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Determining Functionality of Arabidopsis Thaliana Genes in Embryo Development

Determining Functionality of Arabidopsis Thaliana Genes in Embryo Development. Ria Yagnik. T-DNA. intron exon UTR. 151.

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Determining Functionality of Arabidopsis Thaliana Genes in Embryo Development

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

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

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

  4. Determining Genotypes • 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 Genotyping PCR Size ~ 1.2 kb matches expected results Isolated gDNA Size ~ 300 bp ??? try expt. again

  5. Determining Genotypes • 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 Genotyping PCR Size ~ 1.2 kb matches expected results Isolated gDNA Size ~ 300 bp

  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. Where is gene active? • 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) Predicted promoter size: 2573 bp Digested plasmid colonies (contains excess EtBr)

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

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

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

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

  14. Where is gene active?

  15. Where is gene active? • 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) Predicted promoter size: 3016 bp Digested plasmid colonies

  17. Looking at the plant WT Mutant No phenotypic difference

  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

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