Cloning and Subcellular Localization Analysis of A New Gene NAC cDNA in Poncirus trifoliata

Taking the NAC1 cDNA sequence in Arabidopsis thaliana as the template,the homologous gene in the EST database of citrus was searched and screened,and the cDNA sequence of NAC1 gene in citrus was cloned by bioinformatics method.Taking the cDNA sequence in the flower of Poncirus trifoliata(L.) Raf.as the template,the specific primers were designed according to the above cDNA sequence,and the 5′-end and 3′-end sequences of NAC1 gene were obtained by using 5′RACE and 3′ RACE techniques,respectively.Based on these,the corresponding full length cDNA of NAC1 in Poncirus trifoliata was acquired through sequence splicing.This complete cDNA,designated as Pt-NAC1,was 1351 bp in length,it contained a 1047 bp whole open reading frame(ORF),its 5′-end included a putative translation start codon(ATG) at the position of 25 bp,and the length of its 3′-end untranslated region was 280 bp.The deduced amino acid sequence of Pt-NAC1 was 348 residues,which showed 64.8%,57.0%,61.3% identical levels with that of Malus×domestica,Arabidopsis thaliana,Petunia×hybrida,respectively.Bioinformatics analysis showed that the cDNA of Pt-NAC1 had the recognition site of microRNA164,and it also had highly conserved NAC domains.Recombinant plasmid 35S-GW-GFP-FJ619349 was transferred into onion's epidermal cells by using the particle bombardment method,and the subcellular localization analysis results indicated that Pt-NAC1 was all localized at the cell membrane.