Characterization of ScMat1, a putative TFIIH subunit from sugarcane
Characterization of ScMat1, a putative TFIIH subunit from sugarcane
复制标题
DOI：
10.1007/s00299-008-0663-1
复制
发表时间：
2009-04-01
影响因子：
6.2
通讯作者：
Menossi, Marcelo
中科院分区：
文献类型：
--
作者：
Gentile, Agustina;Ditt, Renata F.;Menossi, Marcelo
通用转录因子TFIIH是具有解旋酶、蛋白激酶和DNA修复等不同酶活性的多蛋白复合物。MAT1 （m,nage A trois 1）是具有激酶活性的TFIIH亚基之一，它是周期蛋白依赖性激酶（CDK）激活激酶（CAK）， CDK7-周期蛋白h的第三个亚基。这项工作的主要目的是表征ScMAT1，一个编码MAT1同源的甘蔗基因。Northern blots和原位杂交结果表明，ScMAT1在甘蔗成熟叶片、叶卷和花序中均有表达，在芽和根等其他组织中均无差异表达。此外，ScMAT1在不同应激条件和激素处理下的表达没有差异。原位杂交分析还表明，ScMAT1在叶片发育过程中不同细胞类型中均有表达。为了鉴定与ScMAT1相互作用的蛋白，以ScMAT1为诱饵，采用酵母双杂交法筛选甘蔗叶片cDNA文库。酵母两种杂交种的筛选得到14个阳性克隆。其中之一是参与有毒化合物氧化降解的细胞色素p450家族蛋白。其他被分离的无性系也与植物对胁迫的反应有关。为了确定ScMAT1的亚细胞定位，我们在洋葱表皮细胞中检测了ScMAT1- gfp融合，荧光定位于细胞核，这与ScMAT1作为基础转录因子的假设作用一致。
The general transcription factor TFIIH is a multiprotein complex with different enzymatic activities such as helicase, protein kinase and DNA repair. MAT1 (m,nage A trois 1) is one of the TFIIH subunits that has kinase activity and it is the third subunit of the cyclin-dependent kinase (CDK)-activating kinase (CAK), CDK7- cyclin H. The main objective of this work was to characterize ScMAT1, a sugarcane gene encoding a MAT1 homolog. Northern blots and in situ hybridization results showed that ScMAT1 was expressed in sugarcane mature leaf, leaf roll and inflorescence, and it was not differentially expressed in any of the other tissues analyzed such us bud and roots. In addition, ScMAT1 was not differentially expressed during different stress conditions and treatment with hormones. In situ hybridization analyses also showed that ScMAT1 was expressed in different cell types during leaf development. In order to identify proteins that interact with ScMAT1, a yeast two hybrid assay with ScMAT1 as bait was used to screen a sugarcane leaf cDNA library. The screening of yeast two hybrids yielded 14 positive clones. One of them is a cytochrome p450 family protein involved in oxidative degradation of toxic compounds. Other clones isolated are also related to plant responses to stress. To determine the subcellular localization of ScMAT1, a ScMAT1-GFP fusion was assayed in onion epidermal cell and the fluorescence was localized to the nucleus, in agreement with the putative role of ScMAT1 as a basal transcription factor.