Maize transposition mediated by an arginine-rich intracellular delivery peptide

• Main Authors: Yi-Jhe Cai, 蔡宜哲
• Other Authors: Han-Jung Lee
• Format: Others
• Language: en_US
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/gnr379

碩士 === 國立東華大學 === 生物技術研究所 === 96 === Arginine-rich intracellular delivery (AID) peptide is a new strategy applied to deliver plasmid DNA into plant cells. After simple premixing the AID peptide with DNA, the new formed complex could enter the cell plasma and nucleus. Once exogenous DNA enters the nucleus, gene would be expressed. Although scientists often use agrobacterium method to do transgenic plant, the method still needs to be improved. For example, the big Ti plasmid is difficult to handle and there is unexpected gene integration. Here, we design a two-plasmid system containing transposons. One plasmid is 7.2 kb and contains maize Ac transposase, the other is 5.1 kb and contains maize Ds transposon. For observe gene integration, we subcloned a green fluorescent reporter gene into the Ds transposon. Once Ac transposase is expressed in the Ac plasmid, it will trigger the Ds transposon for transposition and integration into host genomic DNA. In our experiments, AID peptide could successfully deliver Ac/Ds transposon into mung bean root cells. After culturing 48 hours, cells could be detected green fluorescence in confocal microscopy. It could be also observed that DNA expression in Ac plasmid could enhance relative fluorescence. Besides using PCR method to detect endogenous Ds element in mung bean genomic DNA, TAIL-PCR was performed to assay the locus of Ds transposon in genome. The results were only two transposition events and the adjacent sequence was plasmid DNA. We presumed that this phenomenon is interplasmid transposition and the GFP gene didn’t integrate into genomic DNA.