hc70al presentation: gene-knockout analysis arabidopsis thaliana
DESCRIPTION
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? ----------------------- - PowerPoint PPT PresentationTRANSCRIPT
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
UTR ExonPromoter UTRIntron Flanking SequenceExon
(bp) 261 356 54 83 441 40 641
+ 167
FW
- 331
RV
~ 1.6kb Entire Gene (w/o T-DNA)
.5kb (Salk)
2435876 2437500
~1.4kb Between Primers (w/o T-DNA)
5’ 3’
Structure & Location of AT5g07680
UTR Exon Intron UTRIntron Flanking SequenceExon
(bp) 217 187 118 278 123 519 158 19
FW RV
~750bp (w/o T-DNA)
.5kb (Salk)
Exon
2435876 2437500
2436771 2437540
~ 1.6kb Entire Gene (w/o T-DNA)
5’ 3’
Actual T-DNA Locations
AT4G27410 – Different From SALKAT5G07680 – Same as SALK
Salk’s Report for AT4G27410:
UTR ExonPromoter UTRIntron Flanking SequenceExon
(bp) 261 356 54 83 441 40 641
.5kb (Salk)
Actual T-DNA Locations
AT4G27410 – Different From SALKAT5G07680 – Same as SALK
My Results for AT4G27410 Show:
UTR ExonPromoter UTRIntron Flanking SequenceExon
(bp) 261 356 54 83 441 40 641
.5kb (Salk)
.5kb (Salk)
Actual T-DNA Location: AT4G27410 PCR
Odd Results From FW + RV + LBb1:
Plants 6-13
Solution: Separate Primers
2 2 2 4 4 4 5 5 5 6 6 6 7 7 7
LB+FW, LB+RV, RV+FW
Actual T-DNA Location: AT4G27410
UTR ExonPromoter UTRIntron Flanking SequenceExon
(bp) 261 356 54 83 441 40 641
.5kb (Salk)
.5kb (Salk)
Conclusion After- Band-Size Analysis- Sequencing:
Genotypes: First SALK Line
AT4G27410
3
1 2
4
1. 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/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
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
Plants #
10 24 - + (Hetero DNA for Positive Control)
~750bp
T-DNA
Genotypes: Second SALK Line
AT5G07680
3
1 2
4
1. 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/T2. W/T 3. W/T 4. W/T 5. Heterozygous 6. W/T 7. --8. Heterozygous 9. Heterozygous 10. W/T11. W/T12. W/T 13. Heterozygous24. W/T
Phenotypes?
Smaller Plants – NAM Related?
Missing Plants – Embryo Lethal?
Missing Plants – Embryo Lethal?Analysis through Microscopy
Plant #8Heterozygous
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
Plant #8Heterozygous
Silique Image 2
Missing Plants – Embryo Lethal?Analysis through Microscopy
Plant #8Heterozygous
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 FunctionalityPrimary Method T-DNA Knockout
-----------------------• What are the characteristics of the genes
and T-DNA inserts? • Plant Genotypes?• Plant Phenotypes?• What Next?