甜瓜乙烯信号转导途径关键因子基因CTR1的克隆及表达特性分析

在拟南芥中,CTR1在乙烯信号转导途径中发挥重要的负调控作用.目前的研究表明,在拟南芥中只存在一个CTR1基因.利用RT-PCR技术从甜瓜果实中克隆得到CTR1基因cDNA(Cm-CTR1).Cm-CTR1cDNA全长2 613 bp,编码870个氨基酸.氨基酸序列比对和同源性分析表明,所克隆的甜瓜CTR1基因与拟南芥CTR1基因相似度为53.69％.Cm-CTR1的C-末端具有明显的类似于哺乳动物和果蝇中的Raf( retro-viral protein,V-raf)蛋白激酶家族的丝/苏氨酸蛋白激酶(Serine/threonine protein kinase)的特征.该结构具有所有已知蛋白激酶所共有的11个亚结构域,其中包括ATP结合位点和丝/苏氨酸蛋白激酶位点结构域,说明CmCTR1与其他植物CTR1相似.实时荧光定量PCR分析结果表明,从授粉后第15天到第20天,甜瓜果实Cm-CTR1基因表达量显著增加,第20天的表达量是第15天的2.5倍,之后表达水平趋于稳定,第40天到第45天表达量有所下降.     

Biochemical and functional analysis of CTR1, a protein kinase that negatively regulates ethylene signaling in Arabidopsis

CTR1 encodes a negative regulator of the ethylene response pathway in Arabidopsis thaliana. The C-terminal domain of CTR1 is similar to the Raf family of protein kinases, but its first two-thirds encodes a novel protein domain. We used a variety of approaches to investigate the function of these two CTR1 domains. Recombinant CTR1 protein was purified from a baculoviral expression system, and shown to possess intrinsic Ser/Thr protein kinase activity with enzymatic properties similar to Raf-1. Deletion of the N-terminal domain did not elevate the kinase activity of CTR1, indicating that, at least in vitro, this domain does not autoinhibit kinase function. Molecular analysis of loss-of-function ctr1 alleles indicated that several mutations disrupt the kinase catalytic domain, and in vitro studies confirmed that at least one of these eliminates kinase activity, which indicates that kinase activity is required for CTR1 function. One missense mutation, ctr1-8, was found to result from an amino acid substitution within a new conserved motif within the N-terminal domain. Ctr1-8 has no detectable effect on the kinase activity of CTR1 in vitro, but rather disrupts the interaction with the ethylene receptor ETR1. This mutation also disrupts the dominant negative effect that results from overexpression of the CTR1 amino-terminal domain in transgenic Arabidopsis. These results suggest that CTR1 interacts with ETR1 in vivo, and that this association is required to turn off the ethylene-signaling pathway.