Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21335
標題: Molecular cloning and characterization of the primary sigma factor gene from Xanthomonas campestris pv. campestris
十字花科蔬菜黑腐病菌主要 sigma 因子基因之選殖
作者: 曾雅詩
Ya-Shih
關鍵字: 十字花科蔬菜黑腐病菌;Xanthomonas campestris pv. campestris;RNA 聚合■;主要 sigma 因子;轉錄作用.;RNA polymerae;primary
出版社: 分子生物研究所
摘要: 
Sigma 因子為參予原核生物轉錄起始的一種蛋白,當它與 RNA 聚合■的
核心■ 結合後,才能促使 RNA 聚合■辨識正確的啟動子序列,以進行特
異性的轉錄作用。 在細菌體內,通常有多種 sigma 因子存在,主要
sigma 因子負責調控生物體一般基因的轉錄工作,而其它次要的 sigma因
子則是菌體為適應外在環境及形態改變時所必需。本實驗之目的在於選殖
十字花科蔬菜黑腐病菌的主要 sigma 因子基因,並進行 DNA 核■酸定序
與菌體內表達的工作。 在實驗設計上,先以細菌主要 sigma 因子中最具
保留性的區域 (次區域 2.4 及次區域 4.2) 的胺基酸序列,推衍出兩段
退化性寡核■酸引子igDE 及 sig20B; 並利用聚合■鏈鎖反應 (PCR),
增幅兩引子間的片段,並以之為探針,對經不同鑑識■切割後的 Xc 染色
體 DNA,進行南方墨點偵測。 首先回收 SalI切割後的 4.4 kb 左右之
DNA 片段,作為構築選殖基因的第一個部份基因庫,接著利用菌落雜交的
方法分離出含 sigma 因子基因的殖株 #169。然而由鑑識■圖譜和南方墨
點法分析的結果顯示,此殖株內的質體 pYS169只含有 sigma 因子的部份
基因。 於是又回收以經 EcoRI 切割後,約 11 kb 左右的 DNA 片段,作
為構築選殖基因的第二個部份基因庫。利用同樣的菌落雜交方法,分離出
一個含完整 sigma 因子基因的質體 pYS121。然後以 SmaI 切割 pYS121
,構築含完整 sigma 因子基因的次選殖株 pYS121-18 。 此外並構築質
體 pYS169 的刪除變異株,並設計引子對 pYS169 與 pYS121-18 兩股
DNA 進行核酸定序分析。 總計定出 2498 bp DNA 定序,而 sigma 因子
基因的 ORF 起始於第 472 bp,終止於第 2343 bp ,可轉譯出一個具
622 個胺基酸,分子量為 70.641 kDa 的蛋白。最後並進行西方墨點法,
偵測含 sigma 因子基因的 DNA 片段在菌體內的表達。 結果顯示此段
sigma 因子基因所轉譯出之蛋白質與經管柱色層分析法純化之 sigma 因
子分子量相似,而且與 E. coli sigma 70因子基因有 56.2% 的同質性,
因此推斷此一 sigma 因子應為 Xc 的主要 sigma 因子。
In procaryotic system, transcription initiation directed by RNA
polymerase required sigma factor to recognize specific promoter
sequence. Four regions with high conservation were observed
after alignment the amino acid sequences of the sigma factor
proteins. The most conserved regions locate in subregions 2.4
and 4.2, which contact the promoter -10 and -35 regions,
respectively. In order to understand the transcrptional
regulation system of Xanthomonas campestris pv. campestris
(Xc), we designed to clone the sigma factor genes from Xc. Two
degenarate oligonucleotide primers from the subregion 2.4 and
4.2 of the primary sigma factor were designed and used in the
polymerase chain reactions. DNA fragment about 500 bp were
produced and DNA sequence analyses showed that the amino acid
sequence of the amplified DNA fragment has 86.1% identity with
the same region of E. coli sigma 70. A 4.4 kb DNA fragment was
hybridized with 32-p-labele 500 bp DNA fragment in Southern
blot analyses. A colony with plasmid pYS169 was selected from
the constructed Sal-ISalI partial library. Restriction map and
Southern blot analyses indicated that the cloned sigma factor
gene in pYS169 was not with full length. Another partial
library with 11 kb EcoRI-EclRI DNA fragment was constructed and
a colony with plasmid pYS121 was selected. Plasmid pYS121-18,
which contains the full length sigma factor gene in the SmaI-
EcoRI 4.4 kb DNA fragment,was constructed from pYS121.
Restriction map and Southern blot analyses showed that the
overlapping region of the pYS169 and pYS121-18 are consistent.
DNA sequence analyses showed that the cloned sigmafactor gene
encoded a 633 amino acids protein and its molecular weight is
about 70 kDa. Western blot analyses revealed that the cloned
sigma factor gene was able to express in vivo and had similar
molecular weight as the purified Xc RNA polymerase sigma factor.
URI: http://hdl.handle.net/11455/21335
Appears in Collections:分子生物學研究所

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