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

Author:
Wu, Jun, Du, Hongwei, Liao, Xuewei, Zhao, Yu, Li, Liguan, Yang, Liuyan
Source:
Plant molecular biology 2011 v.77 no.1-2 pp. 117-127
ISSN:
0167-4412
Subject:
DNA, biomass, genome, gold, ions, transgenic plants, transposons
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.
Agid:
456188