Cloning And Functional Analysis Of GmFT3a In Soybean

Flowering is one of the most important indicators of the transformation from vegetative to reproductive growth in plants.The flowering time that ranging from emergence to flowering is controlled by both external environments and internal molecular networks.The FT gene family is an important integrator among multiple flowering regulatory pathways in Arabidopsis.Overexpression of FT in Arabidopsis significantly promoted flowering.Recently several flowering related genes have been cloned and their functions have been identified,howere,the molecular network of photoperiodic regulation of soybean flowering is still unclear.In this study,GmFT3 a has been cloned and its molecular function was analyzed to further reveal the molecular mechanism of flowering in soybean.These results will provide solid theoretical fundaments to clarify the molecular function and mechanism of GmFT3 a on photoperiodic regulation of flowering,and create valuable breeding materials for the further improving ecological adaptability of soybean varieties.Firstly,the GmFT3 a gene was cloned from the photoperiod-sensitive Zigongdongdou soybean variety,and its coding region and promoter sequence were analyzed by bioinformatics software and websites.Secondly,a serial of gene expression of GmFT3 a were quantified by real-time fluorescence quantitative method,including different tissues,various varieties belongs to different maturity groups,and the 48 h expression pattern in soybean.Furthermore,the P16318-GmFT3a-GFP fusion expression vector was constructed to utilize the Arabidopsis protoplast transformation for protein subcellular localization,and transgenic technology was used to obtain Arabidopsis thaliana and soybean overexpressing plants,respectively.In addition,the phenotype of GmFT3 a plants and the expression of endogenous known flower-related genes were analyzed to determine their molecular functions in the flowering pathway.The main results are as follows:1.The GmFT3 a gene is located on chromosome 16 of the soybean genome.Its CDS region is 528 bp in length and encodes 175 amino acids.The 27 th to 164 th amino acids are highly conserved family domain of phosphatidylethanolamine-binding proteins(Phosphatidyl Ethanolamine-Binding Protein,PEBP).The tertiary structure of GmFT3 a protein is highly similar to AtFT and GmFT2 a.The GmFT3 a upstream promoter contains a variety of cis-acting elements,including tissue-specific expression elements,photo-responsive elements,gibberellin response elements,and temperature-responsive elements.In soybean,GmFT3 a protein has the highest homology with GmFT3 b followed by GmFT2 a.2.Real-time fluorescence quantitative analysis showed that GmFT3 a gene was expressed in different tissues of soybean but the highest expression in three-leaf compound leaves.The expression analysis within 48 h showed that GmFT3 a had no specific circadian rhythm,and the expression peaked at 4h after illumination;Under the long-day conditions,the expression levels of the maturity group(MG)000 and VII groups were higher than those of the other groups;while under short-day condition,the expression level of GmFT3 a in the MG000 and MG00 varieties were higher than those in the other MG ones.3.The results of subcellular localization of Arabidopsis protoplasts revealed that GmFT3 a protein was expressed in cytoplasm and nucleus,which is in consistent with GmFT2 a,indicating that GmFT3 a may have transcription factor activity.4.Under long-day conditions,the GmFT3 a transgenic Arabidopsis plants showed a very significant earlier flower phenotype,and the flowering time of each line was 3.9 days and 2.3 days earlier than that of wild type Arabidopsis,respectively(P < 0.01).The average flowering time of each overexpressing GmFT3 a line was 3.3 days earlier than the wild type Arabidopsis(P < 0.01);the average number of leaves of each overexpressing Arabidopsis line(Cauline leaf + Rosette leaf)and wild The type of Arabidopsis thaliana decreased by 29.5%,showing a significant decrease(P < 0.01).the expression levels of endogenous genes FT,CO and SOC1 in Arabidopsis plants were significantly increased(P < 0.01),indicating that overexpression of the GmFT3 a gene in Arabidopsis promotes the expression of its endogenous FT gene and other genes involved in flowering regulation.5.Under long-day conditions,the T2 generation of GmFT3 a transgenic soybean showed significant earlier flower phenotype,and the flowering time of each line was 6.2 days earlier than that of the control(P < 0.05);There was no significant difference in flowering time under short-day conditions(P > 0.05);the expression levels of GmFT3 a,GmFT3b and GmFT1 b in T2 transgenic plants were significantly increased under long-day conditions(P < 0.01),the expression levels of GmFT2 a,GmFT5a and GmFT1 a are significantly decreased.(P < 0.01),indicating that GmFT3 a may have functional redundancy or complementarity with other genes of the FT gene family.The promoting and inhibiting gene balanced their expression levels to regulate the flowering process under different conditions.These results provided solid evidence to the theoretical “Teeter board” model of vegetative and reproductive growth in soybean.The significant earlier phenotype of flower and related expression level of other flowering related genes in the transgenic GmFT3 a plants indicated that GmFT3 a possibly promoted the flowering in soybean.These results provides a theoretical reference for the further deconstruction of the molecular mechanisms of the FT gene family in photoperiodic regulation pathways of flowering in soybean.