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標題: 十字花科蔬菜黑腐病菌的蔗糖分解酵素基因之選殖定序與分析
Cloning, sequencing and analysis of the sucrose-hydrolase like gene in Xanthomonas campestris pv. campestris
作者: 廖俊儒
Liao, Chun-Ju
關鍵字: Xanthomonas campestris
sucrose hydrolase
出版社: 植物學系
摘要: 中文摘要 Xanthomonas capmestris pv. campestris 是一種重要的革蘭氏陰性菌, 在農業上它是引起嚴重的十字花科蔬菜黑腐病之病源菌, 在工業上則是被用來生產具廣泛應用價值的 xanthan gum (又稱為黃原膠的一種胞外多醣)的菌種。 在培養基上, X. campestris 能利用蔗糖作為碳源而生長良好, 且可產生大量的胞外多醣; 在感染植物時, 寄主經光合作用產生並輸送至各處的蔗糖也會是此菌的直接碳源。 涉及蔗糖之利用的基因已經在幾種革蘭氏陽性菌與陰性菌被廣泛地研究, 但在 X. campestris 卻一直未見有這方面的報告被提出。本實驗以Xanthomonas capmestris pv. campestris 為實驗菌種,以跳躍子pUT-Tn5(pfm)/Tc致突變法獲得一株完全不具蔗糖分解酵素的突變株Xc17suc-41,再利用基因間的同質性重組交換完整的選殖出X. c. pv. campestris的蔗糖分解酵素基因。依核甘酸定序的結果可選殖到一個包括跳躍子嵌入位置的open reading frame (ORF),全長共1194 bps, 可轉譯出397個胺基酸,推測分子量為43046.50 Daltons 的蛋白產物。將此蛋白產物的胺基酸序列進行比對,發現與glycoside hydrolase family 13的酵素有相當高的同質性。胺基酸序列中包括4個 “consreved” 區域與4個重要的胺基酸,Asp-41是整個酵素的催化中心;Glu-83具有催化酸基形成的功能,並主導蔗糖的水解;Asp-153與His-152則可能扮演穩定酵素結構的角色。從互補試驗中得知,互補株Xc17suc-41(p2GPSNCIS)對蔗糖的分解能力與野生株相仿,證明選殖到的基因確實是Xc17的蔗糖分解酵素。
Xanthomonas campestris pv. campestris is an important gram-negative bacterium, agriculturally, it is the causative agent of severe black rot in crucifers, industrially, it has been used in production of exopolysaccharide xanthan gum which has wide applications. X. campestris grows very well in the culture medium with sucrose as carbon source and produces great amounts of exopolysaccharide. On the other hand, when infecting host plants, it may use sucrose,a translocatable form of carbohydrate, in host plants as an immediate carbon source. Genes involved in sucrose utilization have been studied extensively in many gram-positive and gram-negative bacteria, however, there is no similar genes reported for X. campestris thus far. Bacterial strains used in this study is Xanthomonas capmestris pv. campestris, strain 17, we have used the transposon pUT-Tn5 (pfm) / Tc to generate a mutant, Xc17suc-41, which lost the sucrose-hydrolytic activity. Furthermore, homologous recombination method was used to clone the intact gene of sucrose-hydrolytic enzyme of Xc17. Accroding to the results of nucleotide sequence analysis , the cloned fragment contains an open reading frame, ORF-suc, of 1194 nucleotidesin length, which may encode a putative polypeptide containing 397 amino acids and with an estimated molecular weight of 43046.50 Daltons. The alignment analysis of amino acid sequences indicated that the aminoacid sequence of ORF-suc was closely homologous to the enzyme of glycoside hydrolase family 13. It cantained four conserved regions and four critical amino acids : Asp-41 is proposed as the catalytic neucleophile ; Glu-83 as the general acid-base catalyst; Asp-153 and His-152 are needed to maintain nomail activity levels. The former residue plays a major role in substrate distortion and the histidines in the transition stabilization at sudsite —1. In the complementary test , the ability to hydrolyze sucrose of complementary strain Xc17suc-41(p2GPSNCIS) is similar to that of wild-type 。 This result demonstrated that the cloned gene is really the sucrose-hydrolase like gene of Xc17.
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