| Summary: | 碩士 === 國立臺灣大學 === 分子與細胞生物學研究所 === 95 === Abstract
Starch is synthesized in leaves during photosynthesis and is degraded during night. Arabidopsis sex4 mutants have a starch excess phenotype. SEX4 gene encodes a dual specificity protein phosphatase and is involved in starch degradation; however, its role is poorly understood. Recent evidence showed that circadian regulation plays a role in the oscillation of transcription level of starch degradation related genes, including SEX4 gene. The transcripts for these genes increased during the day and decreased at subsequently night. In this study, putative regulatory elements of SEX4 promoter were analyzed by electrophoretic mobility shift assay (EMSA). EMSA with nuclear extract prepared from Arabidopsis mature leaves and short DNA fragments of SEX4 promoter showed that there were four primary regulatory elements bound specifically with nuclear proteins. The positions at -501 to -452 (AA’ fragment) and -456 to -407 (BB’ fragment) upstream of SEX4 transcriptional start site showed a reciprocal competition in the in vitro binding assay, suggesting that AA’ and BB’ may share a specific unknown protein for interactions. The position at -186 to -137 (HH’ fragment) was identified as a tissue specific binding site. In addition, transcription factors CCA1 and LHY1 bound to AAAATATCT element at position +36 to +57bp 5’-UTR of SEX4 in vitro. mRNA oscillation levels of SEX4 were eliminated in cca1/lhy null mutants, suggesting that CCA1 and LHY may play roles to regulate SEX4 gene expression. Yeast one hybrid screening showed that a CCAAT box subunit protein, SCAT could interact with AA’ and BB’elements in SEX4 promoter. Possible mechanisms controlling SEX4 expression have been discussed in this study.
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