cDNA Cloning, Sequence Analysis and Tissue Expression Detection of APETALA1 Gene (AP1) in Paeonia lactiflora Pall. Petals of Different Development Stages
Abstract：In order to understand the biological function of APETALA1 gene (AP1) in Paeonia lactiflora Pall., the present study designed primer based on conserved domain of higher plant AP1 and RACE (rapid-amplification of cDNA ends) cloned AP1 (GenBank No. KC354376) from Paeonia lactiflora. The molecular structure and function of AP1 protein were analyzed and predicted by using bioinformatics. Moreover, the differential expression of AP1 gene in the inner and outer petals among different development stages was systematically analyzed. Primers were designed based on the conserved sequence of higher plants AP1 gene and were used in amplifying the Paeonia lactiflora AP1 by RACE technology. Sequencing assays showed that the full-length cDNA of AP1 gene consisted of 1 236 bp and contained a 726 bp ORF encoding 242 amino acids. Bioinformatics analysis demonstrated that AP1 protein was unstable and hydrophilic, meanwhile no signal peptide and transmembrane structure indicating AP1 protein of non-secretory. α-helices and random coils were its main elements of the secondary structure of AP1 protein. AP1 protein had one N-glycosylation site located in 89th amino acid, one O-glycosylation site located in 206th amino acid, 13 potential phosphorylation sites including 8 conservative binding sites of specific protein kinase such as protein kinase C(PKC), epidermal growth factor receptor (EGFR), casein kinaseⅡ(CKⅡ), DNA-protein kinase (DNAPK), protein kinase A (PKA), protein kinase G (PKG), ataxia-telangiectasia mutated protein (ATM), p38-mitogen activated protein kinases (p38MAPK). AP1 protein had 2 super family conservative domains of MADS and K-box, which located in the region 2nd ~74th and 89th~173rd amino acid, respectively. Gene expression profile showed the expression level of AP1 gene was different in the inner and outer petals from a flower color chimaera cultivar Jinhui among 4 developmental stages (flower-bud stage, initiating bloom stage, bloom stage and wither stage). Especially, AP1 expressed very significantly different between the inner and outer petals in wither stage (P＜0.01), which furtherly indicated that the expression level of AP1 gene might be related to Paeonia lactiflora flower organ development. This study successfully obtained the full-length cDNA of AP1 gene and revealed the physicochemical property, structure and potential function of AP1 protein, which lays a foundation for further studying on the function of Paeonia lactiflora AP1.
