植物葉綠體的蛋白質主要是由細胞核中的基因轉錄轉譯後，經由轉運蛋白機組(Toc/Tic complex)，送入葉綠體當中。在阿拉伯芥的研究中，Toc33 對於調控葉綠體發育扮演著重要的角色，因為它位於葉綠體外膜上，負責辨識與協助光合作用相關的蛋白質轉送入葉綠體內。然而，關於TOC33 基因表現的調控機制目前仍尚未明瞭。我們於先前研究中發現一個獨特的轉錄因子CIA2 (chloroplast import apparatus 2)，能在葉子中專一性地增加TOC33 基因表現量。為了要進一步了解CIA2 蛋白質與其他的核蛋白調節TOC33 基因表現的機制，我們以基因轉殖系統研究TOC33 啟動子的功能性區域。從轉殖植物偵測報導基因的表現量以及酵素活性發現TOC33 五端未轉譯區域中的第一內插子可能會影響CIA2 蛋白質調控TOC33 基因表現。以啟動子刪除分析(promoter deletion)指出在-616 到-517 之間可能具有與CIA2 蛋白質相關的正向調節因子而在-710 到-616 之間則可能具有其他核蛋白所調控的正向調節因子。這些研究不只可以了解CIA2 蛋白質與其他核蛋白影響TOC33 基因表現的調節機制，更可以了解葉綠體發育的調控過程。

The majority of the chloroplastic proteins are encoded by nuclear genome and then imported into chloroplasts via Toc/Tic complex (translocon at the outer/inner envelope membrane of chloroplast). In Arabidopsis thaliana, Toc33 plays an important role in regulating chloroplast development because it predominantly recognizes and assists the translocation of the photosynthesis-related proteins on
chloroplast membranes. However, the regulatory mechanism of TOC33 expression remains unclear so far. We have previously characterized a transcription factor, CIA2 (chloroplast import apparatus 2), which specifically up-regulate the TOC33 in leaves. To further understand how CIA2 and other nuclear proteins regulate the expression of TOC33 gene, the function of TOC33 promoter sequence are analyzed in detail by stable and transient assays. Quantitative RT-PCR and activity assay of reporter gene suggest that the upregulatory efficiency of TOC33 gene expression by CIA2 is
affected by the 1st intron in the 5’UTR of TOC33. Furthermore, 5’ promoter deletion reveals that two regulatory regions in TOC33 promoter sequences, 33CAE1 (located on -710 to -616) and 33CAE2 (located on -616 to -517). 33CAE1 and 33CAE2 are putative nuclear proteins and CIA2 binding sites, respectively. These results not only
reveal the molecular mechanism how CIA2 and other nuclear proteins modulate TOC33 expression, but also provide more understanding of the regulatory processes during chloroplast development.

1. Agne B and Kessler F. Protein transport in organelles: The Toc complex way of preprotein import. FEBS Journal 276: 1156–1165, 2009.
2. Chen YJ and Sun CW. Transgenic study of chloroplast translocation regulation in Arabidopsis thaliana. Botanical Studies 51: 147-153, 2010.
3. Chung B.YW, Simons C, Firth AE, Brown CM and Hellens RP. Effect of 5'UTR introns on gene expression in Arabidopsis thaliana. BMC Genomics 7: 120, 2006.
4. Clough SJ and Bent AF. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant Journal 16: 735-743, 1998.
5. Constan D, Patel R, Keegstra K, and Jarvis P. An outer envelope membrane component of the plastid protein import apparatus plays an essential role in Arabiodopsis. The Plant Journal 38: 93-106, 2004.
6. Higo K, Ugawa Y, Iwamoto M and Korenaga T. Plant cis-acting regulatory DNA elements (PLACE) database. Nucleic Acids Research 27: 297-300, 1999.
7. Jarvis P. Targeting of nucleus-encoded proteins to chloroplasts in plants. New Phytologist 179: 257-285, 2008.
8. Jarvis P and Robinson C. Mechanisms of protein import and routing in chloroplasts. Current Biology 14: 1064-1077, 2004.
9. Jarvis P, Chen LJ, Li H, Peto CA, Fankhauser C, Chory J. An Arabidopsis mutant defective in the plastid general protein import apparatus. Science 282: 100-103, 1998.
10. Jefferson RA. Assaying chimeric genes in plants: the GUS gene fusion system. Plant Molecular Biology Reporter 5: 387-405, 1987.
11. Jeong YM, Mun JH, Lee I, Woo JC, Hong CB and Kim SG. Distinct roles of the first introns on the expression of Arabidopsis profile gene family members. Plant Physiology 140: 196-209, 2006.
12. Kessler F and Schnell DJ. The function and diversity of plastid protein import pathways: a multilane GTPase highway into plastids. Traffic 7: 248-257, 2006.
13. Li HM and Chiu CC. Protein transport into chloroplast. The Annual Review of Plant Biology 61: 21.1-21.24, 2010.
14. Mascarenhas D, Mettler IJ, Pierce DA, and Lowe HW. Intron-mediated enhancement of heterologous gene expression in maize. Plant Molecular Biology 15: 913-920, 1990.
15. Murashige T and Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15: 473-497, 1962.
16. Norris SR, Meyer SE and Callis J. The intron of Arabidopsis thaliana polyubiquitin genes is conserved in location and is a quantitative determinant of chimeric gene expression. Plant Molecular Biology 21: 895-906, 1993.
17. Robson F, Costa MM, Hepworth SR, Vizir I, Pineiro M, Reeves PH, Putterill J and Coupland G. Functional importance of conserved domains in the flowering-time gene CONSTANS demonstrated by analysis of mutant alleles and
transgenic plants. Plant J. 28: 619–631, 2001.
18. Rose AB, Elfersi T, Parra G and Korf I. Promoter-proximal introns in Arabidopsis thaliana are enriched in dispersed signals that elevate gene expression. Plant Cell
20: 543-551, 2008.
19. Salome PA, To JPC, Kieber JJ and McClung CR. Arabidopsis response regulators ARR3 and ARR4 play cytokinine independent roles in the control of circadian
period. Plant Cell 18: 55–69, 2006.
20. Soll J and Schleiff E. Protein import into chloroplasts. Nature Reviews Molecular Cell Bioligy 5: 198-208, 2009.
21. Sun CW, Chen LJ, Lin LC and Li HM. Leaf-specific up-regulation of chloroplast translocon genes by a CCT motif-containing protein, CIA2. Plant Cell 13: 2053–2061, 2001.
22. Sun CW, Huang YC and Chang HY. CIA2 coordinatedly up-regulates protein import and synthesis in leaf chloroplasts. Plant Physiology 150: 879-888, 2009.
23. Bailey TL and Elkan C. Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proceedings of the Second International Conference on Intelligent Systems for Molecular Biology 2: 28-36, 1994.
24. Yu TS and Li HM. Chloroplast protein translocon components atToc159 and atToc33 are not essential for chloroplast biogenesis in guard cells and root cells.
Plant Physiology 127: 90-96, 2001.
25. Yuan YX, Wu J, Sun RF, Zhang XW, Xu DH, Bonnema G and Wang XW. A naturally occurring splicing site mutation in the Brassica rapa FLC1 gene is associated with variation in flowering time. Journal of Experimental Botany 60: 1299-1308, 2009.