Cloning And Characterization Of ZmTFL1 Gene Related To Photoperiod Sensitivity In Maize

Tropical and sub-tropical germplasms contain abundant hereitary variation ,which could broaden the temperate germplasm of maize ,when they planted in temperated zone. However, photoperiod sensitivity is the main restriction of its use in temperate zone.So,clone and analyse the fuction of photoperiod-sensitive genes is an important way to research the molecular mechanism of maize photoperiod-sensitivity. We cloned the ZmTFL1 gene by the method of orthologus gene cloning in a photoperiod-sensitive tropical maize (Zea mays L.) inbred line CML288 and a photoperiod-insensitive temperate inbred line HuangZao4,which was the cognate of TFL1 gene(Arabidopsis) and FDR1/DR2 gene(rice).Then we studied the expression pattern of ZmTFL1 in shoot apical meristems (SAM) and leaves under long day and short day conditions in CML288 and HuangZao4,and researched the relationship between ZmTFL1 ,ZCN8,DLF1 and ZAP1,in order to investigate the function and mechanism of ZmTFL1 during the floral transition.We constructed the overexpression vector PBI- ZmTFL1 ,then transformed it into the Arabidopsis mutant tfl1-14 with Floral Dip method,and observed the phenotype of transformant plant,analysed the expressive connection of relatived genes had in transformant and tfl1-14.The main results were as follows.1.We had cloned the maize ZmTFL1 gene by orthologus gene cloning ,which GeneBank accession number was DQ925416. ZmTFL1 gene sequence has four exons and three introns,and contained a predicated 522bp open reading frame in the CDS sequence, which could encode a peptide with 173 amino acids. By phylogenetic analysis, we found the protein sequence shared 91% homology to rice FDR1/FDR2,and 85% and 83% homology to barley HvTFL1 and ryegress LpTFL1,respectively ,but only 70% homology to Arabidopsis TFL1. The predicated protein contained a PEBP domain, especially had the specific His88 and Asp144 residues of TFL1 sub-family.So , ZmTFL1 gene belonged to the member of maize PEBPgene family.2.We used fluorescence quantitative RT-PCR to research the expression pattern of ZmTFL1 in shoot apical meristem (SAM) and leaf under long day and short day conditions in CML288 and HuangZao4, and the relationship between ZmTFL1 ,ZCN8,DLF1 and ZAP1during the flowering switch.The results showed that ZmTFL1 gene mainly expressed in the SAM,but had faint expression in leaf ,implying ZmTFL1 gene played role in the SAM.During the floral development, ZmTFL1 gene was a repressor,and ZCN8 was a promoter.They competed for combining with DLF1,to inhibit or activate the expression of floral meristem gene ZAP1,and regulated the process of flowering.3.We have constructed the overexpression vector PBI- ZmTFL1,and successfully transformed the mutant tfl1-14. Three transformants were obtained.Compared to the mutant tfl1-14,two transfomants flowered 11d later, added about 5 leaves,but one transfomant maintained the vegetation growth untill senescence.All the transformants had indefinite inflorescence,more density trichome and enhanced height.The research on expression pattern suggested that, during the switch to flower,the faint expression of FT could enough to activate AP1 in the mutant ,but in the transformants,due to the ZmTFL1 gene could remedy the loss function of TFL1,the higher expression of FT was needed to upregulate the expression of AP1.So,ZmTFL1 gene could repress flowering,and had similar role to Arabidopsis TFL1,it's the TFL1-like gene in maize.