Corr-Menguy Fabienne, Cejudo Francisco J, Mazubert Christelle, Vidal Jean, Lelandais-Brière Christine, Torres Gisele, Rode André, Hartmann Caroline
Institut de Biotechnologie des Plantes, Université Paris Sud, Orsay, France.
Plant Mol Biol. 2002 Nov;50(4-5):687-98. doi: 10.1023/a:1019906031305.
A cDNA coding for phytocystatin, a protease inhibitor, was isolated from wheat embryos by differential display RT-PCR and the corresponding full-length cDNA (named WC5 for wheat cystatin gene 5) subsequently obtained by RACE. The deduced primary sequence of the protein suggests the presence of a 28 amino acid N-terminal signal sequence and a 100 amino acid mature protein containing the three consensus motifs known to interact with the active site of cysteine peptidases. Northern and western analysis revealed a spatio-temporal pattern of the cystatin gene expression during caryopse development. In the embryo, WC5 was only expressed during early embryogenesis whereas, in seed covering layers, WC5 expression was restricted to the maturation stage of grain development. In addition, immunolocalization experiments showed that cystatin accumulated in the aleurone layer of the maturating seed and in the parenchymal tissues of the embryo scutellum. A recombinant form of the wheat cystatin was shown to be able to inhibit peptidase activities present in whole seed protein extracts. In addition, immunological techniques allowed us to identify two putative target peptidases. The possible roles of the cystatin protein are discussed in relation with tissular localization and putative peptidase targets during seed maturation.
通过差异显示反转录聚合酶链反应（differential display RT-PCR）从小麦胚中分离出编码植物胱抑素（一种蛋白酶抑制剂）的互补脱氧核糖核酸（cDNA），随后通过快速扩增cDNA末端（RACE）获得了相应的全长cDNA（命名为WC5，即小麦胱抑素基因5）。该蛋白质推导的一级序列表明存在一个28个氨基酸的N端信号序列和一个100个氨基酸的成熟蛋白，该成熟蛋白包含已知与半胱氨酸肽酶活性位点相互作用的三个共有基序。Northern杂交和蛋白质免疫印迹分析揭示了在颖果发育过程中胱抑素基因表达的时空模式。在胚中，WC5仅在胚胎发育早期表达，而在种皮中，WC5的表达仅限于籽粒发育的成熟阶段。此外，免疫定位实验表明，胱抑素在成熟种子的糊粉层和胚盾片的薄壁组织中积累。小麦胱抑素的重组形式能够抑制全种子蛋白提取物中存在的肽酶活性。此外，免疫技术使我们能够鉴定出两种假定的靶肽酶。结合种子成熟过程中的组织定位和假定的肽酶靶标，讨论了胱抑素蛋白的可能作用。
