Cloning and expression analysis of APETALA2 gene from Poncirus trifoliata based on EST database.

Based on the relative conservation of plant homologous genes,a full-length citrus homologue of APETALA2 was bioinformatically cloned by search of citrus EST database via Arabidopsis thaliana corresponding sequence. Accordingly,the 5'-and 3'-end sequences were obtained from cDNA of opening flower of Poncirus trifoliata (L.) Raf. by RACE with two gene-specific primers designed on the basis of the citrus sequence. The 1 980 bp complete cDNA,designated as Pt-AP2,contained an open reading frame (ORF) of 1 539 nucleotides and 289 bp of 5'-untranslated region (UTR) and a 152 bp 3'-UTR. The sequence has been deposited in GenBank database with the accession number of EU883665. The deduced amino acid sequence of Pt-AP2 (512 residues) showed 59.1%,59.7%,63.8% identity with those of Malus domestica,Arabidopsis thaliana,Petunia hybrida,respectively. Pt-AP2 amino acid sequence contained a putative nuclear localization signal sequence (KKSR) and two highly conserved AP2 domains. The semi-quantitative RT-PCR and SYBR Green I Real-time RT-PCR were employed to analyze the expression of Pt-AP2 in different organs,revealing similar expression profiles in leaf,stem,root,flower,fruit,in which the flower and fruit exhibited the highest and the lowest expression,respectively.