Regulatory mechanism of TOC33 expression in Arabidopsis

• Main Authors: Kuan-Ting Chen, 陳冠廷
• Other Authors: Chih-Wen Sun
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/54126953955516089300

碩士 === 國立臺灣師範大學 === 生命科學系 === 102 === Most of chloroplastic proteins are encoded by nuclear genome and then imported into chloroplast by TOC/TIC complex. TOC proteins, located at outer membrane of chloroplast, are responsive for recognizing and importing chloroplastic proteins. In Arabidopsis, TOC33 and TOC34 are homologous proteins. Nevertheless, they are involved in transporting photosynthetic proteins and housekeeping proteins, respectively. According to our previous microarray and biochemical analyses, chloroplast import apparatus 2 (CIA2, a nuclear transcription factor) can regulate the expression yield of TOC33 but not TOC34. Besides, CIA2-like (CIL) is a homologous protein of CIA2 in Arabidopsis which shares 65% identity. To understand how the TOC33 expression is regulated during gene transcription, promoter deletion and plant transient assays are used to characterize the critical sequences on TOC33 promoter. So far, the reporter activity assay indicate that TOC33 has one positive and two negative regulatory sequence, located on -810 to -710 and -710 to -411 respectively. Then, we analyze these critical sequences by PLACE and AGRIS database to find the cis-acting elements and the protein binding sites. The results indicate these critical sequences might be light-regulated or dehydration-regulated. Moreover, based on DIURNAL database and our reverse-transcription PCR (RT-PCR) results, TOC33, CIA2 and CIL express rhythmically in white light. Therefore, the rhythmic expression pattern and expression yields of TOC33 in wild-type and cia2-related mutant plants under different light treatments are further verified by real-time quantitative RT-PCR (qRT-PCR). The results illustrate that CIA2 and CIL increase the expression yield of TOC33 but has little effect to regulate the rhythmic pattern of TOC33. Furthermore, similar expression level of TOC33 under red light and white light treatment implies that phytochrome might be involved in the regulation of TOC33 expression.