Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21582
標題: Purification of heat shock protein HrcA and cloning of heat shock gene hsp20 of xanthomonas campestris pv. campestris
十字花科蔬菜黑腐病菌熱休克調節蛋白HrcA之大量表現純化及熱休克蛋白基因hsp20之選殖
作者: 蔡宛儒
關鍵字: 十字花科蔬菜黑腐病菌
出版社: 分子生物學研究所
摘要: 
過去的研究顯示,HrcA是一個熱休克蛋白,具有負向調控的功能。本實驗室已經選殖並定序完成Xanthomonas campestris pv. campestris 17 ( Xc17 )的hrcA基因。因此本研究的目的之一是表現純化Xc17的HrcA蛋白,並觀察在in vivo及in vitro的情況下,HrcA蛋白與CIRCE element間的作用情形。首先,以蛋白質表現載體大量表現HrcA蛋白,並純化之。由於純化後的蛋白多以inclusion body形式存在,因此以透析的方式renature。接著將已知含有CIRCE element之DNA片段利用PCR的方式進行增殖。所拓增的DNA片段包括X. campestris pv. phaseoli 及A. tumefaciens的 groES基因上游包含CIRCE element及啟動子部分。利用gel retardation方法觀察HrcA與CIRCE element之間的結合情形,結果並沒有明顯的band shift產生,亦即純化的HrcA並沒有與包含CIRCE element的DNA片段結合。在in vitro的實驗中,我們將含有CIRCE element之DNA片段構築到以b-galactosidase為reporter之啟動子表現載體上,分別送入Xc17及Xc17 hrcA-中,觀察啟動子的表現,以了解HrcA蛋白與CIRCE element間的作用。結果,不管是否經過熱休克處理Xc17 hrcA-比Xc17的b-galactosidase的活性表現高,顯示在菌體內HrcA與CIRCE element可能是有作用,但並沒有顯著效應。
本實驗室先前利用二維電泳法,觀察到Xc17經熱休克處理後會明顯表現一小分子熱休克蛋白,分子量約20 kDa。本研究之另一個目的是由Xc17中選殖出完整的hsp20基因並探討。根據推測蛋白的保守區域序列,先設計degenerate的引子,以PCR的方式選殖出一段約200 bp的片段,將該片段接入載體後利用電孔法送入Xc17中,進行single crossover。以抗生素篩選得到一個轉殖株,命名為Xc17:: pOK-hsp20。再以適當限制酵素切割轉殖株之染色體後,使其自我黏接,再轉殖送入E. coli。利用抗生素篩選,得到3個質體供定序。定序後得到hsp20基因,全長為477 bp,可轉譯出158個胺基酸,蛋白分子量為17.8 kDa,經比對後與 Xylella fastidiosa之小分子熱休克蛋白相似度為87.8%。在hsp20轉譯起始密碼ATG上游6~10 bp處有一個類似ribosomal binding site,在轉譯終止密碼TAA下游有一個類似terminator的序列。Primer extension分析結果發現hrcA轉錄起始點位於轉譯起始點上游第64 bp的位置。在轉錄起點上游可發現類似σ32啟動子認辨的 —10與 —35序列。將hsp20上游片段選殖於啟動子選殖載體pFY7上進行啟動子活性測試。結果顯示所選殖的片段有啟動子活性,且經熱休克處理後其活性最高會增加5~5.8倍。在定序過程中得到hsp20上游約600 bp的核酸序列,經分析比對後,推測hsp20上游有一個轉錄方向與之相反的peroxidase基因。

Heat shock protein HrcA acts as a repressor interacting with the CIRCE element at the transcriptional level in the majority of microorganisms which do not include E. coli. The hrcA in Xanthomonas campestris pv. campestris 17 ( Xc17 ) has been cloned and sequenced. To understand the interaction between HrcA and CIRCE elements, the Xc17 hrcA was cloned into pET-21a vector for over-expression in E. coli. Since the over-expressed HrcA protein was present as insoluble form, dialysis was performed to renature the HrcA. The DNA fragments carrying the CIRCE elements upstream of groES of X. campestris pv. phaseoli and A. tumefaciens were amplified by PCR. However, when the PCR fragments were incubated with the His-bind purified HrcA for gel mobility shift assays, no interaction was observed. To analyze the effects of HrcA on CIRCE element in vivo, the PCR fragments were cloned in the promoter-proving vector pFY13-9, which use the promoter-less lacZ as the reporter, and transformed into the wild-type Xc17 and hrcA mutant. The levels of b-galactosidase in hrcA mutant were higher than that in Xc17, indicating that the expression is repressed by HrcA. After heat shock treatments, the promoter activity increased by 30%, implicating that HrcA interacts with CIRCE elements in vivo, although the effect is not strong.
It was previously found that following heat shock treatments, Xc17 de novo synthesizes a protein of about 18 kDa. N-terminal sequencing data suggested this protein to be a Hsp20 homologue of bacteria. In this study, the Xc17 hsp20 gene was cloned and sequenced. The results indicated that it is 477-bp long able to code for a polypeptide of 158 amino acids with a calculated molecular weight of 17.8 kDa. The deduced amino acid sequence has 87.8 % similarity to the low molecular weight heat shock protein in Xylella fastidiosa. Primer extension analysis showed that the transcription start site is located 64 bp upstream of the hsp20 start codon. Consensus —10 and —35 sequences similar to that recognized by s32 in many other bacteria are present upstream of the transcription start site. Presence of promoter activity at the hsp20 upstream region was demonstrated by transcriptional fusion assay. In addition, the promoter activity was found to increase by 5.8 folds after heat shock. An open reading frame homologous to peroxidase gene which transcribed divergently is present upstream of the Xc17 hsp20.
URI: http://hdl.handle.net/11455/21582
Appears in Collections:分子生物學研究所

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