植物葉綠體為光合作用及其他重要生化代謝發生之場所。然而葉綠體DNA 經常暴露於嚴峻的環境下，其中包含活性氧化物（reactive oxygen species）以及紫外線（UV），會使基因組不穩定並造成DNA的損傷，而雙股DNA的斷裂為最嚴重的形式。RecA為涉及葉綠體同源重組修復的重要蛋白質。本研究針對菸草細胞核編碼的4個 RecA 基因，RecA1S、RecA1L、RecA2S 及 RecA2L 進行大量表現，用以研究 RecA 蛋白質在細胞的位置及探討其對葉綠體DNA 修復的影響。分別構築由35S 啓動子調控RecA-mGFP重組基因之表現，利用農桿菌介導法進行菸草的轉殖，經篩選後分別獲得 26株 RecA1S、23株RecA1L 及25株 RecA2S轉殖植株，並利用螢光顯微鏡觀測 GFP 的表現位置，在RecA1L-15、19、23轉殖植株觀測到GFP 的表現，初步確認其表現位置在葉綠體。進一步再以共軛焦顯微鏡觀測RecA1L-15植株，確認RecA-mGFP在葉綠體中表現。此外，利用 RT-PCR在RecA1L-15及19品系中偵測到RecA1L-mGFP之mRNA表現。另外，已將 RecA 大量表現之轉殖植株與造成葉綠體 DNA 受損之轉殖植株進行雜交，正探討大量表現 RecA 蛋白質與葉綠體修復DNA之間的關係。

Chloroplast, an endosymbiont from cyanobacteria, is the place for photosynthesis and many other biochemical metabolisms to occur. Although the chloroplast DNA （cpDNA） encodes about 110-120 genes, most chloroplast proteins are encoded by the nuclear genome. Hence the chloroplast is in close communication with the nucleus to maintain the normal cellular functions. However, cpDNA is often exposed to severe stress environments, including reactive oxygen species and UV, which may cause the instability of chloroplast genome if the damaged cpDNA could not be repaired properly. Homologous recombination （HR） is an important mechanism for the accurately repairing of damaged cpDNA, and RecA protein play a key role in this repairing process. In this study, we aim to overexpress four RecA genes, RecA1S, RecA1L, RecA2S and RecA2L in tobacco to address the cellular localization of RecA protein and to increase the HR-mediated repairing efficiency in transgenic tobacco. We constructed binary vectors with the recombinant RecA-mGFP fusion genes driven by CaMV 35S promoter. Agrobacteria-mediated method was used to transform leaf tissues of tobacco. After selected with hygromycin, and followed the regeneration, 26 RecA1S-mGFP、23 RecA1L-mGFP and 25 RecA2S-mGFP transgenic T0 plants were obtained, respectively. Transgene integration into nuclear genome in 14 RecA1L-mGFP、9 RecA1S-mGFP transgenic plants was confirmed by PCR analysis. The expression of GFP in chloroplasts could be observed in three transgenic plants (RecA1L-15, -19 and -23) under the fluorescence microscope. In addition, the cellular localization of RecA-mGFP fusion protein in the chloroplasts of transgenic plant (RecA1L-15) was observed by confocal microscope. Furthermore, the expression of RecA1L-mGFP mRNA was confirmed in transgenic plants (RecA1L-15 and -19) by RT-PCR analysis. The transgenic tobacco of overexpressing RecA-mGFP gene was crossed with the transgenic plant of overexpressing TALEN genes to test whether the overexpression of RecA proteins could enhance the HR-mediated process in repairing damaged cpDNA.

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