An improved particle bombardment for the generation of transgenic plants by direct immobilization of relleasable Tn5 transposases onto gold particles

An improved particle bombardment for the generation of transgenic plants by direct immobilization... We have developed a modified particle bombardment method for plant transgenesis. An intein-tag and a 6×Cys-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 6×Cys-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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant Molecular Biology Springer Journals

An improved particle bombardment for the generation of transgenic plants by direct immobilization of relleasable Tn5 transposases onto gold particles

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Publisher
Springer Journals
Copyright
Copyright © 2011 by Springer Science+Business Media B.V.
Subject
Life Sciences; Plant Pathology; Plant Sciences; Biochemistry, general
ISSN
0167-4412
eISSN
1573-5028
D.O.I.
10.1007/s11103-011-9798-5
Publisher site
See Article on Publisher Site

Abstract

We have developed a modified particle bombardment method for plant transgenesis. An intein-tag and a 6×Cys-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 6×Cys-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.

Journal

Plant Molecular BiologySpringer Journals

Published: Jun 4, 2011

References

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