第四十五卷(2016)
應用多源基因體學方法自乳牛瘤胃篩選脂解酵素基因
廖仁寶 陳若菁 蔡新興 蕭宗法 吳明哲
行政院農業委員會畜產試驗所
本研究目的在建立乳牛瘤胃微生物多源基因庫，並以功能導向方式篩選脂解酵素基因。乳牛 瘤胃內容物以磁珠震盪方式萃取多源基因體 DNA，利用質體建構多源基因庫。結果篩選出 2 株具脂解活性的株系，經 DNA 定序、ORF 分析及序列比對後發現，此 2 株系的插入片段 具有 2 個完整脂解酵素基因（LipoR1、LipoR2)。LipoR1 基因長 1,155 bp，可轉譯出 384-aa 蛋白質；LipoR2 基因長 960 bp，可轉譯出 319-aa 蛋白質。2 個預測的脂解酵素胺基酸序列 經與資料庫比對分析後，發現與現有蛋白質比對最高分數者之相同性百分比分別為 39% 與 86%。依據Arpigny & Jaeger 之細菌脂解酵素分類，其中 LipoR1 屬於第一類，而 LipoR2 則 可能成為新的類別。
關鍵語：脂解酵素基因、多源基因體學、瘤胃
Use of metagenomic approach to screen for lipolytic genes from dairy cattle rumen
R. B. Liaw, J. C. Chen, S. S. Tsay, T. F. Shiao and M. C. Wu
Livestock Research Institute, Council of Agriculture, Executive Yuan
The purpose of this study was to construct a metagenomic library from dairy cattle rumen microorganisms, and to screen for lipolytic genes using function-driven approach. The metagenomic DNA was extracted from dairy cattle rumen contents using bead-beating method. Then the metagenomic library was built with a plasmid system. As a result, two clones with lipolytic activity were obtained. By DNA sequencing, ORF finding, and sequence comparing, a total of 2 lipolytic genes (LipoR1 and LipoR2) were found. The lipoR1 gene was 1,155 bp, encoding 384-aa protein; the lipoR2 gene was 960 bp, encoding 319-aa protein. Both proteins showed 39% and 86% identity with the highest score to the closest in the protein database. Based on the lipolytic enzyme classification proposed by Arpigny & Jaeger, LipoR1 was affiliated to Family 1, and LipoR2 maybe belonged to the new family.
Key Words: Lipolytic gene, Metagenomics, Rumen
  
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