綠竹筍乙烯受體 BoERS1 結構與功能之研究

Abstract

An ethylene receptor gene named BoERS1 was cloned from a bamboo (Bambusa oldhamii) cDNA library. The open reading frame of BoERS1 was 1899 bp which encoded a 632-amino acid polypeptide. The encoded BoERS1 contained three domains, a sensor domain with three transmembrane regions, a GAF domain and a histidine kinase domain that contained all the conserved motifs (H, N, G1, F, and G2) that are present in the histidine kinases of the bacterial two-component systems and shared 90% sequence similarity with other ethylene receptors in plants such as rice or maize. According to real-time PCR analysis, the levels of BoERS1 mRNA in the shoots of field-grown bamboo were elevated along with the growth of the emerging shoots, especially in internodes and shoot meristems. Furthermore, the expression levels of BoERS1 were decreased under benzyladenine (BA, a cytokinin) and ABA treatments in multiple shoots of bamboo. The upstream sequences of BoERS1 were obtained using TAIL-PCR (thermal asymmetric interlaced polymerase chain reaction) and some cis-acting elements related to phytohormones, light, and dehydration were found. With in vitro kinase assay, the recombinant histidine kinase domain of BoERS1 (Ala 331 to Gly 611, BHK) showed autophosphorylation activity in the presence of Mn2+ and Mg2+, but not in the presence of Ca2+ and H2O. LC-ESI-MS/MS analysis indicated that four amino acid residues of BHK, namely Thr 442, Ser 444, Ser 489 and Ser 503, were phosphorylated by an in vitro kinase assay. Site-directed mutagenesis of these amino acids did not affect the phosphorylation activity of BHK. It indicated BHK was multiphosphorylated or had other phosphorylation residues. The model of BHK was built according to the structure of AtETR1 (PDB ID: 4PL9) and a histidine kinase of Thermotoga maritima (PDB ID: 2C2A). The three dimensional model of BHK had two flexible loops, namely L1 and L2. It is interesting to note that Ser 489 and Ser 503 were located in the both ends of L1 which was unique to the plant histidine kinase-containing enzymes and the phosphorylation may regulate the interactions between BoERS1 and other proteins; meanwhile L2 may be a gatekeeper of ATP binding pocket and regulate the entry of ATP. The identification of multiple phosphorylation sites on BoERS1 provides a new avenue for future structure–function studies of the ethylene receptor protein family.