An Arabidopsis thaliana T-DNA mutagenized population (GABI-Kat) for flanking sequence tag-based reverse genetics

被引:615
作者
Rosso, MG [1 ]
Li, Y [1 ]
Strizhov, N [1 ]
Reiss, B [1 ]
Dekker, K [1 ]
Weisshaar, B [1 ]
机构
[1] Max Planck Inst Plant Breeding Res, GABI Kat, D-50829 Cologne, Germany
关键词
FST; gene function; insertion mutagenesis; knock-out; reverse genetics;
D O I
10.1023/B:PLAN.0000009297.37235.4a
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The GABI-Kat population of T-DNA mutagenized Arabidopsis thaliana lines with sequence-characterized insertion sites is used extensively for efficient progress in plant functional genomics. Here we provide details about the establishment of the material, demonstrate the population's functionality and discuss results from quality control studies. T-DNA insertion mutants of the accession Columbia (Col-0) were created by Agrobacterium tumefaciens-mediated transformation. To allow selection of transformed plants under greenhouse conditions, a sulfadiazine resistance marker was employed. DNA from leaves of T(1) plants was extracted and used as a template for PCR-based amplification of DNA fragments spanning insertion site borders. After sequencing, the data were placed in a flanking sequence tag (FST) database describing which mutant allele was present in which line. Analysis of the distribution of T-DNA insertions revealed a clear bias towards intergenic regions. Insertion sites appeared more frequent in regions in front of the ATG and after STOP codons of predicted genes. Segregation analysis for sulfadiazine resistance showed that 62% of the transformants contain an insertion at only one genetic locus. In quality control studies with gene-specific primers in combination with T-DNA primers, 76% of insertions could be confirmed. Finally, the functionality of the GABI-Kat population was demonstrated by exemplary confirmation of several new transparent testa alleles, as well as a number of other mutants, which were identified on the basis of the FST data.
引用
收藏
页码:247 / 259
页数:13
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