNA 序列分析結果顯
示 ORF 291 具有三個與 E. coli 的 rpoH 基因相同的特性:(1) 同時具
有可被 (70 及 ( E 辨識的啟動子序列,(2) 在 ATG 下游第 25 至 43
的核酸序列與 16S rRNA 有很高的互補性,類似 downstream box 序列
,(3) 於 ATG 上游的第 59 鹼基 至 ATG 下游第 254 鹼基 的 313 鹼基
mRNA 可形成穩定的二級結構,因此確定 ORF 291為 Xc11 製造 sigma 32
的 rpoH 基因。為得到大量的 sigma 32 蛋白,將 rpoH 基因黏接到高表
現載體 pET-21b 中,以 IPTG 誘發其大量表現後,回收蛋白以製備抗
sigma 32 的抗體,並以西方墨點法偵測 rpoH 基因在菌體內的表現。此
外,為了解 rpoD 基因上游區域是否存在有與 E. coli 類似的大分子操
作組 (Macromolecular synthesis operon, MMS operon),故同時亦對其
上游區域進行 DNA 序列分析,在 rpoD 基因上游的 4028 bp 中共找到四
個 ORF,依序為 ORF 377,ORF 211,ORF 306 及 ORF 146。其中 ORF
377 及 ORF 306 的轉錄方向與 rpoD 相反,ORF 211 與 ORF 146 則與
rpoD 的轉錄方向相同;胺基酸序列比對的結果顯示 ORF 377 和 E. coli
的 ribA 基因有 46.9% 的同質性;ORF 211 與 E. coli 的 pat 基因則
有 41.5% 的同質性;ORF 306 與 E. coli 的 htrB 基因的同質性較低,
只有 25%;而 ORF 146 和 E. coli 的ORF 0145 同質性為 55.9%。根據
DNA 序列分析結果,並未找到與大分子操作組相關的基因序列,再以引子
延伸法分析,在 ATG 上游的第 36 鹼基找到轉錄起始點,並在 rpoD 上
游第 44 至 49 及第 74 至 79 個核酸的位置可找到能被 (70 所辨認
的啟動子序列,因此證明 Xc11 的 rpoD 基因是以單一基因形式存在,並
可能受自己的基因產物所調控,本研究並將 rpoD 基因黏接到高表現載體
pET-21b 以方便得到大量的 sigma 70 蛋白做進一步的研究。
Appears in Collections:分子生物學研究所

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