[Construction of an activation tagging library of Arabidopsis and cloning for mutant genes].
Activation tagging is an important strategy in plant genomics by generating gain-of-function mutants. In this work, a library of Arabidopsis mutants was constructed by in planta transformation mediated by Agrobacterium tumefaciens containing an activation tagging vector pSKI015 with herbicide Basta as a selection marker (Fig. 1). Among 20000 independent transformants, 38 lines, i.e. about 0.2% of T(1) progeny, show visible morphological phenotypic variations (Fig. 2). Results of Southern blot analysis revealed that most of the transformants have more than three copies of T-DNA insertion (Fig. 3). Plasmid rescue and TAIL-PCR were used to recover the flanking genomic sequences of mutated target genes as the first step towards mutant gene cloning (Fig. 4, 5).
Li
Zhi-Miao
ZM
Institute of Vegetable Science, Zhejiang University, Hangzhou 310029, China.
Zhang
Hai-Kuo
HK
Cao
Jia-Shu
JS
He
Zu-Hua
ZH
D017360:Arabidopsis / Q000235:genetics* / Q000254:growth & development; D003001:Cloning, Molecular; D018506:Gene Expression Regulation, Plant; D015723:Gene Library; D009154:Mutation / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000235:genetics / Q000254:growth & development; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction