1. Determining Functionality of Arabidopsis Thaliana Genes in Embryo Development Ria Yagnik

2. AT2G33480 Gene Info 1196 base pairs (bp) long 3 exons, 2 introns Oriented 5’  3’ with respect to chromosome 268 amino acids Part of the NAC protein family Sub-family = NAM (no apical meristem) Is a transcription factor Protein Info 1 68 252 330 583 673 1042 1196 intron exon UTR 5’ Chromosome/gene direction 3’ T-DNA 151 Courtesy of:

3. Primers and Band Sizes FW »»«« RV LBb1 »» Note: primers are not to scale with gene Predicted wild-type band size (FW/RV) = 1181 bp Predicted T-DNA band size (RV/LBb1) = 1009 bp T-DNA

4. Determining Genotypes Isolated gDNA Genotyping PCR DNA was isolated from plant leaves and fractionated on a gel to confirm the presence of both DNA and RNA A 0.2 concentration of that DNA stock was then amplified using PCR, gene specific primers and Hox7D primers as a control Size ~ 1.2 kb matches expected results Size ~ 300 bp ??? try expt. again

5. Isolated gDNA Genotyping PCR DNA was isolated from plant leaves and fractionated on a gel to confirm the presence of both DNA and RNA A 0.2 concentration of that DNA stock was then amplified using PCR, gene specific primers and Hox7D primers as a control Size ~ 1.2 kb matches expected results Size ~ 300 bp Determining Genotypes

6. Separation of Primers FW/RV RV/LBb1 FW/LBb1 Thus, T-DNA is oriented in the reverse direction, contrary to what SALK thought

7. Where is gene active?

8. As seen by the GeneChip data, mRNA of my gene is present in smaller amounts in the leaf than the silique However, the leaf amplification is much greater than the silique, suggesting more leaf RNA was present to become a greater quantity of cDNA This could be due to various factors, such as age of the leaf/silique, quality of RNA extracted, etc.

9. Cloning the Promoter Region Band locations 3.5 kb (TOPO vector) 2.6 kb (promoter region) Various other band fragments (different stages of partial digestion) Digested plasmid colonies (contains excess EtBr) Predicted promoter size: 2573 bp

10. Looking at the plant No observable phenotypic difference WT Mutant

11. AT5G13180 Gene Info 1273 base pairs (bp) long 3 exons, 2 introns Oriented 5’  3’ with respect to chromosome 252 amino acids Part of the NAC protein family Sub-family = NAM (no apical meristem) Is a transcription factor Protein Info 1 67 248 343 590 673 1003 1273 intron exon UTR 5’ Chromosome/gene direction 3’ T-DNA 53 Courtesy of:

12. Primers and Band Sizes FW »»«« RV LBb1 »» Note: primers are not to scale with gene Predicted wild-type band size (FW/RV) = 991 bp Predicted T-DNA band size (RV/LBb1) = 624 bp T-DNA

13. Determining Genotypes Isolated gDNA Genotyping PCR Size ~.9 kb matches expected results Size ~.6 kb matches expected results Size ~ 6 kb confirms mutant

14. Where is gene active?

15. In the GeneChip data, we see that the mRNA of my gene is present in smaller amounts in the leaf and silique However, the leaf amplification (left) is much greater than the silique (lane 3), suggesting more leaf RNA was present to become a greater quantity of cDNA This could be due to various factors, such as age of the leaf/silique, quality of RNA extracted, etc.

16. Cloning the Promoter Region Band locations 3.5 kb (TOPO vector) 3.0 kb (promoter region) Various other band fragments (different stages of partial digestion) Digested plasmid colonies Predicted promoter size: 3016 bp

17. Looking at the plant No phenotypic difference WT Mutant

18. The Big Question Is my gene critical for embryo development? Answer: From current research, AT2G33480 and AT5G13180 of Arabidopsis thaliana do not appear to be critical in the formation of embryos or seeds. However, further research must be done (for example, using multiple knockouts to account for redundancy) before their application during development can be fully determined.

19. Acknowledgements I would like to acknowledge the many many individuals who provided countless hours of their own time helping me with this project. Tomokazu Kawashima Brittan Starr Scales Mike Gaviño as well as our amazing HC70AL Dr. Anhthu Bui class: Emily, Yosuke, Rena, Jon Dr. Xingjun Wang Eric, Combiz, Tim, Joanna, and of course Yuya, Mike and Garen Dr. Bob Goldberg