Cloning, Expression And Biological Function Analysis Of Tall Fescue FaFT Gene

As one of the key pasture and turfgrass seeds,tall fescue features high value of economic development.However,it requires long-day(LD)photoperiod conditions to differentiate flowers and buds,and then produce seeds.Currently its seeds are mainly import from Europe and the US with long exposure to sunshine,whereas China cannot produce them efficiently.Therefore,we further study on the roles of photoperiod-dependent floral reversion and flower bud differentiation of tall fascue,which will have a great significance to promote localized seed production.FLOWERING LOCUS T(FT)and FLOWERING LOCUS D(FD)genes play the important roles during flowering.In this study,the full-length c DNA sequence of FaFT gene was successfully obtained from ‘Qian Cao NO.1' by homologous cloning,and was further analyzed by bioinformatics and phylogenetic tree.In addition,the expression characteristics of FaFT and Fa FD also were exhibited by real-time PCR,yeast two hybrid system,bimolecular fluorescence complementation and transgenic Arabidopsis.The main research results are as foll-ows:1.The FaFT genome sequence was 5045 bp,G+C was 58.33%,containing 2 introns and 3 exons.The full length c DNA sequence of FaFT was 1077 bp,with an open reading frame of 537 bp,ranging from 154 bp to 689 bp,and encoding 178 amino acid residues.FaFT protein was a hydrophilic protein,which contains abundant secondary structures such as alpha helix and random coils,and has a conserved domain PEBP.The results of multiple comparisons showed that the homology between tall fescue FaFT and ryegrass was the highest,which was up to 98.84%,followed by Brachypodium distachyon was 97.73%.The phylogenetic tree results showed that the tall fescue FaFT and the ryegrass Lp FT were in the same branch,and the evolutionary relationships between them were close.2.The expression of FaFT gene is tissue-specific,which can maintain a certain circadian rhythm and is regulated by photoperiod.No matter long-day or short-day,its expression level was the highest in leaves,followed by stems and roots;The expression level of FaFT under LD was higher than that under SD,but the expression peak both appeared at ZT8;When changing long day to continuous light and continuous darkness,the peak of expression appeared at ZT0,while the peak of expresison from short day to continuous light and continuous dark appeared at ZT4 and ZT12,respectively;the peak of expression appeared during ZT4?ZT16 in the light/dark cycle mode.3.The fusion expression vector 1300-GFP-FT of tall fescue FaFT gene and green fluorescent protein was constructed to transform tobacco,and the transient expression of the fusion vector was detected under a laser confocal microscope.The results showed that the protein encoded by the tall fescue FaFT gene was localized in the nucleus.The results of yeast two-hybrid system and two-molecule fluorescence complementation confirmed the interaction between FaFT and Fa FD.4.Agrobacterium-mediated transformation of Arabidopsis thaliana found that: under LD,the transgenic Arabidopsis thaliana overexpressing the FaFT gene showed early flowering,which was about 4 days earlier than the wild type and about 3 days earlier than the mutant.After functional compensation of the mutant atft,the wild-type flowering phenotype was restored,but the atft had no flowering phenomenon and remained in the vegetative growth period;the number of rosette leaves in Arabidopsis thaliana overexpressing the FaFT gene were more than those of the wild-type and mutant.This indicated that the FaFT gene promoted early flowering of Arabidopsis thaliana and restored the late flowering phenotype of atft.