細胞穿透蛋白(CPP)具有DNA的結合能力，可將外源DNA傳送入植物細胞內。本研究利用CPP結合DNA的能力與蛋白質輸入葉綠體的通道，將外源DNA送入並表現於葉綠體中。利用大腸桿菌蛋白質表現系統表現並純化由葉綠體訊息胜肽(TP)和CPP (R9)所組合的重組蛋白(TP-CPP)，而藉由Gel retardation assay可以確定此重組蛋白具有結合DNA的能力，將重組蛋白結合帶有受葉綠體啟動子調控的GFP表現卡匣的DNA片段再與純化的植物葉綠體在試管中混和反應後，利用螢光或共軛焦顯微鏡觀察GFP在葉綠體的表現。實驗結果顯示TP-CPP重組蛋白可藉由蛋白質輸入通道攜帶DNA進入葉綠體，再利用葉綠體的轉錄與轉譯系統進行表現，並在重組蛋白與DNA的N/P比值介於1至1.5時具有較佳的效率，且當DNA片段較小時效率較高，然而其效率仍低(1~6/107個)，仍有待進一步改善。未來，期望可以將此TP-CPP重組蛋白運用在葉綠體轉殖技術與葉綠體蛋白質傳輸機制的研究上。

Cell penetrating peptide (CPP), an Arg-rich small peptide can carry DNA or protein into plant cells. To explore the possibility of CPP as a DNA delivery system through Toc-Tic translocon complex to chloroplasts, the recombinant protein (TP-CPP) gene which the transit peptide (TP) from RbcS, and OsRpoTp or N-terminal of GST as a control fused with nona-arginine (R9) were respectively generated. The recombinant TP-CPP genes were cloned into pET21b vectors, and the recombinant proteins were expressed and purified from E.coli BL21 (DE3). In addition, the chloroplast gene expression vector pRCG1, carrying GFP reporter gene under the regulation of psaA promoter was previously constructed. The recombinant fusion proteins (TP-CPP) showed the DNA-binding ability by gel retardation assay. After the recombinant TP-CPP proteins were pre-mixed with pRCG1 or DNA only carrying GFP expressing cassette, the protein-DNA complex was incubated with intact chloroplasts for in vitro import assay. Subsequently, the chloroplasts expressing GFP were examined under the fluorescent or laser scanning confocal microscope. Our results demonstrated that TP-CPP can bind with DNA to form complex and be able to pass through the chloroplast translocons. Although the import efficiency is low (about 1~6 x 10-7), under the N/P (protein/DNA) ratio ranged from 1 to 1.5, the highest efficiency was observed. In addition, the efficiency for smaller cargo is better than that of large one. The efficiency of TP-CPP mediated DNA delivery to chloroplasts is still need to be improved. Hopefully, this novel delivery system can be applied in plastid transformation and the study of protein import process in the future.

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