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?