| Higher plant has a set of meticulous signal regulating and controlling system during long-term evolution process, urges itself to accomplish its growing and generative propagation. Flowering is a central part of individual development of higher plants, including control of flowering time, flower meristem formation and development of flower organs and so on, which is a multi-factor system control process. In Arabidopsis thaliana, variousflowering pathways integrate to activate the downstream flower meristem genes, such as LEAFY (LFY) and APETALA1(API). LFY and API mainly involve the floral meristem formation, the regulation of flowering time and flower organ development.This study focuses on the cloning and functional identification of LFY homologous genes and homologous API gene in wild soybean, which is expected to be a foundation of the further research on the mechanism of flower development in wild soybean.From the floral organs of wild soybean, GsLFY and GsAPl was cloned and sequenced. GsLFY mRNA is1224bp in length and encodes407amino acid residues. GsLFY encoding protein consists of a LFY/FLO domain, belonging to LFY/FLO family. GsAP1mRNA is711bp in length, and encodes a protein with236amino acid residues. GsAPl encoding protein sequence contains a MADS-box domain, belonging to MADS-box superfamily. The full-length genomic DNA sequence of two genes were cloned and sequenced. GsLFY gDNA of2585bp contains three exons and two introns and the intron positions are conserved among various plant LFY genes. GsAPl gDNA of5400bp contains eight exons and seven introns.The phylogenetic tree based on deduced amino acid sequences of genes plant LFY and AP1genes showed that the LFY and API of Lotus corniculatus, Glycine soja and Medicago sativa clustered into one branch, and the topologic structure is agreed well with evolutionary relationship.Expression levels of GsLFY and GsAP1in different tissues and different developmental stages of were analyzed by real-time fluorescence quantitative PCR technique. The results showed that GsLFY was expressed in the roots, flowers and seeds but not expressed in the stems, leaves, shoot-tips; in four whorls of floral organs GsLFY was expressed in the sepals and stamens but not in the petals and carpels. GsAP1was expressed mainly in flowers and weakly expressedin the roots, stems and seeds; in four floral organs, GsAP1was expressed in the petals and sepals and the expression level in the sepals was significantly higher than that in the petals. The expression pattern of GsAP1agrees with the expression characters of Class A genes. The expressions of two genes in flowers at different developmental stages share a similar pattern, decreasing during the course of the development of flower buds. During the different developmental stages of seeds, it was observed that GsLFY expression was increased tillthe35DAF (days after flowering) and then decreased but gradually increased thereafter. GsAP1expression level reached the maximum at40-45DAF and followed by the decrease.The transcriptional activation assay showed that GsLFY and GsAP1encoding products obviously had the transcriptional activation activities in yeast.The transient expression analysis in onion epidermal cells suggested that GsLFY and GsAP1proteins both located in the nucleus.The transgenic tobacco plants overexpressing GsLFY or GsAP1were generated. The real-time fluorescence quantitative RT-PCR analysis suggested that GsLFY and GsAP1expressions in transgenic tobacco plants were significantly increased compared to wild-type plants. The flowering time of GsLFY and GsAP1transgenic plants were29.08and65.7days earlier than wild-type plants under normal conditions, respectively. |
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