Study of relationship between development of embryo specific endopeptidase activites and induction of seed germination in maize plant.

玉米植株胚特异性内肽酶活性发育与种子萌发诱导关系的研究。

• 批准号: 09660071
• 负责人: MITSUHASHI Wataru
• 金额: $ 2.18万
• 依托单位: YAMAGATA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09660071/
• 关键词: ENDOPEPTIDASE; DRY SEED; EMBRYO; GERMINATION

Very high proteinase activity was observed in maize (Zea mays L dentcorn cv. W64A) dry seeds. Major activity was shown as one band which has 0.45 of Rf value on a native PAGE after activity staining. The activity located specifically in embryo from dry seed, but not in endosperm and scutelum. The enzyme may have an important role during early stage of germination, because of the activity disappeared until 48 hours though the seed will germinate after 72 hours after imbibition. The role of this enzyme is interested in induction of seed germination, because timing of development of the activity is concomitant with degradation of LEA proteins in the embryo.It was very hard to detect the activity stably in crude extract. Many effectors affected the activity in crude extract. For example, monovalent cation, especially, sodium ion, disappeared the activity dramatically. And, the activity increased significantly by treatment of EDTA.However, the activation may not due to the chelate metallo-ion. The effect of specific inhibitors for protease showed the enzyme is a trypsin-like serine-endopeptidase.Sedimentation by Ammonium sulfate from crude extract was effectively to have it's stabale activity. Purification of the enzyme was attempted by using ion-exchange chromatography, gel-filtration, hydrophobic chromatography, and native PAGE.Combination of these chromatography always gave the same 15-20 peptides which have 45-65 kD of molecular masses on SDS-PAGE.Affinity chromatography also gave the same pattern of peptides. These results indicate that the enzyme may make a complex with these peptides at least in vitro.

在玉米（Zea mays L dentcorn cv. W 64 A）干种子。主要活性显示为活性染色后在非变性PAGE上Rf值为0.45的一条带。这种活性只存在于干种子的胚中，而不存在于胚乳和盾片中。种子吸胀后72 h才萌发，但48 h后酶活性消失，说明该酶在种子萌发早期可能起重要作用。该酶的作用是诱导种子萌发，因为活性的发展时间是伴随着胚中莱亚蛋白的降解，在粗提物中很难稳定地检测该酶的活性。粗提物中有多种效应物影响其活性。例如，一价阳离子，特别是钠离子，使活性急剧消失。EDTA处理后酶活力明显提高，但这种激活作用可能不是由于螯合金属离子的作用。蛋白酶的特异性抑制剂的作用表明该酶是一种类似胰蛋白酶的丝氨酸内肽酶，粗提液经硫酸铵沉淀可有效地保持其稳定的活性。用离子交换层析、凝胶过滤、疏水层析和非变性聚丙烯酰胺凝胶电泳等方法对该酶进行了纯化，得到了15-20个分子量为45-65 kD的肽段，亲和层析也得到了相同的肽段。这些结果表明，至少在体外，酶可以与这些肽形成复合物。