Cloning, Expression Profiles And Functional Analysis Of GmTOC1s And GmLCLs Genes In Soybean (Glycine Max L.)

Three genes encoded major components of the central oscillator in Arabidopsis are CIRCADIAN CLOCK ASSOCIATED 1 (CCA1). LATE ELONGATED HYPOCOTYL (LHY) and TIMING OF CAB 1 (TOC1). CCA1 and LHY are closely related MYB-like transcription factors, both of which are regulated by circadian clock with an expression peak at dawn. The LHY/CCA1 and TOC1 genes were proposed to be compose of a feedback loop.AtLHY/CCA1 proteins directly bind to the evening element (EE) on the promoter region of TOC1, resulting in repression of its transcription during the daytime. Soybean (Glycine max L.). a palaeotetraploid, is a typical short-day plant. A study on the functions of soybean LHY/CCA1/TOC1 may be helpful to elucidate the molecular mechanism underlying flowering control in soybean and the evolution and diversity of circadian clock system in planta.In this study, four homologues of TOC1 and their promoters were cloned from soybean cv. Kennong 18. The analysis by bioinformatics showed that GmTOC1s and AtTOCl are grouped in the same phylogenetic clade. suggesting their close relationship each other. The intron-exon structure indicated that four copies of GmTOC1s and AtTOCl have two exons. which encoded a conserved regulating domain, and another exon encoded conserved CCT domain, inferring that GmTOC1s and AtTOC1 had the similar function.The promoters of four TOC1 homologues shared common cis-elements related to light regulation and ones induced by hormone and environment stesses. On the other side, all of GmTOC1s promoters were identified by their specific elements in comparison with AtTOC1 promoter. EE in GmTOCIs had two types. one including GmTOCl-3 was identical to that of AtTOC1 (AAAATATCT), the other homologues including GmTOC1-1. GmTOC1-2. GmTOC1-4 had the sequence of AAAATATNN. Four EEs may have similar function. even though the difference of two bases.Four genes of soybean GmLCLs were cloned and bioinformatically analyzed. Their spatio-temporal expression profiles across different developmental stages and in various tissues/organs were constructed by quantitative real-time RT-PCR (qRT-PCR). Furthermore, functional analysis of GmLCL1 and GmLCL2 genes were carried out in transgenic Arabidopsis. The main points we got were showed as follows:1. All of GmLCLs genes had conserved MYB DNA binding domain and conserved serine residue in upstream of MYB DNA binding domain;2. The mRNA expression level of four copies of GmLCLs was higher in leaves of different developmental phases except flowering phase, suggesting that GmLCLs had important functions in the early and middle development stages;3. The expression patterns of GmLCLs protein in different developmental phases and tissues were coordinate with that of GmLCLs mRNA;4. GmLCLs protein expression level in the leaves of different soybean cultivars was negatively related to the latitudes, which the cultivars originated, in long day conditions, but no significantly difference in short day conditions;5. The transient expression of GmLCLl and GmLCL2 in Arabidopsis proplast showed the similar subcellular location(cell nucleus) of their proteins to that of Arabidopsis LHY/CCA1;6. The transgenic Arabidopsis plants of GmLCL1 and GmLCL2 presented late flowering phenotype, which may be result from dysfunction of biological clock by overexpression of GmLCLl and GmLCL2.