Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21129
標題: 十字花科黑腐病菌α-澱粉脢基因之表現與調控
Expression and Regulation of Xanthomonas campestris pv. campestris α-Amylase Gene
作者: 邵源源
Shao, Yuan-Yuan
關鍵字: Xanthomonas
黃原菌
promoter
amylase
啟動子
澱粉脢
出版社: 分子生物研究所
摘要: α-amylase, which hydrolyzes starch to a mixture of maltose, maltotriose and relatively small amount of glucose, is one of the extracellular proteins produced by Xanthomonas campestris pv. campestris. The gene encoding the α-amylase (amy) has been cloned from strain Xc11. The purpose of this study was to locate and analyze the promoter and the regulatory region of Xc α-amy from Xanthomonas campestris pv. campestris 11. To achieve this end, the upstream region of the amy gene was cloned and sequenced. The total length ofthe DNA sequenced was 1091 bps, which ranged from -836th to +255th bp relative to the translational initiation site (+1). The sequence analysis illustrated that the upstream region of α-amy contained a putative upstream activator-binding site ΩUAS, potential promoter -10/-35 sequences, two inverted repeats ΩI , ΩII, and Shine-Dalgarno sequence (SD). Sequence comparison result revealed that the -376th to -482th bp upstream of Xcα-amy showed extensive homolgy to the -229th to -123th bp of Xc putative sigma factor gene (rfaY). The 0.8 kb insert DNA fragment of PE800 carrying the putative promoter sequence was cloned into the promoter proving vector pFY7 which uses bacterial luciferase as the reporter, forming plasmid pAMY800. Xc17(pAMY800) was able toemit bioluminescence suggesting that the 0.8 kb insert DNA carried promoter sequences. To understand the effect of carbohydrates on the expression of α-amy promoter, Xc17(pAMY800) was incubated in minimal medium containing one of the eight different carbon sources. Results showed that expression of α-amy promoter could be induced by the presence of maltose, starch, glucose, and sucrose in the medium, but functioned poorly in the medium containing galactose, lactose, fructose, or glycerol medium.. The time course of luciferase activity expressed by Xc17(pAMY800) coincided with that of the amylase activity expressed by Xc17 grown in LB, maltose or galactose medium. However, it seemed that glucose would inactivate upon the enzyme activity of α- amylase, but not on the expression by α-amy promoter. To determine the border region of the Xc α-amy promoter and regulatory region, PCR technique and Bal31 exonuclease deletion system were used to construct the 5'' deletion mutants; and methods of site-directed mutagenesis and hydroxylamine random mutagenesis were used to generate mutation clones in this regulatory region. Totally 14 mutation clones were selected and assayed by the system using expression of bioluminescence as as a function of promoter activity. Results showed that sequences around the ΩUAS palyed an important role in maltose induction and galactose repression. The minimal 5'' end for α-amy regulation was located around -187. And the 5'' upstream sequence essential for expression of Xc α-amy promoterefficiently was located around -133th to -119th bp, sequence around the putative -35 region. In addition, our data also showed that the amy promoter can not function efficiently in E. coli.
Xanthomonas campestris pv. campestris (Xc)可分泌產生胞外酵素 α- amylase,將可溶性 starch 水解成 maltose、部分 maltotriose 以及微 量之 glucose。為探討 Xc α-amy gene 啟動子之表現與調控,首先進行 Xc α-amy gene 上游核甘酸之定序。得到之核甘酸序列包含 α-amy gene coding region 之 255th bp 起至 5'' 端,全長共 1091 bps。分析 結果顯示,α-amy 基因之調節區域可能包含 putative upstream activator-binding site ΩUAS, potential promoter -10/-35 cononical sequences, 2 段 inverted repeats ΩI 及 ΩII, 與 Shine-Dalgarno sequence (SD)。本研究中,利用報導基因 luxAB 反應 Xc α-amy gene 啟動子表現,選殖了調節區域並作進一步分析。實驗結 果顯示,在 maltose medium 中 Xc α-amy 啟動子及調控區域因受到誘 發而表現最高;在 starch、glucose 及 sucrose medium 中,亦有良好 之螢光表現;在 lactose、galactose、fructose 或 glycerol medium 中之螢光表現則非常低。推測 maltose 為一重要之 inducer 直接或間接 誘發 positive regulator 調控α-amy 之表現。 Xc17(AMY800) 在 glucose 中生長時,其螢光表現較 α-amylase 酵素活性為高,故在轉錄 層面,其對α-amy 有誘發 之功能,但在轉錄後或轉譯後,則可能有 glucose inactivation 之作用。因此 glucose 對 Xc α-amy gene 之調 控則可能是多層面的。利用刪短與突變等方式測試 α-amy 調節區域之核 甘酸序列之功能及重要性後,結果顯示 α-amy 轉錄起始點 (+1) 上游 -152th 至 -117th bp 處,可能為啟動子之邊界區域。當除去 putative -35 region,仍可有極弱之表現。此外, putative -35 region 內之 -158th 及 -147th bp 位置的 G,對啟動子之效率亦十分重要。可能由於 這是 -35 region ,核甘酸序列之改變影響轉錄因子與 DNA 的接合能力 。當 ΩUAS 之 inverted repeat 上游一段序列被刪除時,maltose 誘發 之現象即開始減弱。以 site-directed mutagenesis 將 ΩUAS 之對稱性 破壞後,α-amy 受 maltose 及 glucose 誘發之現象與原來相同,但在 galactose medium 中表現受 repression 之現象則被解除。故 ΩUAS 之 對稱性可能與 galactose 有關之 negative regulator 認辨所須。將 pAMY800 送入 E. coli 中,螢光表現測試之結果顯示,Xc α-amy 在 E. coli 不表現。
URI: http://hdl.handle.net/11455/21129
Appears in Collections:分子生物學研究所

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