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HC70AL Presentation: Gene-Knockout Analysis Arabidopsis Thaliana

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? -----------------------

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HC70AL Presentation: Gene-Knockout Analysis Arabidopsis Thaliana

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  1. HC70AL Presentation:Gene-Knockout AnalysisArabidopsis Thaliana • Genes • AT4g27410 • AT5g07680 • Salk Lines • # 063576 • # 006735 Presented By Tim Schulz

  2. 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?

  3. Structure & Location of AT4g27410 .5kb (Salk) 2437500 2435876 (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)

  4. Structure & Location of AT5g07680 .5kb (Salk) 2437500 2435876 (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)

  5. 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

  6. 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

  7. Actual T-DNA Location: AT4G27410 PCR Odd Results From FW + RV + LBb1: Plants 6-13

  8. Solution: Separate Primers LB+FW, LB+RV, RV+FW 2 2 2 4 4 4 5 5 5 6 6 6 7 7 7

  9. 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

  10. Genotypes: First SALK Line AT4G27410 Plant 1. W/T2. Heterozygous3. W/T 4. W/T 5. W/T6. Homozygous7. W/T8. W/T 9. Heterozygous 10. W/T11. W/T12. 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

  11. Genotypes: Second SALK Line AT5G07680 Questions: Missing Plants? Smaller Plants?

  12. Phenotypes? Smaller Plants – NAM Related? Missing Plants – Embryo Lethal?

  13. Genotypes: Second SALK Line Plants # 1 2 3 4 5 6 8 9 10 11 12 13 - +

  14. Smaller Plants: Mutants?

  15. Smaller Plants: Mutants?

  16. Smaller Plants: Mutants?The Test: #10 & #24 Plants # 10 24 - + (Hetero DNA for Positive Control) • ~750bp  T-DNA

  17. Genotypes: Second SALK Line AT5G07680 1 2 Plant 1. W/T2. W/T 3. W/T 4. W/T 5. Heterozygous 6. W/T7. --8. Heterozygous 9. Heterozygous 10. W/T11. W/T12. W/T 13. Heterozygous24. 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

  18. Phenotypes? Smaller Plants – NAM Related? Missing Plants – Embryo Lethal?

  19. Missing Plants – Embryo Lethal?Analysis through Microscopy Plant #8 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

  20. Missing Plants – Embryo Lethal?Analysis through Microscopy Plant #8 Heterozygous Silique Image 2

  21. Missing Plants – Embryo Lethal?Analysis through Microscopy Plant #8 Heterozygous Silique Image 2 (opened)

  22. Missing Plants – Embryo Lethal?Heterozygous-Embryo Development Early Heart

  23. Missing Plants – Embryo Lethal?Heterozygous-Embryo Development Heart

  24. Missing Plants – Embryo Lethal?Heterozygous-Embryo Development Late Heart

  25. Missing Plants – Embryo Lethal?Heterozygous-Embryo Development Torpedo

  26. Missing Plants – Embryo Lethal?Heterozygous-Embryo Development Walking Stick

  27. Missing Plants – Embryo Lethal?Heterozygous-Embryo Development U-Turn

  28. Where to go from here… • Promoter + GFP • Multiple Knockouts (determined via microarrays) • Other, non-knockout related experiments

  29. 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?

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