Molecular Cloning and Characterization of the GoMADS-box2 Gene from Guzmania Ruiz & Pav

Abstract

The MADS-box gene plays a crucial role in the formation of the floral meristem, development of floral organs and florescence regulation in plants. Based on a full-length cDNA library of floral organs and bracts in ostara , a Guzmania cultivar, four EST sequences from the same contig that included the MADS-box gene were obtained by large-scale random sequencing. Using the primer walking sequencing method, a 985 bp full-length cDNA sequence of the MADS-box gene was identified, which was named GoMADS-box2 gene (GenBank accession No.:JN936044). It has a 675 bp open reading frame that encodes 225 amino acid residuals, and its theoretical molecular weight and isoelectric point were predicted to be 26.3 kD and 9.36, respectively, by using a ProtParam analysis. The putative GoMADS-box2 protein contains a MIKC domain, which only exists in plant MADS-box proteins. The SPOMA program predicted that the secondary structure of the protein was composed of alpha helices (56.00%), random coils (24.44%), extended strands (13.78%) and beta turns (5.78%). Based on the 3D structure of the 1TQE P chain, a tertiary structure model was predicted by the ESyPred 3D program. Molecular phylogenetic tree analysis revealed that the cloned gene belongs to the AP3-type family of genes as well as B-class genes, which might control the development of the petals and stamens. Finally, real-time quantitative PCR analysis indicated that the GoMADS-box2 gene could be expressed in all of the tested tissues, and the amount expressed in the flower was much higher than that of other three tissues; while the expre ssion levels of the scape and leaf were nearly equal and the expression level in the bra ct was the lowest that was close to zero.