Evaluation of Co-expressed Accessory Proteins in the Enhancement of T-DNA Transfer Efficiency Using Agroinfiltration Analyses on N. benthamiana

• Main Authors: Yu-Cheng Dai, 戴于政
• Other Authors: Wei-Ming Leu
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/29357580976993164303

碩士 === 國立中興大學 === 生物科技學研究所 === 97 === Agrobacterium-mediated transformation of higher plants is a well-known and powerful tool for transgene delivery to plant cells. The use of plants as expression systems for valuable recombinant proteins often involves transfer and integration of a transferred DNA, known as T-DNA, from Agrobacterium into the plant genome. In the bacteria side, various virulent proteins are involved in the assembly and export of T-DNA. After entering of T-DNA into plant cells, various accessory proteins including VirE2, VirE3, VirF, etc. imported from Agrobacterium and BTI, RAB8, VIP1, H2A, KU80, etc. provided by plant cells, are required for T-DNA’s long journey from the cell peripheral into nucleus for chromosome integration. Previous reports indicated that the transformation efficiency of T1 transgenic Arabidopsis, in which certain accessory proteins were overproduced, could be greatly promoted. Based on the sequence information, their orthologous versions were isolated from rice and named as ET (enhance transformation) genes. To evaluate that can the transformation efficiency be increased by rice ET genes in a heterologous system, Agroinfiltration of N. benthamiana was chosen to use. Two cultures of A. tumefaciens containing ET and mGFP5 genes, respectively, were mixed and co-infiltrated into the young leaves of N. benthamiana. Three days after inoculation, leaves were cut and fluorescent images were recorded. Quantification of GFP expressions revealed that three ET genes did enhance fluorescent signals to a significant level, as comparing with control experiment by using an empty vector. This result demonstrated that the transiently expressed rice ET proteins can facilitate T-DNA transferring into plant nucleus. Such a transient co-expression strategy may be used to promote the permanent transformation frequency of recalcitrant plants.