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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
Environmental Adaptation
Biodiversity
Pest protection
UV protection
Powerful antioxidants
(Dyer et al., 2013; Miller et al., 2011; Seeram, 2008)
Flavonoid biosynthetic Pathway
Determines color by producing anthocyanins
Chalcone isomerase (CHI)
Flavanone 3 beta-hydroxylase (F3H)
Dihydroflavonol reductase (DFR)
Anthocyanidin synthase (ANS)
UDPG-flavonoid glucosyl transferase (UF3GT)
(Miller et al., 2007)
Hydrangea macrophylla
Factors affecting color:
• pH
• Sugar content
• Metal content
• Temperature
• Light
(Tanaka et al., 2008)
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
Material
• Collected 5 samples from 5 different flowers
for both white and pink hydrangeas
Methods
1. RNA extraction with Trizol
Transferred the material to 1ml Trizol extraction buffer. Added chloroform,
phenol:chloroform:isoamyl alcohol (25:24:1), isopropanol, and centrifuged the
samples at 14,000rpm for 10 min at 6°Cin after each was added.
Methods
2. DNA Digestion
Purpose: Purification of RNA extract
White Molecular
Marker
Molecular
Marker
750bp
500bp
250bp
Pink
Methods
3. Gel Electrophoresis
Purpose: To check quality and size of RNA
White Molecular
Marker
Molecular
Marker
750bp
500bp
250bp
Pink
Methods
4. cDNA library construction
Purpose: To turn RNA into complimentary DNA
Methods
5. Polymerase Chain Reaction (PCR)
Purpose: Amplification of genes
Results and Discussion
M
DFR
F3H
CHI
ANS UFGT
M
DFR
F3H
CHI
ANS
1000bp
500bp
Pink Hydrangea
Gene Expression
White Hydrangea
Gene Expression
UFGT
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
Future Research
• Quantitative PCR
• Gene Sequencing
Acknowledgments
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Dr. Khairy Soliman, mentor, AAMU
Danial Hassani, mentor, NFU
Dr. Tongming Yin, NFU
Dr. Moss
Dr. Wang
National Science Foundation
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
Chemistry 56 (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
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