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克隆了水稻的WIP1基因,该基因属于Bowman-Birk蛋白酶抑制剂(BBI)家族.RNA杂交实验结果显示该基因的表达受伤害和茉莉酮酸的诱导,表明它在植物防御反应中起着重要的作用.将此基因克隆到表达载体pET28a,并在大肠杆菌中表达融合蛋白.分别用胰凝乳蛋白酶和胰蛋白酶为底物检测纯化的融合蛋白的抑制活性,发现融合蛋白具有胰凝乳蛋白酶的抑制活性,但没有对胰蛋白酶的抑制活性;同时发现融合蛋白C端的融合残基对蛋白酶抑制活性有重大影响,具有C端(His)6纯化标签的融合蛋白不具有胰凝乳蛋白酶的抑制活性,而有天然C端序列的融合蛋白具有胰凝乳蛋白酶的抑制活性,这暗示了过长的C末端序列可能会影响WIP1蛋白的正确折叠.蛋白酶抑制活性分析表明水稻Bowman-Birk抑制剂家族的成员可能在结构和功能上均发生了分化.
The WIP1 gene was cloned from rice and belongs to the Bowman-Birk family of protease inhibitors (BBI). The results of RNA hybridization showed that the expression of this gene was impaired and induced by jasmonic acid, indicating that it plays an important role in plant defense response The gene was cloned into the expression vector pET28a and expressed in E.coli.The inhibitory activity of the purified fusion protein was detected by using chymotrypsin and trypsin as substrate respectively and the results showed that the fusion protein had chymotrypsin But not trypsin. At the same time, it was found that the fusion residues at the C-terminal of the fusion protein had a significant effect on the protease inhibitory activity. The fusion protein with the C-terminal (His) 6 tag did not have the inhibitory activity of chymotrypsin , While the fusion protein with the native C-terminal sequence has the inhibitory activity of chymotrypsin, suggesting that an overly long C-terminal sequence may affect the correct folding of the WIP1 protein.Protease Inhibitory Activity Assays showed that the Bowman-Birk inhibitor family of rice Members may be structurally and functionally differentiated.