Characterization and expression analysis of WOX5 genes from wheat and its relatives

Abstract

The WUSCHEL (WUS)-related homeobox (WOX) gene family plays an important role in coordinating gene transcription in the early phases of embryogenesis. In this study, we isolated and characterized WOX5 from common wheat and its relatives Triticum monococcum, Triticum urartu, Aegilops speltoides, Aegilops searsii, Aegilops sharonensis, Aegilops longissima, Aegilops bicornis, Aegilops tauschii, and Triticum turgidum. The size of the characterized WOX5 alleles ranged from 1029 to 1038 bp and encompassed the complete open reading frame (ORF) as well as 5' upstream and 3' downstream sequences. Domain prediction analysis showed that the putative primary structures of wheat WOX5 protein include the highly conserved homeodomain besides the WUS-box domain and the EAR-like domain, which is/are present in some members of the WOX protein family. The full-length ORF was subcloned into a prokaryotic expression vector pET30a, and an approximate 26-kDa protein was successfully expressed in Escherichia coli BL21 (DE3) cells with IPTG induction. The WOX5 genes from wheat-related species exhibit a similar structure to and high sequence similarity with WOX5 genes from common wheat. The degree of divergence and phylogenetic tree analysis among WOX5 alleles suggested the existence of three homoeologous copies in the A, B, or D genome of common wheat. Quantitative PCR results showed that TaWOX5 was primarily expressed in the root and calli induced by auxin and cytokinin, indicating that TaWOX5 may play a role related to root formation or development and is associated with hormone regulation in somatic embryogenesis.

Keywords: 1-naphthoxyacetic acid; 2,4-D; 2,4-dichlorophenoxyacetic acid; 6-BA; 6-benzyl-adenine; AP; BLAST; CDA; CIA; CTAB; EAR; EC; ERF-associated amphiphilic repression; Expression; HD; IAA; IPTG; MS; NAA; NEC; NJ; ORF; PAGE; Phylogenetic analysis; QHB; RAM; RT-qPCR; Real-time quantitative PCR; SAM; SDS; SNPs; TAIL-PCR; USDA-ARS; UTR; United States Department of Agriculture—Agricultural Research Service; WOX; WOX5; WUS; WUS-related homeobox; Wheat; Wuschel; adaptor primer; basic local alignment search tool; callus in differentiation medium after days; callus in induced medium after days; cetyltrimethyl ammonium bromide; embryogenic callus; homeodomain; indole-3-acetic acid; isopropyl β-d-thiogalactopyranoside; murashige and skoog medium; neighbor-joining; non-embryogenic callus; open reading frame; polyacrylamide gel electrophoresis; quiescent center-specific homeobox; real-time quantitative PCR; root apical meristem; shoot apical meristem; single nucleotide polymorphism; sodium dodecyl sulfate; thermal asymmetric interlaced PCR; untranslated transcribed region.