Molecular Cloning And Function Identification Of LEAFY Homologue Gene From Dendranthema Lavandulifolium

LFY plays important roles in floral development. As a floral meristem identity gene, LFY not only control the transition from vegetative to inflorescence and floral meristem in plants, its overexpression can induce the plant to flower early. LFY also functions as upstream regulator of floral organs identity genes and is a key intergrator of the outputs of floral inductive pathways to induce floral meristem transformation.Using 5'RACE and 3'RACE, we have obtained the LFY homologue in the Dendranthema lavandulifolium(Fisch.ex Tranutv). The completed cDNA sequence is. 1435bp and includes a 1239bp open reading frame that encodes 412 amino acids and a terminating codon (GenBank accession number: AY559245, named as DFL). The DFL protein aligns well with the sequences of other lfy-like proteins. It shares with other angiosperm LFY proteins special regions such as an amino-terminal Pro- rich region , an acidic domain, a short putative Leu-zipper and a basic region formed by a core of Arg and Lys residues. Some motif indicates their possible role as transcriptional activators. The DFL protein is 63% identical to LFY and 70% identical to FLO.DFL contains two introns of 583bp and 1091bp. The number and position are both conserved with those described in other species. Genomic DNA Southern hybridization analysis suggest the existence of a single DFL gene in Dendranthema lavandulifolium genome.To determine the spacial-temporal expression pattern of DFL in D. lavandulifolium development, RT-PCR and In situ hybridization techniques were applied. RT-PCR experiment results show that DFL is expressed at low levels in the leaf and young stem, but strongly expressed in flower bud, there is no expression in roots. In situ hybridization were carried out to localize the DFL transcripts during flower bud development. Expression of DFL was detected in meristematic tissues throughout the developmemt. ThemRNA levels detected varied in different stages. Accumulation of DFL transcripts in sepal primordia, apex of whole sepal formed and in stamen primordia. It is also expressed in mature leaf primordia and in developing leaves.A full-length cDNA of DFL and its antisense nucleotide sequence have been transformed into Nicotiana tabacum under the control of a cauliflower mosaic virus 35S promoter. The transgenic tobacoo was identified by molecular biology method such as antibiotics PCR and by histochemical staining of GUS. The success of transform into tobacoo established the foundation to transform into D. morifolium (Ramat.) Tzvel).