Journal article
The ben1-1 brassinosteroid-catabolism mutation is unstable due to epigenetic modifications of the intronic T-DNA insertion
G3 (Bethesda, Md.), Vol.3(9), pp.1587-1595
09/04/2013
Handle:
https://hdl.handle.net/2376/104352
PMCID: PMC3755919
PMID: 23893742
Abstract
Loss-of-function genetic analysis plays a pivotal role in elucidating individual gene function as well as interactions among gene networks. The ease of gene tagging and cloning provided by transfer DNA (T-DNA) insertion mutants have led to their heavy use by the Arabidopsis research community. However, certain aspects of T-DNA alleles require caution, as highlighted in this study of an intronic insertion mutant (ben1-1) in the BEN1 (BRI1-5 ENHANCED 1) gene. As a part of our analysis of brassinosteroid catabolic enzymes, we generated a genetic triple-mutant from a cross between the bas1-2 sob7-1 double-null (T-DNA exonic insertion mutants of phyB-4 ACTIVATION TAGGED SUPPRESSOR 1 and SUPPRESSOR OF phyB-4 7) and ben1-1. As previously described, the single ben1-1 line behaves as a transcript null. However, in the triple-mutant background ben1-1 was reverted to a partial loss-of-function allele showing enhanced levels of the wild-type-spliced transcript. Interestingly, the enhanced expression of BEN1 remained stable when the ben1-1 single-mutant was reisolated from a cross with the wild type. In addition, the two genetically identical pretriple and posttriple ben1-1 mutants also differed phenotypically. The previously functional NPTII (NEOMYCIN PHOSPHOTRANSFERASE II) T-DNA marker gene (which encodes kanamycin resistance) was no longer functional in the recovered ben1-1 allele, though the length of the T-DNA insertion and the NPTII gene sequence did not change in the pretriple and posttriple ben1-1 mutants. Methylation analysis using both restriction endonuclease activity and bisulfite conversion followed by sequencing showed that the methylation status of the T-DNA is different between the original and the recovered ben1-1. These observations demonstrate that the recovered ben1-1 mutant is epigenetically different from the original ben1-1 allele.
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Details
- Title
- The ben1-1 brassinosteroid-catabolism mutation is unstable due to epigenetic modifications of the intronic T-DNA insertion
- Creators
- Kulbir Singh Sandhu - Department of Crop and Soil Sciences, Washington State University, Pullman, Washington 99164Pushpa Sharma KoiralaMichael M Neff
- Publication Details
- G3 (Bethesda, Md.), Vol.3(9), pp.1587-1595
- Academic Unit
- Crop and Soil Sciences, Department of
- Publisher
- United States
- Identifiers
- 99900546689301842
- Language
- English
- Resource Type
- Journal article