| SBP (SQUAMOSA PROMOTER BINDING PROTEIN) family is a transcription factor family only exists in plant kingdom. AmSBPl and AmSBP2 of Antirrhinum majus are the first members of SBP family discovered in plants, and they were named after their in vitro binding activity with the promoter of SQUAMOSA. All the SBP proteins contain a highly conserved SBP-domain constituted by approximately 76 aminoacid residues and responsible for the interaction with the cis-regulatory elements located upstream of target genes. In general, the coding sequences of the SBP domains are known as SBP box and the genes encoding the SBP transcriptional factors are termed as SBP-box genes. Previous researches showed that SBP transcriptional factors play important roles in many aspects of plant growth and development.In the present work, the genomic DNA sequences of a number of SBP-box genes were identified from the tobacco databases maintained in Sol Genomics Network (http://solgenomics.net/) by BLAST search, and the Gene Scan Web Server (http://genes.mit.edu/GENSCAN.html) was used to predict the intron-exon structures. By RT-PCR (reverse transcription-polymerase chain reaction) method, the coding sequences of 15 SBP-box genes were cloned from tobacco genotype Qinyan95 and were authenticated by sequencing. In order to accurately reflect the potential functions, the SBP-box genes isolated from tobacco were named according to the orthologs of Arabidopsis and tomato uniformly, and NtabSPL was adopted as the basic name of these genes. In fifteen NtabSPL genes,13 possess a 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, N. sylvestris, N. tomentosiformis and N. otophora. The results showed that 6 NtabSPL genes (including NtabSPL2, NtabSPL3a, NtabSPL6-1, NtabSPL6-2, NtabSPL6-3 and NtabSPL9a) were derived from a progenitor of N. sylvestris and 9 NtabSPL genes (including NtabSPLLb, NtabSPL6-4, NtabSPL6-5, NtabSPL6-6, NtabSPL6-7, NtabSPL7, NtabSPL8, NtabSPL9b and NtabSPL13) were derived from a progenitor of N. tomentosiformis, indicating N. tabacum is an allotetraploid came from 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.To gain further insight into the evolutionary relationship of SPL genes among tobacco, tomato and Arabidopsis, phylogenetic analysis was carried out based on the aminoacid sequences of the highly conserved SBP domain using UPGMA (Unweighted Pair-Group Method with Arithmetic means) algorithm. The resultant phylogenetic tree reveals that the SPL proteins of tobacco, tomato and Arabidopsis can be categorized into eight subfamilies, and the SPL genes in the same group bear a similar structure, including the number of exons, the size of the genomic sequences and the length of the open reading frame. It is worth noting that N. tabacum contains 7 NtabSPL6 genes originated from two parental genomes and NtabSPL6-2 contains a special GC-AG intron.In addition, the sequences of Arabidopsis Pri-miRl56a was amplified from genomic DNA of Col-0 ecotype. After restriction digestion, the PCR fragment was ligated into pRI 101-AN to produce a plant expression vector. Subsequently, the recombinant plasmid was introduced into Agrobacterium tumefaciens strain LBA4404 and transgenic tobacco plants were regenerated via leaf disc transformation method based on tissue culture and resistance screening. Compared to wild-type tobacco plants, the transgenic tobacco plants habouring the Arabidopsis Pri-miRl56a showed a number of changed phenotypic characteristics, including dwarfism in size, delay in floral transition and increases in leaf number and biomass accumulation. The transcript abundance of NtabSPL3 was reduced remarkably in transgenic tobacco plants containing Arabidopsis Pri-miRl56a in comparison with the wild-type tobacco plants, suggesting overexpression of Pri-miR156a in tobacco could downregulate the expression level of NtabSPL3a, repress the transition from vegetative growth to reproductive growth, and postpone the flowering induction process. Meanwhile, constitutive expression of miR156 could inhibit the activities of seven NtabSPL6 genes obviously, suggesting the function of miR 156is conservative in tobacco and Arabidopsis. |
