Gene identification with sequenced T-DNA tags generated by transformation of Arabidopsis cell suspension.
A protocol for establishment and high-frequency Agrobacterium-mediated transformation of morphogenic Arabidopsis cell suspensions was developed to facilitate saturation mutagenesis and identification of plant genes by sequenced T-DNA tags. Thirty-two self-circularized T-DNA tagged chromosomal loci were isolated from 21 transgenic plants by plasmid rescue and long-range inverse polymerase chain reaction (LR-iPCR). By bidirectional sequencing of the ends of T-DNA-linked plant DNA segments, nine T-DNA inserts were thus localized in genes coding for the Arabidopsis ASK1 kinase, cyclin 3b, J-domain protein, farnesyl diphosphate synthase, ORF02, an unknown EST, and homologues of a copper amine oxidase, a peripheral Golgi protein and a maize pollen-specific transcript. In addition, 16 genes were identified in the vicinity of sequenced T-DNA tags illustrating the efficiency of genome analysis by insertional mutagenesis.
Mathur
J
J
Max-Planck Institut für Züchtungsforschung, Köln, Germany.
Szabados
L
L
Schaefer
S
S
Grunenberg
B
B
Lossow
A
A
Jonas-Straube
E
E
Schell
J
J
Koncz
C
C
Koncz-Kálmán
Z
Z
D017360:Arabidopsis / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics*; D018744:DNA, Plant / Q000235:genetics*; D017343:Genes, Plant; D005822:Genetic Vectors; D018745:Genome, Plant; D016254:Mutagenesis, Insertional; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction; D012231:Rhizobium / Q000235:genetics; D016324:Sequence Tagged Sites; D014170:Transformation, Genetic
D017931:DNA Primers; D004269:DNA, Bacterial; D018744:DNA, Plant; C036483:T-DNA