Studies On Molecular Mechanism Regulating The Expression Of E1 In Soybean [Glycine Max(L.) Merr.]

Maturity gene E1 loci significantly control flowering time and maturity in soybean. However, the molecular mechanism regulationg the expression of E1 has largely unsolved. Therefore, the aim of this study is to reveal molecular mechanism govering E1 expression level and function of E1 by studying the circadian rhythm of E1 under various light/dark regimes, various genetic factors regulating the expression levels of E1, and characterization of e1-b3 a, a new mutation of E1.1. Definitize the expression rhythm of E1In this study, we revealed that the expression of E1 is promoted by light(long days), and somewhat, but not tightly, associated with the circadian clock. The diurnal expression pattern was similar to the typical bimodal as expression described previously, except that the first peak appeared around 2hr after dawn in this study. The slightly discrepancy for the first peak might be ascribed to the sampling intervals(2hr in this study vs 4hr in Xia et al.’s experiment). The second peak in LD was associated with the starting of the dark phase at 16 hr. During 8 to 12 hr and 20 to 24 hr, the expression of E1 was at the bottom. The expression of E1 was promoted by continuous light, but is suppressed by continuous dark. The contrasting difference of E1 expression occurred between long day and short day. And continuous light could promote the expression of E1. On the contrary, continuous dark conditions could suppress the expression of E1. However, the long night might be the key factor leading to a suppressed E1 expression. The rhythm could not be kept for more than one subjective day, indicating that E1 is somewhat, but not tightly, associated with the circadian clock.2. Bioinformatics analysis of upstream sequence of E1, 5’ flanking sequences of E1 and screen gene expression library of soybean by yeast one-hybridThe 5’ flanking sequences of E1 and the two homologous genes contain multiple cis-elements involving in light reaction and circadian rhythms. Upstream sequences of E1 and its homologous genes were compared, and the sequence similarity of-326bp~-1bp region is over 85%. Analysis of GUS expression driven by deletion derivatives of the 5’-flanking region of E1 in Arabidopsis cis-elements related to light reaction are existing in the sequence-288bp~-1bp 5’ flanking region of E1. Sequence1(-288bp~-154bp) and sequence3(-364bp~-257bp) were used as baits to screen gene expression library of soybean by yeast one-hybrid in order to find protein binding on 5’ flanking region of E1, and several candidate protein have been identified.3. Influence of different genetic factor imposing on expression of E1Flowering time and other agronomic traits of two populations derived from cross of Kariyotaka × Kenfeng 18 were conducted at Harbin and Hailun, Heilongjiang province. The effect of differentg genotype at E2、E3 and E4 loci on the expression level of E1 was analyzed. Transcriptional abundance of E1 was significantly correlated with flowering time. In Harbin and Hailun,expressive abundance of E1 in recessive homozygous is higher than that in dominant homozygous on E2, E3 and E4 loci. The impacts of E3 and E4 on trancript abundance of E1 gene were less than E2 in Harbin(45o70’N, 126o64’E), but E2 had higher impact than E3 or E4 in Hailun, a relatively higher latitude than Hailun(47o26’N, 126o38’E).4. Subcellular localization of e1-b3 a and analysis of its functionIn early flowering cultivar Yanhuang 3, we identified a 5bp(3bp SNP and 2bp deletion) mutation that occurs in the B3 domain of E1, leading to a premature termination, referred to as e1-b3 a. e1-b3 a genotype flowered 30 days earlier than E1 genotype in Harbin(45o70’N, 126o64’E) and 10 days earlier in Huaian(33o57’N, 119o04’E). Subcellular localization analysis showed that the putative truncated protein e1-b3 a was predominately distributed in nuclei, which is similar to E1 protein, but not to e1-as protein. The characterization of e1-b3 a demonstrated the B3 domain is essential for the function of E1 as a flowering repressor.E1 underwent strong selection pressure. e1-as is a typical leaky allele, having partial function as flowering suppressor. The mechanism leading to the loss function in e1-b3 a is not the same as e1-as since e1-b3 a is distributed mainly in the nuclei, as does the E1 protein. Multiple allelic variations and flucturated expression levels of E1 endow soybean genetic flexibility to adapt different ecological niche or geographic latitude.