孫智雯Chih-Wen Sun陳冠廷Kuan-Ting Chen2019-09-052016-7-122019-09-052014http://etds.lib.ntnu.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=id=%22GN060143031S%22.&%22.id.&http://rportal.lib.ntnu.edu.tw:80/handle/20.500.12235/104074植物葉綠體蛋白質主要是由細胞核中的基因轉錄轉譯後，透過轉運蛋白機組(TOC/TIC complex)送至葉綠體內。TOC蛋白位在葉綠體外膜上，負責辨識及運送蛋白質。在阿拉伯芥的研究中，TOC33及TOC34是同源蛋白，但TOC33負責辨識及協助光合作用相關蛋白的運輸；TOC34則參與辨識及運送一些維持葉綠體基本功能的蛋白。根據我們之前的基因微陣列及生化實驗結果，發現CIA2 (是一個細胞核內的轉錄因子)會調節TOC33基因表現但不會影響TOC34基因表現。而CIL是一個在阿拉伯芥中與CIA2胺基酸有65%相同的同源蛋白。為了瞭解表現TOC33基因的調節機制，啟動子刪除及植物暫時性基因轉殖實驗先用來分析TOC33啟動子上的轉錄關鍵序列，分析報導基因表現的結果發現TOC33轉錄起始位點前-810至-710及-710至-411之間分別可能會促進及抑制基因的表現量。接著透過生物資訊網站PLACE及AGRIS來分析比對這些關鍵序列是否有已知的順式作用元素及蛋白結合位，預測結果顯示TOC33啟動子序列上存在許多可能的順式作用元素，其中TOC33關鍵序列會受到光線或缺水等環境因子調節。此外，根據DIURNAL基因資料庫及反轉錄聚合酶連鎖反應實驗結果，發現TOC33、CIA2及CIL在白光下具有規律性的表現。因此，我們也利用不同光源處理野生型及突變株植物，並以qRT-PCR來分析TOC33基因的表現規律性或表現量是否會直接或間接受到CIA2及CIL調節。結果顯示CIA2及CIL不會調節TOC33基因的表現規律性，但會增加TOC33基因的表現量。此外，TOC33基因在單獨紅光處理與白光處理下呈現相同的表現量，暗示光受體光敏素亦參與調節TOC33基因的表現。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.TOC33TOC34CIA2CIL植物暫時性基因轉殖qRT-PCRTOC33TOC34CIA2CILplant transient assaysqRT-PCR