紅梗水芋中黏質多醣類其生合成主要酵素尿嘧啶雙磷酸葡萄糖去氫酶

紅梗水芋中黏質多醣類其生合成主要酵素尿嘧啶雙磷酸葡萄糖去氫酶 紅梗水芋中黏質多醣類其生合成主要酵素尿嘧啶雙磷酸葡萄糖 去氫酶基因之選殖及現 Cloning and Expression of an Enzyme that Biosynthesizes Polysaccharide in Mucilage from Colocasia esculenta (Taro): UDP-glucose dehydrogenase 研究生: 陳柏宏 Po-Hong Chen Tzou-Chi Huang 指導教授: 黃卓治 博士 吳美莉 博士 Mei-Li Wu [摘要] 存在於植物中的多醣物質除了合成植物細胞壁的半纖維素以及球莖中儲藏的澱粉外,最主要的就是維持植物細胞滲透壓的水溶性黏質,mucilage,。黏質當中最主要的聚合體為阿拉伯半乳糖蛋白,arabinogalactan-protein, AGPs,,AGPs蛋白質鏈可藉由羥基化作用並在高基氏體中連接上多醣類。植物多醣類化合物一般結構中含有許多經過醣基化的取代基,glycosyl residues,,再以不同連結方式結合不同種單醣類修飾而成。這些經過修飾的多醣類有一共同的前趨物—尿嘧啶雙磷酸葡萄糖醛酸,UDP-glucuronic acid, UDPGA,,其會再經植物轉化代謝形成多醣類之次級中間產物,如,尿嘧啶雙磷酸阿拉伯糖,UDP-arabinose,、尿嘧啶雙磷酸半乳糖醛酸,UDP-galacturonce acid,以及尿嘧啶雙磷酸木糖,UDP-xylose,等,最後再形成最終存在於植物中的多醣類,如,arabinogalactans、arabinans、xylans、xyloglucans…等。而UDPGA的生合成,主要又是由尿嘧啶雙磷酸葡萄糖去氫酶,UDP-glucose dehydrogenase, UDPGDH,催化所產生,因此對於植物來說,UDPGDH是合成其生理所需多醣類的重要酵素。我們已將紅梗水芋,Colocasiaesculenta,中合成UDPGDH之基因利用退化性引子,degenerate primer,以RT-PCR的方式選殖出來,並將此長度為1,443bp cDNA序列註冊於NCBI genebank之中,Accession number,AY222335,。此酵素具有一個催化活性中心部位,Gly267~Asp276,、一個NAD結合部位,Gly8~Gly14,。將此一序列與其他植物甚至動物相同基因來比較,皆有很高的相似度,此一全長之cDNA可以轉譯成為480個胺基酸,預測的蛋白質大小為52.9 kD,同時利用pET21b載體以及大腸桿菌BL21 DE3為宿主進行蛋白質表達,並以UDP-glucose做為基質而另外加入NAD+做為輔酶測量由E.coli表達系統所表達出的UDP-glucose dehydrogenase,確實具有酵素活性。另一方面,我們也利用即時螢光定量PCR,real-time PCR,比較Ugd基因於不同的植物器官及水份逆境下之表現量,發現此基因於根部的表現量遠超過莖及葉,而因水芋根部黏質分泌旺盛,所以此關鍵酵素被認為能催化產生黏質多醣類並扮演植物 保護之重要角色。 關鍵字: 黏質、阿拉伯半乳糖蛋白、尿嘧啶雙磷酸葡萄糖醛酸、紅梗水芋、尿嘧啶雙磷酸葡萄糖去 氫、半纖維素 【Abstract】 The main polysaccharides include hemicellulose in cell wall and starch in corm, but appreciable quantities of water-soluble mucilage are also present in plant. Compositional analysis of purified mucilage showed that the main polymer present was an arabinogalactan-protein (AGPs). AGPs protein backbone are hydroxylated and linked to the polysaccharides in Golgi apparatus. Many of the glycosyl residues found in polysaccharides in plant are derived from the sugar precursor UDP-glucuronic acid (UDPGA), which can be converted in to UDP-arabinose, UDP-galacturonic acid, UDP-xylose, and the other components including arabinogalactans, arabinans, and xyloglucans. The enzyme controlling the biosythesis of UDP-glucuronic acid, UDP-glucose dehydrogenase (EC 1.1.1.22) (UDPGDH or Ugd), and that is the key regulator in the polysaccharides biosynthesis pathway in plant. Recently, we cloned the Ugd gene from Colocasia esculenta (1,443 bp) and register the sequence on NCBI GeneBank (accession: AY222335). The enzyme contains the characteristic motifs UDP-glucose dehydrogenase, including the catalytic center and a NAD-binding site. The sequence is highly conserved within the plant and animal sequence. The full length cDNA encodes a protein of 480 amino acids with a predicted size of 52.9 kD. We use the E.coli expression system to express the target protein, and the enzyme assay to confirm enzyme activity. We also use real-time PCR to confirm that enzyme is highly expressed in young root, but lower expression levels were observed in expanding tissues of the epicotyl, stem and in young leaves. The key enzyme: UDP-glucose dehydrogenase considering that biosynthesizes polysaccharide in mucilage and play an important role in plants. Keyword,mucilage arabinogalactan-protein (AGPs) UDP-glucuronic acid (UDPGA) Colocasia esculenta (taro) UDP-glucose dehydrogenase (37238; UDPGDH) hemicellulose