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Cloning genes involved in the flavonoid pathway of pink and white Hydrangea macrophylla

Cloning genes involved in the flavonoid pathway of pink and white Hydrangea macrophylla. Melissa DellaTorre , Bowdoin College Danial Hasani , mentor, NFU Dr. Khairy Soliman , mentor, AAMU Dr. Tongming Yin, mentor, NFU. Importance of Anthocyanins. Attract pollinators

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Cloning genes involved in the flavonoid pathway of pink and white Hydrangea macrophylla

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  1. Cloning genes involved in the flavonoid pathway of pink and white Hydrangea macrophylla Melissa DellaTorre, Bowdoin College DanialHasani, mentor, NFU Dr. KhairySoliman, mentor, AAMU Dr. Tongming Yin, mentor, NFU

  2. Importance of Anthocyanins • Attract pollinators • Environmental Adaptation • Biodiversity • Pest protection • UV protection • Powerful antioxidants (Dyer et al., 2013; Miller et al., 2011; Seeram, 2008)

  3. Flavonoid biosynthetic Pathway Determines color by producing anthocyanins Chalconeisomerase (CHI) Flavanone 3 beta-hydroxylase (F3H) Dihydroflavonolreductase (DFR) Anthocyanidin synthase (ANS) UDPG-flavonoid glucosyltransferase (UF3GT) (Miller et al., 2007)

  4. Hydrangea macrophylla Factors affecting color: • pH • Sugar content • Metal content • Temperature • Light (Tanaka et al., 2008)

  5. Objective • Determine which major flavonoid pathway genes are present in the pink and white hydrangea flower • Help to discover which gene is responsible for the color change

  6. Material • Collected 5 samples from 5 different flowers for both white and pink hydrangeas

  7. Methods 1. RNA extraction with Trizol Transferred the material to 1ml Trizol extraction buffer. Added chloroform, phenol:chloroform:isoamylalcohol (25:24:1), isopropanol, and centrifuged the samples at 14,000rpm for 10 min at 6°Cin after each was added.

  8. Methods 2. DNA Digestion Purpose: Purification of RNA extract White Molecular Molecular Pink Marker Marker 750bp 500bp 250bp

  9. Methods 3. Gel Electrophoresis Purpose: To check quality and size of RNA White Molecular Molecular Pink Marker Marker 750bp 500bp 250bp

  10. Methods 4. cDNA library construction Purpose: To turn RNA into complimentary DNA

  11. Methods 5. Polymerase Chain Reaction (PCR) Purpose: Amplification of genes

  12. Results and Discussion M DFR F3H CHI ANS UFGT M DFR F3H CHI ANS UFGT 1000bp 500bp Pink Hydrangea Gene Expression White Hydrangea Gene Expression

  13. Conclusion • There are copy number differences in CHI for pink and white hydrangea flowers • Ex Taq caused negative results for F3H and UFGT • Too many cycles resulted in extra band for ANS

  14. FutureResearch • Quantitative PCR • Gene Sequencing

  15. Acknowledgments • Dr. KhairySoliman, mentor, AAMU • DanialHassani, mentor, NFU • Dr. Tongming Yin, NFU • Dr. Moss • Dr. Wang • National Science Foundation

  16. References Bailey D. (1992) Hydrangeas. Introduction to Floriculture p365-383 Clegg M., Durbin M. (2000) Flower color variation: A model for the experimental study of evolution. PNAS 97(13):7016-7023 Dyer A., Boyd-Gerny S., McLoughlin S., Rosa M., Simonov V., Wong B. (2013) Parallel evolution of angiosperm color signals: common evolutionary pressures linked to hymenopteran vision 198:301-307 Miller R., Owens S., Rorslett B. (2011) Plants and Color: Flowers and Pollination. Optics and Laser Technology. 43(2):282-294 Seeram, Navindra P. (2008). Berry Fruits: Compositional Elements, Biochemical Activities, and the Impact of Their Intake on Human Health, Performance, and Disease. Journal of Agricultural and Food Chemistry56 (3): 627–9 Toyama-Kato Y., Yoshida K., Fujimon E., Haraguchi H., Shimizu Y., Kondo T. (2003) Analysis of metal elements of hydrangea sepals at various growing stages by ICP-AES. Biochemical Engineering Journal. 14(3):237-241

  17. Favorite Cultural Experiences TV Tower, Shanghai Massacre Site, Nanjing

  18. Favorite Cultural Experiences Dumpling Banquet, Xian Confucius Temple, Nanjing

  19. Favorite Cultural Experiences Silk Factory, Nanjing Underwater World, Nanjing

  20. Favorite Cultural Experiences Great Wall, Beijing Kung Fu Show, Beijing

  21. THANK YOU

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