Journal
PLANT MOLECULAR BIOLOGY
Volume 77, Issue 1-2, Pages 117-127Publisher
SPRINGER
DOI: 10.1007/s11103-011-9798-5
Keywords
Tn5 transposase; Intein-tag; 6xCys-tag; Transformation frequency; Single copy insertion
Categories
Funding
- Fundamental Research Funds for the Central Universities [021114340007]
- National Natural Science Foundation [20637030]
- National Science and Technology Surporting Item [2006BAJ08B01-02]
- Science and Technology Department of Jiangsu Province of The People's Republic of China [BS2007160]
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We have developed a modified particle bombardment method for plant transgenesis. An intein-tag and a 6xCys-tag were successively fused to the N-terminus of a hyperactive Tn5 transposase. The modified transposase was immobilized on bare gold microscopic particles via covalent binding of a 6xCys-tag sulfydryl groups to the gold surface. The tethered transposase can bind the transposon DNA in vitro to form the transposome in the absence of Mg2+ ions. After bombardment of the gold particles carrying the transposomes into the plant cells, the transposomes will be released from the carrier due to the activated self-cleavage function of intein-tag. Our data showed this procedure integrated foreign DNA into the plant genome with an increased transformation frequency as compared to the conventional particle bombardment method. A single copy insertion can also be obtained by decreasing of the assembled transposon DNA amount in relation to plant cell biomass.
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