Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/20882
標題: 十字花科黑腐病菌 groESL 基因之選殖及特性之研究
Cloning and Characterization of the groES and groEL genes from Xanthomonas campestris pv. phaseoli
作者: Hsu, Chia-Chih
許佳智
關鍵字: 監護子
chaperonin
熱休克
緊急求救系統
黃原菌
heat shock
SOS response
GroESL
Xanthomonas
出版社: 分子生物學研究所
摘要: Abstract In this study, the purpose is selection and cloning the genes which induction or regulation of SOS response from Xanthomonas campestris pv. phaseoli (Xcp). These genes were selected by E. coli ER1992 strain. Chromosome DNA library from Xcp , in which the genes induction of SOS response is disrupted by the integration of plasmid pBluescript SK, was ligated and introduced into E. coli ER1992. After transformed into E. coli ER1992, to express the genes, which carrie the gene for the lacZ gene under the control of SOS inducible genes by din promoter. The plasmid-harboring cells cultivated in LB plates containing 1Mm of X-gal were presence blue colonies when SOS response was induced in ER1992 strain. 1036 blue transformed colonies were selected through this way. For decreese the selection of target genes, lvir phage infection was used to cloning the genes not be able to suppress of lvir phage infected with E. coli but also inhibit the production of plaque. From above described, five clones were showed to blue colonies in the ER1992 host, and can not be infected by lvir phage. In this article, we only investigated the clone carrie the pXCPA32-2 plasmid. The nucleotide sequences of the 4.6 kb insert DNA fragment were determined. Results of the DNA sequence analysis revealed two open reading frames that contain GroE chaperonin genes. GroE chaperoningenes exhibit the same operon structure, the first 288 nt ORF encoded a GroES homologue of 95 residues with a predicted size of 9.98 kDa and a calculated G+C content of 62.85 %, that exhibited 50 % sequence identity with GroES from E. coli, respectively. The second 1638 nt ORF encoded a 545-aa polypeptide with a deduced size of 57.3 kDa and a calculated G+C content of 63.79 %, that exhibited 77 % sequence identity with GroEL from E. coli, respectively. In Northertn blot, the groES and groEL DNA fragment from PCR product wwere used as probes. The Northern analysis revealed that different probes hybridized to a 2.2-kb transcript, suggesting that in Xcp groES and groEL are cotranscribed as a single message and regulated from the same promoter. To establish whether the putative promoter, identified by sequence comparison upstream of groES or groEL, behaves as a functional promoter in Xcp, the transcription star site of groE operon was determined by primer extention using a synthetic oligonucleotide that binds to the 5'' end of groES and groEL. A particularly strong signal was obtained with RNA from 37℃ heat-shock for 10 min, confirming the effect of temprature increase on groE transcription. The results showed that the putative transcription star site for the groE operon corresponds to a G 113 bases upstream of groES. Thus, the promoter sequence of -10 and -35 region is supported by our primer extension, in addition, the determined promoter sequence is a specific promoter that is recognized by the heat shock sigma factor, sigma -32(sH). Two weak signals obtained that upstream of groES and the promoter -10 and -35 region were recognized by sigma -24(sE)factor. Western immunoblotting was employed to characterize the relationshop of GroES and GroEL proteins expression level betweennormal growth temprature and after heat shock. The result indicated that groES and groEL genes induced by shift the temperature to 37℃. The western blot analysis revealed that Xcp GroES and GroEL can be detected at normal growth temperature(28℃)and a large increase in the protein level of GroES and GroEL after temperature shift from 28℃ to 42℃ for 60 min. To sum up the results, we suggesting that Xcp groESL regulated under heat shock, and both are heat shock proteins.
中 文 摘 要 本研究是以能從 Xanthomonas campestris pv. phaseoli(Xcp)篩選出具有誘導或調控緊急求救反應系統 (SOS response) 的基因為目的。 這些基因的篩選工作是藉由E. coli ER1992菌株來進行, 當經過誘導之後會使得受 din 啟動子調控的 lacZ 基因表現, 經過此篩選過程在含有 X-gal 的平板上一共得到1036個藍色的轉形株, 為縮小目標基因的選擇, 進一步利用 lvir 噬菌體的感染作用, 篩選出會抑制 lvir 噬菌體與 E. coli 感染作用的進行, 而無法產生溶菌斑的選殖基因。 一共取得 5 株既可在 ER1992 宿主內表現為藍色菌落, 又不受 lvir 噬菌體感染的選殖株。 本實驗僅就其中之一株所含之 pXCPA32-2 質體作進一步探討, 經定序後發現含有兩個 Open Reading Frames(ORFs) , 分別為 ORF95和 ORF545。 這二個 ORFs的基因順序及的胺基酸序列經比對後, 發現與 E. coli 的 GroE 操縱組具有高度的相似性。第一個 ORF (ORF95) 其 G+C 含量為 62.85%, 可轉錄出 95 個胺基酸的 GroES 蛋白, 分子量為 9.98 kDa的蛋白。 第二個 ORF (ORF545), 其 G+C 含量為 63.79 %, 可轉錄出 545 個胺基酸的 GroEL 蛋白, 分子量為 57.3 kDa 的蛋白。將 Xcp 與 E. coli 的 GroES 及 GroEL 進行胺基酸的相似性比較, 結果發現分別有 50 % 及 77 % 的相同性。此外, 本研究並將 Xcp 的 groES、 groEL 基因黏接到高表現載體 pET21b 與 pET21bT, 經大量表現後純化得到 GroES 及 GroEL蛋白, 並進一步製備抗 GroES 及 GroEL 蛋白的抗體, 以進行西方墨點法偵測蛋白表現情形。 利用 groES 及 groEL 的基因片段分別進行北方墨點法偵測, 其結果顯示出不同探針對在熱休克時抽取之 RNA 在相同的 2.2 kb 位置有放射性訊號, 此結果透露出 Xcp 的 groES 及 groEL 基因同屬於一個操縱組 (operon), 並且受到熱休克的調控。 為了推斷 groESL 操縱組的轉錄起始點位置, 於是進行引子延伸的分析, 此部份的結果發現,在 groES 基因轉譯起始點上游第 113 個鹼基出現明顯的放射性訊號, 並在上游區域發現一可被熱休克 sH(s32) 因子所辨認的同質性高的-10 及 -35 區域啟動子序列。 此外, 另有兩個較弱的轉錄起始訊號, 分別位於轉譯起始點上游第 97(G)及 98(G)鹼基位置, 其上游亦可找到類似熱休克轉錄因子 sE(s24) 辨認的啟動子序列。 利用北方墨點法來探討 groEL 基因的表現與熱休克的關係, 結果發現 GroE 操縱組基因的表現會受熱休克作用而誘導增加, 並在 37 ℃ 熱休克 20 分鐘後表現量達到最高。 以西方墨點法進行偵測蛋白表現後發現, Xcp 在正常生長溫度 28 ℃ 下, 即可偵測到 GroES 及GroEL 蛋白的表現, 在 42 ℃ 的熱休克下, 隨著熱休克的時間增加至60 分鐘, 其表現量亦隨之增加。
URI: http://hdl.handle.net/11455/20882
Appears in Collections:分子生物學研究所

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