| Summary: | 碩士 === 國立成功大學 === 生命科學系碩博士班 === 100 === Flowering is a crucial stage in plant life cycle for reproduction. Previous studies in Arabidopsis thaliana have revealed four major pathways to regulate flowering: the photoperiod, vernalization, gibberellins dependent and autonomous pathways. Besides the four major pathways, FRI (FRIGIDA) pathway may also participate in flowering regulation. In Arabidopsis FRI pathway, PAF1-like (RNA polymerase II associated factor 1) complex and histone methyltransferase EFS (early flowering in short days) can induce FLC gene expression and further delay flowering. Phalaenopsis orchid is one of the most important commercial floral crops in Taiwan, but usually has a long juvenile period. Therefore, studying the flowering genes of orchid will contribute to orchid industry for flowering time regulation. In previous research, a partial sequence from BAC end sequence in P. equestris BAC clone Pe-NCKU-HBAC-1031M24 was with high similarity to Arabidopsis EFS gene. To investigate the chromosomal location of EFS gene in P. equestris and P. aphrodite subsp. formosana, EFS gene-containing BAC DNA together with either 45S rDNA or telomere DNA were used as probes in fluorescence in situ hybridization (FISH). The FISH results showed that EFS gene had a single signal which was close to telomere on both P. equestris and P. aphrodite subsp. formosana chromosome. The distance between EFS signal and telomere was about 1.13 μm and 1.28 μm in P. equestris and P. aphrodite subsp. formosana, respectively. These results suggested that EFS can be served as a chromosome-specific marker in P. equestris and P. aphrodite subsp. formosana karotyping. Furthermore, EFS gene-containing BAC clone was sequenced by 454 pyrosequencing and assembled into 14 contigs. The structure of EFS gene is composed of 17 exons in both P. equestris and P. aphrodite subsp. formosana. The EFS cDNA sequences derived from the BAC clone and P. equestris genome are both 5,802 bp in length, encoding 1,934 amino acids, while the length in P. aphrodite is 5,799 bp, encoding 1,933 amino acids, named PaEFS. The identity of EFS amino acid sequence derived BAC clone with that of P. equestris and P. aphrodite subsp. formosana was 99% and 97.1%, respectively. The published EFS gene amino acid sequences of other species were 22.5-25.4% identical with that of P. equestris. Molecular phylogenetic analysis revealed that PeEFS was clustered together with other monocot species. All amino acid sequences of EFS gene contained the conserved CW, AWS, SET and post-SET domains. Phylogenetic analysis based on amino acid sequences of EFS functional domains among eight Phalaenopsis species showed that the clustering was in accordance with subgenus. Moreover, EFS genes of eight Phalaenopsis species were clustered in SDG family class II by comparison of Arabidopsis SET domain group family. In conclusion, EFS gene was highly conserved among Phalaenopsis species and single locus of EFS gene can be used as a marker for chromosome identification in Phalaenopsis species.
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