Characterization and phylogenetic analysis of fifteen NtabSPL genes in Nicotiana tabacum L. cv. Qinyan95

Fifteen SPL (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE) genes were identified and characterized in Nicotiana tabacum L. cv. Qinyan95. The exon-intron structures of these genes were determined according to the coding sequences confirmed by RT-PCR and the genomic DNA sequences downloaded from the databases in Sol Genomics Network, and thirteen of them were found to carry the response element of miR156. To elucidate the origin of the validated NtabSPL genes, multiple alignments of the nucleotide sequences encompassing the open reading frames were conducted by using the orthologs in N. tabacum, Nicotiana sylvestris, Nicotiana tomentosiformis, and Nicotiana otophora. The results showed that six NtabSPL genes were derived from a progenitor of N. sylvestris, and nine NtabSPL genes were derived from a progenitor of N. tomentosiformis, further corroborating that N. tabacum came from the interspecific hybridization between the ancestors of N. sylvestris and N. tomentosiformis. In contrast to previous statements about highly repetitive sequences, the genome of N. tabacum mainly retained the paternal-derived SPL genes in diploidization process. Phylogenetic analyses based on the highly conserved SBP (SQUAMOSA PROMOTER BINDING PROTEIN) domains and the full-length amino acid sequences reveal that the SPL proteins of tobacco, tomato, and Arabidopsis can be categorized into eight groups. It is worth noting that N. tabacum contains seven NtabSPL6 genes originated from two parental genomes and NtabSPL6-2 possesses a GC-AG intron. In addition, transgenic tobacco plants harboring Arabidopsis Pri-miR156A were generated by Agrobacterium-mediated transformation method, and the constitutive expression of miR156 could obviously inhibit the activity of the NtabSPL genes containing its target site, suggesting the function of miR156 is conservative in tobacco and Arabidopsis.