烟草乙烯受体类似物NTHK2全长基因的克隆及其激酶结构域生化特性

应用5＇-RACE方法克隆到烟草NTHK2的全长cDNA.其全长cDNA共有3 216bp,其中5'非编码区为509bp,3＇非编码区为427bp,编码区为2 280bp,编码产物为760个氨基酸.NTHK2氨基酸序列与植物中的许多杂合型的两组分乙烯受体基因有较高的同源性,具有推测的组氨酸激酶结构域和接受域;但是,在激酶结构域中没有保守的组氨酸,而是被一个天冬氨酸残基所替代.为了研究其生化特性,在酵母中以融合蛋白的形式表达了激酶结构域.体外激酶分析表明,当有Mg2+存在的情况下NTHK2能够自我磷酸化.进一步的研究应阐明NTHK2在植物体内是否能够作为乙烯受体,参与乙烯的信号传导过程.

Cloning of the Full-length Gene for Tobacco Ethylene Receptor NTHK2 and Characterization of Its Kinase Domain

Previously the partial sequence of an ethylene receptor gene NTHK2 was isolated from tobacco (Nicotiana tabacumL. Var. Xanthi) plants and it was wound and drought inducible. In the present study full_length cDNA of NTHK2was cloned by 5′_RACE method.NTHK2 gene has 3 216 bp, with 509 bp of 5′_non_coding region and 427 bp of 3′_non_coding region, and encodes an ethylene_receptor homolog of 760 amino acids. NTHK2 protein has a putative signal peptide, three transmembrane domains, a histidine kinase domain and a receiver domain. In the putative histidine kinase domain, the histidine at the phosphorylation site was replaced by an asparagine. To study the biochemical property of NTHK2, its kinase domain was expressed as a fusion protein with glutathione S_transferase (GST) using yeast Schizosaccharomyces pombe as an expression system. In vitro kinase assay showed that NTHK2 kinase domain can autophosphorylate in the presence of Mg2+, indicating that NTHK2 may function as a kinase. Further studies will elucidate the function of NTHK2 in plant.