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標題: Cloning of bacteriocin genes from Xanthomonas and expression in E. coli
Xanthomonas 細菌素基因的篩選及在 E. coli 中表現
作者: 廖珮鑾
Liao, Pei-Luan
關鍵字: bacteriocin
出版社: 分子生物學研究所
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摘要: This study, the glyA, glyB and glyC genes of Xanthomonas campestris pv. glycines YR32 (XcgYR32) were PCR amplified, cloned into pSK-T vector and sequenced. The putative ORF of the cloned glyA gene has 1,122 base pairs and from which a 374 amino acids protein with molecular weight of 39.2 kDa could be encoded. The putative ORF of glyB, which overlaps by 30 base pairs with glyA, encoded a 152 amino acids protein with a deduced size of 14.9 kDa. The glyC gene is located upstream of the glyA and encoded 152 amino acids with molecular weight of 14.9 kDa and shows 96.7% identity with that of X. campestris pv. glycines 8ra (Xcg8ra). However, a TAA stop codon was found at position 24 of XcgYR32 GlyC. The DNA fragments of this genes were subcloned into pSK vector in different gene organization and orientation. The individual constructs was then transformed into E.coli and bacteriocin activities of transformants were determined. Results showed that bacteriocin activities could be identified in the transformants with both of the glyA and glyB genes and primarily in cell extracts. No specific translation product from glyC in E. coli LCD44 was identified by maxicell experiment. Four protein fusion constructs connecting the glyA and glyB in one open reading frame were created in this study. However, expression of this chimeric constructs in E.coli resulted in formation of insoluble recombinant proteins and no bacteriocin activity could be detected. Bacteriocin activity produced Xanthomonas fragariae (Xf), a phytophathogenic bacterium of strawberry plants, was identified in this study. The antimicrobial activity of Xf was effective to most of the tested Xanthomonas species. Maximal bacteriocin activity in culture fluid was obtained in the early stationary phase of growth of the Xf. The bacteriocin of Xf was stable at temperature up to 65℃ for 10 minutes. The bacteriocin produced by Xf was partial purified from culture supernatant by ammonium sulfate precipitation and gel filtration chromatography. The molecular weight of the proteins with bacteriocin activity collected from gel filtration column was calculated to be approximately 200 kDa. However, SDS-PAGE analysis indicated that the bacteriocin of Xf might exist as a protein complex from small molecular weight subunits.
本研究將 Xanthomonas campestris pv. glycines YR32 (XcgYR32) 合成細菌素 Glycinecin A之glyA 及 glyB 和上游之 glyC 基因利用引子設計及 PCR 增幅進行選殖與定序。glyA 基因全長包含1,122個核苷酸,轉譯出之GlyA 含有374個胺基酸,分子量約為 39.2 kDa;glyB 基因全長包含 456 個核苷酸,轉譯出之 GlyB 含有 152 個胺基酸且與 glyA 基因重疊 30 個核苷酸,分子量約為14.9 kDa;glyC 基因轉譯出152 個胺基酸且與 Xanthomonas campestris pv. glycines 8ra (Xcg8ra) 的胺基酸序列相似度達 96.7 %,但在第 24 個胺基酸位置出現轉譯終止碼 TAA。將上述基因構築於表現載體,觀察在E. coli 中 Glycinecin A 的活性表現,發現含有 glyA 與 glyB 基因之轉殖株胞內萃取液抑菌活性優於胞外培養液抑菌活性。Maxicell 實驗沒有發現特殊的 GlyC 轉譯產物。設計引子將 glyA、glyB 進行黏合,經轉譯後形成單一蛋白 chimeric A,但四種重組 chimeric A 所表現之蛋白活性多為不可溶且未見抑菌效果。 Xanthomonas fragariae 是以草莓為宿主的植物病原菌,所產生的細菌素對於大部分 Xanthomonas 菌屬具有抑菌效果。當菌株培養至 12 小時即達穩定生長期,且胞外液抑菌活性也達最大值。Xf 細菌素最高溫度耐受度為 65℃ 下作用10分鐘。收集 Xf 細菌素需利用硫酸銨鹽將 Xf 胞外液蛋白沉澱後再以分子篩進行純化,純化後所得具活性的細菌素蛋白分子量估算高達200 kDa。但利用SDS-PAGE 分析卻發現Xf 細菌素可能是由小分子量蛋白所構成。
其他識別: U0005-2708200610270500
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