Validation of a Putative TIR3aos Mutant Line of Arabidopsis thaliana

This project validates the previous generation of a putative tir3aos mutant in Arabidopsis thaliana. tir3 is a mutation caused by a single nucleotide change in BIG, a gene found on chromosome three of Arabidopsis. Polar auxin transport, a process essential for plant growth and development, has been shown to be perturbed in tir3 mutants. Due to this and other unique phenotypic changes observed within BIG mutants, BIG has been implicated in playing an interesting physiological role throughout the plant. Validation of the putative tir3aos mutant was done through sequencing and analysis of PCR product generation. Following literature reviews, unique primer pairs were synthesized to encompass the tir3-1 mutation and T-DNA insertion site within the aos gene. In addition, previously designed T-DNA specific primers were used in the validation of the aos mutation. Analysis of generated PCR products proved sufficient to validate presence T-DNA within the aos gene. PCR products surrounding the tir3-1 locus were cloned into PCR 2.1 vector, and sent for sequencing. Interestingly sequence analysis showed that the tir3-1 mutation was not present in the putative tir3aos mutant despite exhibiting a clear tir3 phenotype. A second tir3 allele, tir3-101, was obtained. Unique primer pairs were synthesized and following cloning into the PCR 2.1 vector PCR products were sequenced. Sequence analysis showed presence of the tir3-101 mutation. Together, these two findings proved the generation of a tir3-101aos double mutant. Currently, the f2 generation of a tir3-1 x aos cross is being screened for the production of a tir3-1aos double mutant.