Study the import of DNA into chloroplasts by cell-penetrating peptides

• Main Authors: Yi-KuanChen, 陳怡寬
• Other Authors: Ching-Chung Chang
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/52hpca

碩士 === 國立成功大學 === 生物科技研究所 === 102 === 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.