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HC70AL Presentation: Gene-Knockout Analysis Arabidopsis Thaliana Genes –AT4g27410 –AT5g07680 Salk Lines –# 063576 –# 006735 Presented By Tim Schulz.

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Presentation on theme: "HC70AL Presentation: Gene-Knockout Analysis Arabidopsis Thaliana Genes –AT4g27410 –AT5g07680 Salk Lines –# 063576 –# 006735 Presented By Tim Schulz."— Presentation transcript:

1 HC70AL Presentation: Gene-Knockout Analysis Arabidopsis Thaliana Genes –AT4g27410 –AT5g07680 Salk Lines –# –# 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 UTRExon Promoter UTRIntron Flanking Sequence Exon (bp) FW   RV ~ 1.6kb Entire Gene (w/o T-DNA) .5kb (Salk) ~1.4kb Between Primers (w/o T-DNA) 5’ 3’

4 Structure & Location of AT5g07680 UTRExonIntron UTR Intron Flanking Sequence Exon (bp) FW   RV ~750bp (w/o T-DNA) .5kb (Salk) Exon ~ 1.6kb Entire Gene (w/o T-DNA) 5’ 3’

5 Actual T-DNA Locations AT4G27410 – Different From SALK AT5G07680 – Same as SALK Salk’s Report for AT4G27410: UTRExon Promoter UTRIntron Flanking Sequence Exon (bp) .5kb (Salk)

6 Actual T-DNA Locations AT4G27410 – Different From SALK AT5G07680 – Same as SALK My Results for AT4G27410 Show: UTRExon Promoter UTRIntron Flanking Sequence Exon (bp) .5kb (Salk) .5kb (Salk)

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

9 Actual T-DNA Location: AT4G27410 UTRExon Promoter UTRIntron Flanking Sequence Exon (bp) .5kb (Salk) .5kb (Salk) Conclusion After - Band-Size Analysis - Sequencing:

10 Genotypes: First SALK Line AT4G W/T 2. HET 3. W/T 4. W/T 5. W/T 8. W/T 7. HOMO 6. HOMO 9. HET 10. W/T 11. W/T 12. HET 13. HOMO 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

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 #

14 Smaller Plants: Mutants?

15

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

17 Genotypes: Second SALK Line AT5G W/T 2. W/T 3. W/T 4. W/T 5. HET 8. HET 7. (Too Small) 6. W/T 9. HET 10. W/T 11. W/T 12. W/T 13. HET Plant 1. W/T 2. W/T 3. W/T 4. W/T 5. Heterozygous 6. W/T Heterozygous 9. Heterozygous 10. W/T 11. W/T 12. W/T 13. Heterozygous 24. W/T

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

19 Missing Plants – Embryo Lethal? A nalysis 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? A nalysis through Microscopy Plant #8 Heterozygous Silique Image 2

21 Missing Plants – Embryo Lethal? A nalysis 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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