Resources for targeted insertional and deletional mutagenesis in Arabidopsis.
The maize transposons Activator (Ac) and Dissociation (Ds) are active in many monocots and dicots, including Arabidopsis. We describe a new Ac-derived transposon construct, designated the Ds-loxP T-DNA, which can be used for both insertional and deletional mutagenesis. There are loxP sites in both orientations on both the transposon and the donor site T-DNA and an arrangement of marker genes that permits selection of transposition events, as well as deletions and inversions extending from the donor site to a transposon reinserted on either side of it. We show that Cre-mediated deletions and inversions occur at a high frequency. The tendency of Ac-Ds transposons to reinsert near the donor site can be used to target both insertional and deletional mutagenesis, but efficient exploitation of this property requires a library of mapped marked donor sites distributed in the genome. We have created a population of independent Ds T-DNA transformants and we have mapped an initial set of 75 Ds T-DNA integration sites. We assessed the potential efficiency of targeted mutagenesis by detecting Ds reinsertion events at several loci over a 400 kb interval from each of two donor sites with different Ds T-DNA constructs. The distribution of reinsertion sites is similar around the two tested loci, with roughly 10, 4, and ca. 1% of reinsertions detected within 1-2 kb of sites 10, 100, and 200-400 kb from the donor site, respectively. To facilitate the use of this targeted mutagenesis system. we have constructed a searchable database of the mapped Ds T-DNA integration sites.
Zhang
Sen
S
National Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China.
Raina
Surabhi
S
Li
Hong
H
Li
Jun
J
Dec
Ewa
E
Ma
Hong
H
Huang
Hai
H
Fedoroff
Nina V
NV
D017360:Arabidopsis / Q000235:genetics*; D001287:Attachment Sites, Microbiological / Q000235:genetics; D001483:Base Sequence; D004251:DNA Transposable Elements / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D017353:Gene Deletion; D019426:Integrases / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016254:Mutagenesis, Insertional / Q000379:methods*; D030821:Plants, Genetically Modified; D010957:Plasmids / Q000235:genetics; D014170:Transformation, Genetic; D014764:Viral Proteins / Q000235:genetics
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; C036483:T-DNA; D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases