鵲豆Bowman-Birk 型胰蛋白酶抑制劑基因之選殖

Molecular Cloning of a Bowman-Birk TypeTrypsin Inhibitor from Lablab Purpureus

利用不同種類豆科植物的Bowman-Birk型蛋白酶抑制劑基因序列比對，找出相似度最高5’端核苷酸序列的部分作退化性正向引子的設計，以鵲豆胰蛋白酶抑制劑胺基酸序列的羧基端設計退化性反向引子，以鵲豆葉子的genomic DNA當作模板進行聚合酶連鎖反應，選殖出一種鵲豆胰蛋白酶抑制劑（Lablab purpureus trypsin inhibitor；LPTI）基因，基因含有336 bp且不含內含子（intron），可編碼出一條具有112個胺基酸殘基的多胜肽鏈，其N端含有31個胺基酸殘基組成的訊號胜肽，與其它Bowman-Birk型蛋白酶抑制劑比較胺基酸序列，結果相同度非常高（57%-83%）。利用親緣關係樹分析顯示，LPTI與Phaseolus filiformis及Phaseolus grayanus的Bowman-Birk型蛋白酶抑制劑親緣關係最親近，最後利用SWISS MODEL軟體模擬其3D架構。

Bowman-Birk type protease inhibitor gene sequences has been cloned from different leguminous plants, and was used for nucleotide similarity comparison to identify the part with the highest similarity for use it as 5’-terminal the degenerate forward primers design. Design of the degenerate reverse primers based on the carboxy-terminal amino acid sequences of Lablab purpureus trypsin inhibitor. Then the genomic DNA extracted from Lablab purpureus leaves as used as the template to conduct a polymerase chain reaction, thereby cloning of a Bowman-Birk type protease inhibitor gene sequences of the Lablab purpureus. The LPTI gene contains 336 bp, which can be encoded a polypeptide chain containing 112 amino acids without introns. N-terminal of polypeptide chain contains a signal peptide composed of 31 amino acid residues. There was a very high identify of amino acid sequences compared with other Bowman-Birk type protease inhibitors (57%-83%). The phylogenetic tree analysis shows that LPTI is the closest to the Bowman-Birk type protease inhibitor of Phaseolus filiformis and Phaseolus grayanus. Finally, the SWISS MODEL software as used to predict its 3D framework.