Molecular Cloning, Expression Profiles And Functional Analysis Of FT/TFL1 Genes In Soybean (Glycine Max)

Phosphatidylethanolamine-binding protein(PEBP) gene family members are of important roles in controlling flowering time in plants.As a key member of PEBP gene family,FT(Flowering Locus T) protein or/and mRNA are one of the principal components of florigen.An analysis of soybean PEBP gene family aimed to clarify their definite roles and positions in soybean flowering network may be helpful to elucidate the molecular mechanism underlying flowering control in soybean(Glycine max),a typical short day(SD) crop and a model plant in the early study of photoperiodic flowering.In the present study,19 members of soybean PEBP gene family were cloned and bioinfomatically analyzed. Their spatio-temporal expression profiles across different developmental stages and in various tissues/organs were performed by quantitative real-time reverse transcript PCR(qRT-PCR). Furthermore,functional analysis of three FT-like genes was performed in transgenic Arabidopsis.As the accuracy of qRT-PCR mainly relies on the selection of proper internal reference controls.We perform the gene stability analysis of 14 candidate reference genes in soybean among 116 samples including different developmental stages,various tissues/organs and different photoperiod treatments.The main results were as follows:ⅰACT11,UKN1 and UKN2 were the most stably expressed reference genes among different tissues/organs,whereas SKIP16,UKN1 and MTP showed the most expression stabilities in samples from different developmental stages.In LD and SD photoperiodic treatment,ACT11,TUA5 and TIP41 were the most stably expressed.TIP41,UKN1 and UKN2 showed the most expression stabilities in blue and red light photoperiodic treatment.ACT11,UKN2 and TUB4 performed the best in different cultivars. Generally,SKIP16,UKN1 and UKN2 showed excellent expression stabilities across all the experimental samples.ⅱTo determine the optimal number of genes required for accurate normalization in soybean gene expression studies,a default cut-off value of 0.15 as proposed by geNorm was adopted.For the samples of different cultivars and blue and red photoperiodic treatment,two reference genes are necessary for optimal normalization.For samples of LD and SD photoperiodic treatments,three genes would be necessary and sufficient to achieve accurate normalization.In contrast,for developmental stages and tissue/organ samples,a combination of four genes is recommended for the accurate normalization.ⅲNineteen PEBP genes in soybean were cloned,including 9 FT homologs,4 TFL1 homologs,4 CEN homologs and 1 MFT homolog.They were located on 10 different chromosomes,and FT-like genes seemed proning to parallelly locate in a short section on the same chromosome.In contrast,the different copies of TFL1 and CEN genes were located on the four different chromosomes,respectively. There were 7 pairs of genes which shared high identities of more than 90 percent,and locate on the different chromosomes.This may be due to the characteristics of soybean genome,an ancient tetraploid.ⅳAll the soybean PEBP genes except for CEN4 possessed a conserved exon-intron structure with 4 exons and 3 introns.The second and third exons were most conserved among all the PEBP genes, which is 62bp and 41bp in length,respectively.Most of soybean FT/TFL1 proteins shared almost identical ligand-binding sites with their counterparts in other plants reported.ⅴThe transient expression using particle bombardment in three different systems including onion epidermal cells,Arabidopsis proplast and soybean leaves was employed to investigate the subcellular location of 9 soybean FT homologs.All the analysis reached to the same patterns that all soybean FT homologs were localized in both nuclear and cytoplasm as Arabidopsis FT did.ⅵThe mRNA expression level of GmFTL1/2 and GmFTL3 was positively correlated with the flowering time of different cultivars from different latitudes under long day(LD) conditions.There is no significant differences in FT mRNA expression and flowering time among the different cultivars in short day(SD) conditions.Soybean FT-like genes and TFL1-like genes showed generally opposite expression patterns across different developmental stages.Most of the FT-like genes showed high expression at the anthesis,while TFL1 genes showed high expression at the earlier vegetative stages.The mRNA expression of most of FT genes increased gradually along with the vegetative growth and peaked at the flowering.Overexpression of GmFTL1,GmFTL2 and GmFTL3 in Arabidopsis all resulted in extremely early flowering.These results demonstrated that FT genes may be important in soybean flowering time control and the function of FT was highly conserved in soybean.ⅶThe mRNA expression of a majority of FT homologs was detected with a relatively high abundance in petioles,which was 30～50 folds higher than that in leaves.This indicated that the petiole may function(at least in part) on the floral induction in soybean.