title: Tn5 transposase-assisted transformation of indica rice.
abstract: Here, we describe experiments on Tn5 transposase-assisted transformation of indica rice. Transposomes were formed in vitro as a result of hyperactive Tn5 transposase complexing with a transposon that contained a 19-bp tetracycline operator (tetO) sequence. To form modified projectiles for transformation, the Tn10-derived prokaryotic tetracycline repressor (TetR) proteins, which can bind transposomes via the high affinity of TetR for tetO, were immobilized onto the surface of bare gold microscopic particles. These projectiles were introduced into cells of the indica rice cultivar Zhuxian B by particle bombardment. Once projectiles were inside the cell, tetracycline induced an allosteric conformational change in TetR that resulted in the dissociation of TetR from tetO, and thus generated free transposomes. Molecular evidence of transposition was obtained by the cloning of insertion sites from many transgenic plants. We also demonstrated that the introduced foreign DNA was inherited stably over several generations. This technique is a promising transformation method for other plant species as it is species independent.
authors.affiliation: State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210046, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Wu
authors.forename: Hongwei
authors.initials: H
authors.lastname: Du
authors.forename: Xuewei
authors.initials: X
authors.lastname: Liao
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhao
authors.forename: Liguan
authors.initials: L
authors.lastname: Li
authors.forename: Liuyan
authors.initials: L
authors.lastname: Yang
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics; D003001:Cloning, Molecular; D004251:DNA Transposable Elements / Q000235:genetics*; D018744:DNA, Plant / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D006046:Gold / Q000737:chemistry; D008957:Models, Genetic; D016254:Mutagenesis, Insertional; D009693:Nucleic Acid Hybridization; D012275:Oryza / Q000235:genetics* / Q000502:physiology; D030821:Plants, Genetically Modified / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D013752:Tetracycline / Q000378:metabolism; D014170:Transformation, Genetic; D019895:Transposases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D004251:DNA Transposable Elements; D018744:DNA, Plant; D012097:Repressor Proteins; C083139:Tet O resistance protein, Bacteria; C473503:Tn5 transposase; C034811:tetracycline resistance-encoding transposon repressor protein; D006046:Gold; D019895:Transposases; D013752:Tetracycline