Thesis
Using CRISPR-Cas9 to knockout brassinosteroid inactivation genes in Arabidopsis thaliana
Washington State University
Master of Science (MS), Washington State University
07/2019
DOI:
https://doi.org/10.7273/000004130
Handle:
https://hdl.handle.net/2376/125087
Abstract
Brassinosteroids (BRs) are important plant growth-promoting hormones involved in many processes throughout plant development from seed germination to flowering time. Since BRs do not undergo long-distance transport, cell- and tissue-specific regulation of hormone levels involves both biosynthesis and inactivation. To date, ten BR-inactivating enzymes, with at least five distinct biochemical activities, have been experimentally identified in the model plant Arabidopsis thaliana. Generating higher-order null mutants in these genes has been hampered by epigenetic interactions between T-DNA insertion alleles as well as due to genetic linkage. The focus of this thesis is to target some of these genes using the CRISPR-Cas9 genome editing system. For example, previous studies demonstrated that the bas1-2 sob7-1 ben1-1 triple-null mutant could not be generated due to epigenetic interactions between the exonic T-DNA insertions in bas1-2 and sob7-1 causing the intronic T-DNA insertion of ben1-1 to no longer confer a null-allele. To alleviate this problem, the CRISPR-Cas9 genome editing system was used to create bas1-2 sob7-1 ben1-3 triple-null mutant. This triple-null mutant resulted in an additive seedling long-hypocotyl phenotype. In contrast, the triple-null mutant uncovered a role for BEN1-mediated BR-inactivation in seedling cotyledon petiole elongation that was not conferred by the single ben1-2 null mutant but only in the absence of both BAS1 and SOB7. In addition, genetic analysis demonstrated that BEN1 does not contribute to the early-flowering phenotype that is redundantly controlled by BAS1 and SOB7. While targeting BEN1, an additional BR-inactivating gene UGT73C5 was simultaneously targeted using a multiplex CRISPRCas9 system. This quadruple-null bas1-2 sob7-1 ben1-3 ugt73c5-1 mutant demonstrated additional phenotypic complexity associated with BR-inactivation. The resolution of this complex interaction will likely require also targeting the genetically linked and chromosomally-adjacent UGT73C5 duplicate, UGT73C6. As a part of this thesis, three additional BR-inactivating genes, BAT1 and the BAT1-like genes BAL1 and BAL2 were also targeted using a multiplex CRISPR-Cas9 system in the quadruple-null-mutant background and the Col-0 wildtype background as a part of the Neff Lab's goal of generating higher-order null-mutant combinations for all ten BR-inactivating genes in the Arabidopsis thaliana genome.
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Details
- Title
- Using CRISPR-Cas9 to knockout brassinosteroid inactivation genes in Arabidopsis thaliana
- Creators
- Shelby Ruth Westenskow
- Contributors
- Michael Neff (Advisor) - Washington State University, Crop and Soil Sciences, Department of
- Awarding Institution
- Washington State University
- Academic Unit
- Crop and Soil Sciences, Department of
- Theses and Dissertations
- Master of Science (MS), Washington State University
- Publisher
- Washington State University
- Identifiers
- 99900890781801842
- Language
- English
- Resource Type
- Thesis