Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21755
標題: Characterization of the Xanthomonas campestris pv. campestris lexA1 and lexA2 Regulons
Xanthomonas campestris pv. campestris中受到lexA1與lexA2調控之基因之探討
作者: 王雲慶
Wang, Yun-Ching
關鍵字: 十字花科黑腐病菌
Xanthomonas campestris
緊急求救反應
lexA1
lexA2
recA
SOS response
lexA1
lexA2
recA
出版社: 分子生物學研究所
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摘要: Two lexA genes, named lexA1 and lexA2, had been identified in Xanthomonas campestris pv. campestris strain 17 (Xc17) in our previously studies. Computational search for genes possibly regulated by LexA1 and LexA2 proteins in the available genomic sequence of the X. campestris showed that the sequence in the upstream region of umuDC gene has an identical LexA1 binding motif. Southern blot analysis revealed that umuDC gene only exists in X. campestris ATCC33913 among the ten Xanthomonas strains studied. UmuD gene was demonstrated to be positively regulated by LexA1 protein using UV sensitivity test and promoter activity assay. To elucidate the regulatory mechanisms of the two lexA genes and recA in SOS response in the ten Xanthomonas strains, a currently constructed lexA1 mutant strain (XDA1) and previous constructed lexA2 mutant strain (XDA2) and recA mutant strain (NT3) were used in this study. Growth curves showed no difference among mutants and wild type strains. The XDA1, XDA2 and NT3 mutant strains were found to be UV sensitive because an exposure to 300 J/m2 UV irradiation resulted in respective 220, 200 and 3370-fold decrease in survival rate. LexA1, lexA2 and recA genes should have their importance when DNA was damaged. Because the recA and recX genes are located just downstream of lexA1 in Xc17, the promoter activities of lexA1, recA, and recX were determined. The results indicated that the lexA1 and recA promoters were induced by MMC but recX was not. On the other hand, it has been shown that sulA2, dinP2, and dnaE2 genes were located just downstream of the lexA2 gene. Promoter activity assay indicated that the lexA2 was induced by MMC and dinP2 was slightly induced but sulA2 and dnaE2 were not. Northern blot analysis and RT-PCR indicated that the two lexA genes could be regulated to form operons lexA1-recA-recX and lexA2-sulA2-dinP2-dnaE2 for downstream gene expression. By using the lacZ as a reporter gene to measure the promoter activity of lexA1, lexA2 and recA, the transcriptional activities of these promoters were enhanced by MMC and the induction was time-dependent. After inducing for 3 hours, the recA increased 3.5-fold but the ratio of lexA1 and lexA2 was 1.1~1.4-fold, indicating that the recA promoter was the first target among these three promoters. Because recA is the most important gene in SOS response, the genes affecting recA expression were investigated by Tn5 mutagenesis. To date, recD, recC and piuB mutants were confirmed to be three of the mutagenesis colonies and waited for further studies of the relationship between piuB, recD, recC and recA.
本實驗室在Xanthomonas campestris pv. campestris strain 17 (Xc17)中,發現具有兩套lexA基因,分別命名為lexA1及lexA2,而利用LexA1 Box進行X. campestris基因庫序列的搜尋,在umuDC基因上游有一段吻合的序列,可能受LexA1調控,因此本研究以南方墨點法分析十株不同的Xanthomonas菌株後,確定umuDC僅存在於X. campestris pv. campestris ATCC33913中,此外經由UV敏感性測試及umuD啟動子活性測試,證明umuD基因會受到LexA1的正調控。同樣利用南方墨點法分析十株不同的Xanthomonas菌株後,發現這些菌株都具有兩套lexA基因,為了探討這兩套lexA基因及recA基因在Xanthomonas菌株的調控關係及在SOS反應中所扮演的角色,本研究中構築了lexA1突變株 (XDA1) 及利用先前實驗室已構築之lexA2突變株 (XDA2) 與recA突變株 (NT3) 進行探討。先測試其生長曲線無明顯差異後,再經由UV敏感性測試證明在UV劑量300 J/m2照射下,這三株突變株與Xc17野生株相較起來,XDA1及XDA2存活率分別下降了約220及200倍,而NT3存活率約下降了3370倍,顯見lexA1、lexA2及recA基因對菌體遭受到DNA傷害時的重要性。由於Xc17菌株的lexA1基因下游存在著recA及recX基因,測試三者之啟動子片段的表現活性,確定僅有lexA1及recA啟動子可受mitomycin C (MMC) 誘導而大量表現,而recX啟動子片段則完全不表現。而在lexA2基因的下游則是存在著sulA2、dinP2及dnaE2,同樣測試這四個啟動子片段的表現活性,發現只有lexA2啟動子是可被MMC誘導而提高表現量,dinP2啟動子僅有微量的表現,而sulA2及dnaE2則是完全不表現。所以藉由北方墨點法及反轉錄聚合酶連鎖反應,確認lexA1及lexA2基因可能藉由lexA1-recA-recX及lexA2-sulA2-dinP2-dnaE2以形成operon的方式共同表現來帶動下游的基因,藉此方式來調控下游基因的表現。而利用lacZ報導基因進行lexA1、lexA2及recA啟動子之活性測試,發現在受到MMC 誘導後的不同時間點,這三個啟動子皆能隨著時間的增加而提高表現比值;在誘導3小時後,recA啟動子有3.5倍的增加,而lexA1與lexA2的誘導比值約在1.1 ~ 1.4倍,確定最先啟動的是recA啟動子。由於整個SOS反應中最重要的基因為recA,因此利用Tn5跳躍子隨機破壞造成突變,篩選能影響recA啟動子活性之菌落。目前已從篩選出的11顆突變株中,確認出Tn5是分別破壞了recD、recC及piuB基因,而與recA基因之關係,仍有待進一步探討。
URI: http://hdl.handle.net/11455/21755
其他識別: U0005-2408200615322700
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2408200615322700
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