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Molecular Mechanisms Underlying Differentiation of Microbodies.

微生物分化的分子机制。

基本信息

批准号：
04454017
负责人：
NISHIMURA Mikio
金额：
$ 4.48万
依托单位：
National Institute for Basic Biology
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

项目摘要

A cDNA clone for a leaf-peroxisomal enzyme, glycolate oxidase (E C 1.1.3.1), was isolated by an immunochemical method from a cDNA expression library constructed from poly (A)^+ -RNA of green pumpkin cotyledons. Northern blot analysis showed that the probe could hybridizable with a single 1.5-kb species of mRNA from pumpkin cotyledons and that the amount of the hybridizable mRNA increased dramatically during greening of the cotyledons, Immunoblot analysis showed that the amount of glycolate oxidase was low in darkgrown cotyledons and increased during greening of pumpkin cotyledons. This observation indicates that the induction of a leaf peroxisomal enzyme, glycolate oxidase, during greening of the cotyledons is due to an increase in level of the mRNA.After the function transition of glyoxysomes to leaf peroxisomes during the greening of pumpkin cotyledons, the reverse microbody transition of leaf peroxisomes to glyoxysomes occurs during senescence. Immunocytochemical labeling with protein A-gold was performed to analyze the reverse microbody transition using antibodies against a leaf-peroxisomal enzyme, glycolate oxidase, and ageinst two gloxysomal enzymes, namely, malate synthase and isocitrate lyase. The intensity of labeling for glycolate oxidase decreased in the microbodies during senescence whereas in the case of malate synthase and isocitrate lyase intensities increase strikingly. Double labeling experments with protein A-gold particles of different sizes showed that the leaf-peroxisomal enzymes and the glyoxysomal enzymes coexist in the microbodies of senescing pumpkin cotyledons, indicating that leaf peroxisomal are directly transformed to glyoxysomes during, senescence.

用免疫化学方法从绿色南瓜子叶聚(A)~+-RNA构建的表达文库中克隆了一个叶片过氧化物酶--乙醇酸氧化酶(EC 1.1.3.1)的基因克隆。Northern印迹分析表明，该探针可与单一1.5kb的南瓜子叶基因片段杂交，杂交产物的量在子叶变绿过程中显著增加；免疫印迹分析表明，乙醇酸氧化酶在暗培养的子叶中含量较低，而在南瓜子叶变绿过程中含量增加。这一观察结果表明，在南瓜子叶变绿过程中，叶片过氧化物体酶--乙醇酸氧化酶的诱导是由于mRNA量的增加所致。在南瓜子叶变绿过程中，糖氧体向叶片过氧化物体的功能转变后，叶片过氧化物体向乙醇体的反向微体转变发生在衰老过程中。用蛋白A-金免疫细胞化学标记技术，利用抗叶过氧化物酶、乙醇酸氧化酶和年龄两种谷体酶(苹果酸合成酶和异柠檬酸裂解酶)的抗体来分析反向微体转变。在衰老过程中，微体中乙醇酸氧化酶的标记强度降低，而苹果酸合成酶和异柠檬酸裂解酶的标记强度显着增加。不同大小的蛋白A-金双标实验表明，衰老南瓜子叶的微体中同时存在叶片过氧化物酶和乙氧基体酶，表明叶片中的过氧化物体在衰老过程中直接转化为乙氧基体。