Yamasaki Teruyuki, Deguchi Masaki, Fujimoto Toshiko, Masumura Takehiro, Uno Tomohide, Kanamaru Kengo, Yamagata Hiroshi
Laboratory of Biochemistry, Faculty of Agriculture, Kobe University, Japan.
Biosci Biotechnol Biochem. 2006 May;70(5):1200-9. doi: 10.1271/bbb.70.1200.
The complete nucleotide sequences of the cDNA and its gene that encode a bifunctional alpha-amylase/subtilisin inhibitor of rice (Oryza sativa L.) (RASI) were analyzed. RASI cDNA (939 bp) encoded a 200-residue polypeptide with a molecular mass of 21,417 Da, including a signal peptide of 22 amino acids. Sequence comparison and phylogenetic analysis showed that RASI is closely related to alpha-amylase/subtilisin inhibitors from barley and wheat. RASI was found to be expressed only in seeds, suggesting that it has a seed-specific function. A coding region of RASI cDNA without the signal peptide was introduced into Escherichia coli and was expressed as a His-tagged protein. Recombinant RASI was purified to homogeneity in a single step by Ni-chelating affinity column chromatography and characterized to elucidate the target enzyme. The recombinant inhibitor had strong inhibitory activity toward subtilisin, with an equimolar relationship, comparable with that of native RASI, and weak inhibitory activity toward some microbial alpha-amylases, but not toward animal or insect alpha-amylases. These results suggest that RASI might function in the defense of the seed against microorganisms.
对编码水稻（Oryza sativa L.）双功能α-淀粉酶/枯草杆菌蛋白酶抑制剂（RASI）的cDNA及其基因的完整核苷酸序列进行了分析。RASI cDNA（939 bp）编码一个由200个氨基酸残基组成的多肽，分子量为21417 Da，包括一个22个氨基酸的信号肽。序列比较和系统发育分析表明，RASI与大麦和小麦的α-淀粉酶/枯草杆菌蛋白酶抑制剂密切相关。发现RASI仅在种子中表达，表明它具有种子特异性功能。将不含信号肽的RASI cDNA编码区导入大肠杆菌，并表达为His标签蛋白。通过镍螯合亲和柱层析一步法将重组RASI纯化至同质，并对其进行表征以阐明靶酶。重组抑制剂对枯草杆菌蛋白酶具有较强的抑制活性，呈等摩尔关系，与天然RASI相当，对一些微生物α-淀粉酶具有较弱的抑制活性，但对动物或昆虫α-淀粉酶无抑制活性。这些结果表明，RASI可能在种子对微生物的防御中发挥作用。
