Cloning And Characterization Of Photoperiod Flowering Time Controlling Gene GmELF4 In Soybean

Soybean [Glycine max(L.) Merr.] is an important crop for human consumption and animal feed in China. Flowering time and maturity significantly affect soybean grain yield. To understand flowering pathway in soybean, therefore, functional analyzing the flowering relative genes is valuable. Here we demonstrate that Gm ELF4 is the candidate gene of the QTL controlling flowering in soybean described by Tasma et.al(2001), using bioinformatics, q PCR and molecular cloning. The only difference between Corsoy and Kitamishiro at the coding region of Gm ELF4 was a single nucleotide substitution(SNP)at the DUF1313 domain. The DUF1313 domain of ELF4 is conserved across diverse plant species. Thus, it is likely that the single mutation at the DUF1313 domain may leads to loss of Gm ELF4 functions. Gm ELF4 was present throughout the development of all organs when we analyzed transcription profiles of Gm ELF4 in various tissues of Harosoy plants grown under LD conditions with real-time PCR. The expression level of Gm ELF4 exhibits a diurnal rhythm with the peaks at 16 h after dawn under LD conditions and 12 h after dawn under SD conditions, indicating that Gm ELF4 is involved in photoperiod perception. When the plants grown under LD conditions were transferred to LL conditions, the Gm ELF4 transcript levels continued to oscillate with a period of 24 h, showing that Gm ELF4 was regulated by the circadian clock. In addition, Gm ELF4 was down-regulated by E1 and up-regulated by E3E4 under LD or SD conditions. E2 down-regulated the expression of Gm ELF4 under LD conditions, whereas up-regulated its expression under SD condition. To gain further knowledge regarding the function of the Gm ELF4 protein, we conducted a transient expression assay to determine its subcellular distribution. Confocal imaging showed that the Gm ELF4-e GFP fusion protein was distributed primarily in the cytoplasm. To validate the function of Gm ELF4 for flowering, we introduced the Corsoy Gm ELF4 allele driven by itself promotor into p TF101 and obtained construct p TF101-Gm ELF4. We also obtained RNAi construct based on the CRISPR/Cas9. We are performing the complementation of the gmelf4 phenotype with Corsoy Gm ELF4 allele using soybean cultivar Heinong 37 and RNAi knockdown using soybean cultivar Williams 82. Further study is needed to reveal the role of Gm ELF4 in regulating the flowering mechanism in soybean.