| Summary: | 碩士 === 國立成功大學 === 生命科學系碩博士班 === 94 === Proper timing of the transition from vegetative stage to flowering is critical to the reproductive success of plants. The floral transition in Arabidopsis is regulated by four flowering pathways: the photoperiod, vernalization, autonomous, and gibberellin dependent pathways and SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1) integrates signals from the four pathways. Phalaenopsis orchid is one of the most commercially important floral crops in Taiwan but usually has long juvenile periods. Therefore, studying the flowering time genes of orchid will be contributed to orchid industry. The objectives of this study were cloning and characterization of PeSOC1 from Phalaenopsis equestris. In order to isolate the SOC1 gene from Phalaenopsis equestris, we designed the degenerate primers based on the conserved domain of SOC1 genes from other species. The full length cDNA of SOC1 was obtained by using RACE (rapid amplification of cDNA end). The SOC1 homologous gene from Phalaenopsis equestris was designated as PeSOC1. The full-length cDNA of PeSOC1 was 1227 base pair (bp) and contained a 666 bp open reading frame (ORF) encoding for 221 amino-acid residues. In addition, using the primer which amplified the cDNA of PeSOC1, we obtained the cDNA of SOC1 (designated as OfSOC1) in Oncidium flexuosum. The amino acid sequence of PeSOC1 contained a conserved MADS-box domain and K-box domain. The amino acid sequence of the PeSOC1 protein was 57% identical with the rice OsSOC1 and 97.8% with OfSOC1 (Oncidium flexuosum). Molecular phylogenetic analysis of evolutionary relationship revealed that PeSOC1 was clustered with the monocot genes. Southern blot analysis indicated that PeSOC1 was a single-copy gene when 3′ UTR was used as a probe. Northern blot analysis of Phalaenopsis equestris root, leaf, floral bud and flower indicated that relative high amount of gene expression of PeSOC1 was detected in the leaf in comparison with that of flower. Furthermore, The functional study of PeSOC1 was performed by transforming wild-type, ft and soc1 mutant Arabidopsis plants with PeSOC1 gene. Transgenic plant of T1 and T2 seedlings were shown to rescue the late flowering phenotypes of mutants, suggesting that the PeSOC1 gene is a functional equivalent of AtSOC1. In the vernalization experiment, the transcript levels of PeSOC1 were higher after 8 weeks of cold treatment in Phalaenopsis. Finally, we cloned 2 kb promoter regions upstream of the transcription start site (TSS) of PeSOC1 and identified a CArG box sequence and some photoperiod-related cis-elements. In conclusion, PeSOC1 is the flowering time gene which plays the positive-regulator in Phalaenopsis equestris.
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