從白鳳豆選殖出一種Bowman-Birk型胰蛋白酶抑制劑基因

Molecular Cloning of a Bowman-Birk Type Trypsin Inhibitor gene from Canavalia ensiformis

透過比對不同豆科植物的Bowman-Birk型胰蛋白酶抑制劑基因序列，篩選出5’端及3’核苷酸序列相似度最高的區域，並據此設計退化性正向及反向引子。以白鳳豆葉片的基因組DNA（genomic DNA）作為模板進行聚合酶連鎖反應（PCR）。成功選殖出一種Bowman-Birk型白鳳豆胰蛋白酶抑制劑基因（Canavalia ensiformis trypsin inhibitor, CETI），該基因含有一個起始密碼及終止密碼，全長336 bp，不含內含子（intron），可編碼一條由111個胺基酸殘基組成的多胜肽鏈，其中N端含有40個胺基酸殘基的訊號胜肽（signal peptide）。將CETI與其它Bowman-Birk型胰蛋白酶抑制劑進行胺基酸序列比對，結果顯示其相似度極高（58%-86%）。親緣關係樹分析顯示，CETI與Phaseolus filiformis及Phaseolus grayanus的Bowman-Birk型胰蛋白酶抑制劑親緣關係最為接近。最後，利用SWISS-MODEL軟體模擬其三維結構（3D structure），以進一步探討其可能的功能與結構特性。

By comparing the gene sequences of Bowman-Birk type trypsin protease inhibitors from various leguminous plants, the regions with the highest sequence similarity at both the 5’and 3’ends were identified. Based on these regions, degenerate forward and reverse primers were designed. Genomic DNA from White Jack Bean (Canavalia ensiformis) leaves was used as a template for polymerase chain reaction. A Bowman-Birk type trypsin inhibitor gene from White Jack Bean (Canavalia ensiformis trypsin inhibitor, CETI) was successfully cloned. The gene contains a start and stop codon, with a total length of 336 bp, and is intron-free. It encodes a polypeptide chain of 111 amino acid residues, including an N-terminal signal peptide of 40 amino acids. Sequence alignment of CETI with other Bowman-Birk type trypsin inhibitors revealed a high amino acid sequence similarity (58%-86%). Phylogenetic tree analysis indicated that CETI is most closely related to the Bowman-Birk type trypsin inhibitors from Phaseolus filiformis and Phaseolus grayanus. Finally, the SWISS-MODEL software was used to predict the 3D structure of CETI, providing insights into its potential function and structural characteristics.