Cloning and Subcellular Localization Analysis of Two F-box Genes from Poncirus trifoliata

A bioinformatics strategy is applied to clone a full length cDNA of TIR1 and Pt-F-box gene from citurs by blasting search of EST database with homologous gene cDNA of Arabidopsis thaliana and identified.According to the cDNA of above sequence,the 5′-end and 3′-end sequence were obtained from cDNA library of opening flower of Poncirus trifoliata(L.) Raf.using two gene specific primers by 5′RACE and 3′RACE methods,respectively.The full length cDNA of TIR1 and F-box gene from Poncirus trifoliata was spliced based on 5′-end and 3′-end sequence.The complete cDNA,designated as Pt-TIR1 and Pt-F-box,were 2 048,1 695 bp,respectively.These sequences were deposited in GenBank database,accession numbers FJ502240 and FJ502241,with an open reading frame encoding 569 and 468 amino acids,respectively.T Bioinformatics analysis showed that the cDNA of Pt-TIR1 and Pt-F-box have the recognition sites of microRNA393 and microRNA394.Pt-TIR1 and Pt-F-box with other plant F-boxes have the same amino acid sequences that are highly conserved as the designed F-box domains.Recombinant plasmid 35S-GW-FJ502240/FJ502241-GFP was introduced into onion epidermal cells by the particle bombardment method with a PDS1000/He.Transformed cells were incubated for 24 h at 22℃ in the dark and green fluorescence was monitored under a laser scanning confocal microscope.Subcellular localization results showed that the Pt-TIR1 and Pt-F-box were localized in the nucleus.