HC70AL Presentation: Gene-Knockout Analysis Arabidopsis Thaliana Genes AT4g27410 AT5g07680 Salk Lines # 063576 # 006735 Presented By Tim Schulz
----------------------- Outline Overall: What will knocking out these genes tell us about how they function? ----------------------- What are the characteristics of the genes and T-DNA inserts? Plant Genotypes? Plant Phenotypes? What Next?
Structure & Location of AT4g27410 .5kb (Salk) 2435876 2437500 (bp) 261 356 54 83 441 40 641 Promoter UTR Exon Intron Exon UTR Flanking Sequence 3’ 5’ + 167 FW - 331 RV ~1.4kb Between Primers (w/o T-DNA) ~ 1.6kb Entire Gene (w/o T-DNA)
Structure & Location of AT5g07680 .5kb (Salk) 2435876 2437500 (bp) 217 187 118 278 123 519 158 19 UTR Exon Intron Exon Intron Exon UTR Flanking Sequence 3’ 5’ FW RV ~750bp (w/o T-DNA) 2436771 2437540 ~ 1.6kb Entire Gene (w/o T-DNA)
Actual T-DNA Locations AT4G27410 – Different From SALK AT5G07680 – Same as SALK Salk’s Report for AT4G27410: .5kb (Salk) (bp) 261 356 54 83 441 40 641 Promoter UTR Exon Intron Exon UTR Flanking Sequence
Actual T-DNA Locations AT4G27410 – Different From SALK AT5G07680 – Same as SALK My Results for AT4G27410 Show: .5kb (Salk) .5kb (Salk) (bp) 261 356 54 83 441 40 641 Promoter UTR Exon Intron Exon UTR Flanking Sequence
Actual T-DNA Location: AT4G27410 PCR Odd Results From FW + RV + LBb1: Plants 6-13
Solution: Separate Primers LB+FW, LB+RV, RV+FW 2 2 2 4 4 4 5 5 5 6 6 6 7 7 7
Actual T-DNA Location: AT4G27410 Conclusion After - Band-Size Analysis - Sequencing: .5kb (Salk) .5kb (Salk) (bp) 261 356 54 83 441 40 641 Promoter UTR Exon Intron Exon UTR Flanking Sequence
Genotypes: First SALK Line AT4G27410 Plant 1. W/T 2. Heterozygous 3. W/T 4. W/T 5. W/T 6. Homozygous 7. W/T 8. W/T 9. Heterozygous 10. W/T 11. W/T 12. Heterozygous 13. Homozygous 1 2 1. W/T 3. W/T 5. W/T 7. HOMO 2. HET 4. W/T 6. HOMO 8. W/T 3 4 9. HET 12. HET 11. W/T 10. W/T 13. HOMO
Genotypes: Second SALK Line AT5G07680 Questions: Missing Plants? Smaller Plants?
Phenotypes? Smaller Plants – NAM Related? Missing Plants – Embryo Lethal?
Genotypes: Second SALK Line Plants # 1 2 3 4 5 6 8 9 10 11 12 13 - +
Smaller Plants: Mutants?
Smaller Plants: Mutants?
Smaller Plants: Mutants? The Test: #10 & #24 10 24 - + (Hetero DNA for Positive Control) ~750bp T-DNA
Genotypes: Second SALK Line AT5G07680 1 2 Plant 1. W/T 2. W/T 3. W/T 4. W/T 5. Heterozygous 6. W/T 7. -- 8. Heterozygous 9. Heterozygous 10. W/T 11. W/T 12. W/T 13. Heterozygous 24. W/T 1. W/T 3. W/T 5. HET 7. (Too Small) 2. W/T 4. W/T 6. W/T 8. HET 3 4 10. W/T 12. W/T 9. HET 13. HET 11. W/T
Phenotypes? Smaller Plants – NAM Related? Missing Plants – Embryo Lethal?
Missing Plants – Embryo Lethal? Analysis through Microscopy Heterozygous Silique Image 1 All Alive: No Immediate affect 3:1 Alive/Dead Ratio- Seed Development affected Zygotically 1:1 Alive/Dead Ratio- Female Gametophyte affected, post fertilization
Missing Plants – Embryo Lethal? Analysis through Microscopy Heterozygous Silique Image 2
Missing Plants – Embryo Lethal? Analysis through Microscopy Heterozygous Silique Image 2 (opened)
Missing Plants – Embryo Lethal? Heterozygous-Embryo Development Early Heart
Missing Plants – Embryo Lethal? Heterozygous-Embryo Development Heart
Missing Plants – Embryo Lethal? Heterozygous-Embryo Development Late Heart
Missing Plants – Embryo Lethal? Heterozygous-Embryo Development Torpedo
Missing Plants – Embryo Lethal? Heterozygous-Embryo Development Walking Stick
Missing Plants – Embryo Lethal? Heterozygous-Embryo Development U-Turn
Where to go from here… Promoter + GFP Multiple Knockouts (determined via microarrays) Other, non-knockout related experiments
----------------------- Conclusion Primary Goal Know Gene Functionality Primary Method T-DNA Knockout ----------------------- What are the characteristics of the genes and T-DNA inserts? Plant Genotypes? Plant Phenotypes? What Next?