| Summary: | 碩士 === 國立成功大學 === 生物學系碩博士班 === 92 === Phase change or maturation can be observed in all higher plants and affects reproductive competence, the morphology and growth rate of the vegetative body, as well as the regenerative potential of tissue explants. Efforts to elucidate the underlying mechanism of phase change of plants, or maturation and rejuvenation, have disclosed biochemical correlations, some of which could aid in furthering the investigations. Hence, the aims of this project are to characterize these phase-regulated signal transduction components.
Plants have developed elaborate mechanisms to perceive external signals and to manifest adaptive responses with proper physiological and morphological changes. The mitogen-activated protein (MAP) kinase cascade is one of the well-characterized intracellular signaling modules, and that cross-talk between various signal-transduction pathways might be concentrated at MEK level in plant MAPK cascades. In this study, two novel genes of Sequoia sempervirens, SsMAPKA, and SsMEKA have been identified and characterized.
According to the phylogenetic analysis based on the amino acid sequences, SsMAPKA belongs to the Group A2 MAPK subfamily, and shares high homology with PsD5 (82% identity) from Pisum sativum. Southern blot analysis showed that SsMAPKA is a single copy gene in S. sempervirens. Northern blot analysis showed that SsMAPKA was expressed in all plant organs. The mRNA level of SsMAPKA was accumulated in adult and 1x- to 3x- grafted shoots than in juvenile and 4x- to 5x- grafted shoots. SsMAPKA was fused to glutathione-S-transferase (GST) and the fusion protein was expressed in Escherichia coli. Results showed that the recombinant SsMAPKA was recognized by monoclonal anti-phosphotyrosine antibody. This result indicated that SsMAPKA is a functional protein kinase that has features characteristics of MAP kinases. The promoter of the SsMAPKA gene was isolated from S. sempervirens. The cloned promoter regions upstream of the transcription start site of SsMAPKA gene is 1,838bp. The most prominent matches identified include those to the consensus binding size for ABA-responsive promoters（ACGT or ACGTG）and for pollen-specific regulatory elements（AGAAA）.
Then we made an attempt to isolate the SsMAPKA upstream factor, SsMEKA in signal transduction components. Four types of S. sempervirens PCR product have been isolated that encode putative protein kinases, designated SsMEKA ⅠA, SsMEKA ⅠAs, SsMEKA ⅡA and SsMEKA ⅡAs. According to the phylogenetic analysis based on the amino acid sequences, SsMEKA belongs to the Group A2 MEK subfamily. These kinases exhibit a high degree of homology to NtMEK1（65％ identity）, a tobacco protein that is a membrane of the family of mitogen-activated protein kinase kinase （MAPKKs）, which appears to function in a cell-cycle-dependent manner. One of these isoforms SsMEKA IIA which contained the region that flanked the 42bp intron-like sequence was found in S. sempervirens. Southern blot analysis showed that SsMEKA is a single copy gene in S. sempervirens. Tissue-specific analysis indicated that the basal transcription level of SsMEKA was expressed in all organs in plants. Our results revealed that transcripts of the SsMEKA ⅡA gene accumulated predominantly in adult and 1x- to 3x- grafted shoots, and a small amount of the transcript was found in juvenile and 4x- to 5x- grafted shoots of S. sempervirens. Furthermore, we provide the first report that concentrated MEK by alternative splicing regulating phase change of plants in the MAPK cascades.
In this study, we propose that DNA rearrangement involved in woody plant maturation.
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