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標題: | 阿拉伯芥SEX4基因調節子之分析 Analysis of Regulatory Elements of Arabidopsis SEX4 gene |
作者: | Kuan-Jen Lu 呂冠箴 |
指導教授: | 董桂書(Kuei-Shu Tung) |
關鍵字: | SEX4,調節片段,SCAT,CCA1,LHY, starch excess 4 gene (SEX4),regulatory elements,CCAAT box binding factor subunit B-like protein SCAT,CCA1,LHY, |
出版年 : | 2007 |
學位: | 碩士 |
摘要: | 摘要
植物暫存性澱粉在白天於葉片中合成,並在晚間進行降解。阿拉伯芥基因SEX4突變時,突變株會在夜晚累積過多的澱粉,已知SEX4蛋白質為一雙重特異性蛋白質磷解酵素 (dual-specificity protein phosphatase),並參與在澱粉降解過程中,然而對於SEX4如何參與澱粉降解目前仍不清楚。近來研究指出,一群參與澱粉分解的基因包括SEX4,其基因的轉錄具有日夜週期的變化,白天這些基因的轉錄會增加,夜晚會減少。為進一步了解SEX4基因轉錄的調控機制,本論文利用SEX4基因啟動子片段做為探針進行電泳位移分析Electrophoretic mobility assay (EMSA),鑑定出能與細胞核之蛋白萃取物專一結合的調節片段,並以所鑑定之調節片段進行調控因子的篩選。實驗結果顯示在SEX4基因轉譯起點上游具有四個調節片段,分別位於SEX4轉錄起點前-501~ -452bp (片段AA’), -456~ -407bp (片段BB’), -186~-137bp (片段HH’) 及 +37~ +57bp (片段LL’)。EMSA實驗中AA’和BB’片段能相互競爭葉片組織的某個核蛋白,推測可能與同一個未知的蛋白質結合。HH’調節片段能與葉片及根部的核蛋白結合,顯示出HH’片段上有組織特異性的調節序列。另外LL’片段能與大腸桿菌所表現之重組蛋白質CCA1和LHY結合,由微矩陣資料庫分析得知在cca1/lhy雙突變株中,SEX4 mRNA的轉錄會明顯的減弱,顯示CCA1和LHY可能參與調控SEX4的表現。調控因子篩選方面,則利用BB’片段進行yeast one-hybrid實驗,篩選出一個CCAAT序列結合因子B類似的蛋白質SCAT蛋白基因,並證實在酵母菌中SCAT能與SEX4基因轉錄起點前的AA’和BB’片段結合。 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/29275 |
全文授權: | 有償授權 |
顯示於系所單位: | 分子與細胞生物學研究所 |
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