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Functional Analysis of Arabidopsis CDKA during plant development

拟南芥CDKA在植物发育过程中的功能分析

基本信息

批准号：
18570046
负责人：
SEKINE Masami
金额：
$ 2.57万
依托单位：
Ishikawa Prefectural University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18570046/
关键词：
cell cycle Arabidopsis cyclin gametogenesis plant development 分子生物学

项目摘要

The protein kinase cell division cycle 2 (cdc2) is conserved throughout eukaryotes, and acts as a key regulator of the cell cycle. In plants, A-type cyclin-dependent kinase (CDKA), a homologue of cdc2, has a role throughout the cell cycle. In this study, we show that a loss-of-function mutation in CDKA;1, encoding the only Arabidopsis CDKA, results in male gametophyte lethality. Microspores normally undergo an asymmetric cell division, pollen mitosis I (PMI), to produce bicellular pollen grains. The smaller generative cell undergoes subsequently second mitosis, PMII, to form the two sperm cells, thereby generating tricellular pollen grains. The cdka-1 mutant, however, produces mature bicellular pollen grains, consisting of a single sperm-like cell and a vegetative cell, due to failure of PMII. Thus, CDKA; 1 is essential for cell division of the generative cell in male gametogenesis.In addition, phosphorylation of threonine 161 (T161) in CDKA is required for the activation of its associ … More ated kinase. Western blot analysis revealed that phosphorylation of CDKA T161 increased greatly, in parallel with activation of p13^suct-associated kinase activity when cells were subcultured. Although induced overexpression of a dominant-negative CDKA mutant (D146N) fused with green fluorescent protein (GFP) in tobacco BY-2 cells resulted in elongated cells, overexpression of this CDKA mutant possessing a non-phosphorylatable alanine in place of T161 (T161A) had no effect on cellular growth. However, immunoprecipitates of both GFP-fused CDKAs exhibited no histone H1 kinase activity, suggesting that both mutants formed kinase-inactive complexes. To further elucidate the role of T161 phosphorylation, we used a loss-of-function mutation of Arabidopsis CDKA; 1. Introduction of CDKA; 1(T161E)-GFP, which mimics phosphorylated T161, resulted in successful complementation of the cdka-1 mutation, whereas no recovery was observed when CDKA; 1(T161A)-GFP was introduced. Thus, phosphorylation of T161 in Arabidopsis CDKA;1 is essential for cell division during male gametogenesis. Less

蛋白激酶细胞分裂周期2（cdc 2）在整个真核生物中是保守的，并作为细胞周期的关键调节因子。在植物中，A型细胞周期蛋白依赖性激酶（CDKA），cdc 2的同源物，在整个细胞周期中发挥作用。在这项研究中，我们表明，在CDKA的功能丧失突变;1，编码唯一的拟南芥CDKA，导致雄性配子体致死。小孢子通常经历不对称的细胞分裂，即花粉有丝分裂I（PMI），产生双细胞花粉粒。较小的生殖细胞随后进行第二次有丝分裂，PMII，形成两个精细胞，从而产生三细胞花粉粒。cdka-1突变体，然而，产生成熟的双细胞花粉粒，由一个单一的精子样细胞和营养细胞，由于失败的PMII。因此，CDKA; 1是雄性配子发生中生殖细胞分裂所必需的，另外，CDKA中的苏氨酸161（T161）的磷酸化是激活其相关的细胞分裂所必需的。 ...更多信息磷酸化激酶Western印迹分析显示，细胞传代培养后，CDKA T161磷酸化水平显著增加，同时p13-suck相关激酶活性也被激活。虽然在烟草BY-2细胞中诱导与绿色荧光蛋白（GFP）融合的显性负性CDKA突变体（D146 N）的过表达导致伸长的细胞，但是具有不可磷酸化的丙氨酸代替T161（T161 A）的这种CDKA突变体的过表达对细胞生长没有影响。然而，免疫沉淀的两个GFP融合的CDKA没有表现出组蛋白H1激酶活性，这表明这两个突变体形成激酶失活复合物。为了进一步阐明T161磷酸化的作用，我们使用了拟南芥CDKA的功能缺失突变; 1. CDKA介绍;模拟磷酸化T161的1（T161 E）-GFP导致cdka-1突变的成功互补，而当引入CDKA; 1（T161 A）-GFP时没有观察到恢复。因此，在拟南芥中的T161的磷酸化CDKA;1是必不可少的雄性配子发生过程中的细胞分裂。少