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Title: 水稻(Oryza Sativa L.) Osm34分泌性蛋白質的基因選殖及第三型幾丁質酶基因家族的親緣關係分析
Other Titles: Gene Cloning of Rice (Oryza Sativa L.) Osm34 Secretory Protein and Phylogenetic Analysis of the Class Ⅲ Chitinase Gene Family
Authors: 李嘉雯
Issue Date: Jun-2006
Publisher: 國立臺灣師範大學生命科學學系
Department of Life Science, NTNU
Abstract: 本研究從水稻細胞的培養液中,分離出一分子量為34kDa的分泌性蛋白質(Osm34),經由N端胺基酸微定序分析及基因選殖的結果顯示:不包含分泌訊息序列的Osm34 cDNA長度為1045bp,可轉譯出含有282個胺基酸,等電點為4.94的蛋白質。Osm34的胺基酸序列中,除了具有醣苷水解酵素第18家族成員所特有的兩個保守序列「104RVAVSMGG111」和「146LDGIDIDYE154」之外,胺基酸序列一致度(identity)的比對結果也顯示該蛋白質為一個由我們最先鑑定的水稻第III型幾丁質酶,我們已將其序列註冊於GenBank,編號為AF330230。水稻基因組資料庫中,共登錄有32個具保守序列的第III型幾丁質酶。我們對這些幾丁質酶進行多序列比對與親緣關係分析,結果顯示這些幾丁質酶可被分類至IIIa和IIIb兩個亞型(subc1asses)。III a亞型共包含有29個蛋白質,其胺基酸序列的一致度介於34-85%之間;IIIb亞型雖僅含有3個蛋白質,但其胺基酸序列的一致度高達50-83%;不同亞型之間,胺基酸序列的一致度則低於25%。由前人的研究(Bokma et al., 2002)得知,第III型幾丁質酶保守序列「DXXDXDXE」中的D和E為影響酵素活性的兩個關鍵胺基酸,但我們發現在IIIa亞型的29個幾丁質酶中,有20個成員的這兩個關鍵胺基酸發生變異,可能會導致酵素活性或受質專一性的改變。若將此一因素列入考量,我們建議將IIIa亞型幾丁質酶進一步區分為兩個次亞型(sub-subclasses),兩個關鍵胺基酸維持不變的九個成員為IIIa-1次亞型,而兩個關鍵胺基酸從D和E變異成F/Y和D的20個成員則為IIIa-2次亞型。如此,屬於III a-1次亞型的幾丁質酶彼此間胺基酸序列一致度也可提高到48-69%,而同屬IIIa-2次亞型內的幾丁質酶其胺基酸序列一致度則增加為43-85%。
In this study, we report the identification of a novel class III chitinase (Osm34) secreted by suspension-cultured rice cells. The Osm34 gene had been cloned and sequenced and its cDNA sequence was submitted to GenBank (accession no. AF330230). Since two amino acid segments conserved for bacterial and fungal chitinases are also present in Osm34 amino acid sequence (104RVAVSMGG111 and 146LDGIDIDYE154), this plant class III chitinase is classified as a member of glycosyl hydrolase family 18 as well. Recently, some researchers have suggested that the plant class III chitinases to be further divided into 2 subclasses, IlIa and IIIb. However, this proposal has not been widely accepted. In this study, we are the first group to analyze the phylogeny of 32 putative class III chitinases identified in the rice genome database. Our results suggested that among the 32 examined chitinases, 29 of them, sharing identity from 34% to 85%, can be grouped into subclasses IIIa, while the rest 3, sharing identity of 50-83% are to be placed into subclasses IIIb. Meanwhile, the amino acid sequence identity between the two subclasses is less than 25%. The amino acid residues of aspartic acid (D) and glutamic acid (E) in the conserve region of class III chitinases (DXXDXDXE) are thought to be the most critical amino acids for enzyme activity (Bokma et al., 2002). We discovered that 20 of rice subclasses IIIa chitinases have been mutated in these residues, while 9 of them remain unaltered. According to this variation, we suggest that the rice IIIa subclass chitinases might be further divided into two sub-subclasses. Nine members with unchanged conserve region belong to sub-subclass IIIa-1 and the other 20 members with D→F/Y and E→D mutation are classified into sub-subclass IIIa-2. By this way, the amino acid sequence identity within sub-subclass IIIa-1 and IIIa-2 would be raised to 48-69% and 43-85%, respectively.
Other Identifiers: AC0F0B93-CBD3-72CB-2D27-64B293F2044E
Appears in Collections:生物學報

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