1. Cloning and
Bioinformatics Analyses
of Novel Plant GAPDH Genes
Valeria Rivera
Dr. Michael Rubin
RISE Program, Department of Biology,
University of Puerto Rico at Cayey

2. GAPDH
 Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
 This gene codes for a protein that catalyzes a reaction of
an important pathway known as glycolysis, which is
involved in energy production in carbohydrate
metabolism.
 The human genome has an orthologous GAPDH gene.
 The gene is expressed abnormally in 21 types of cancers
and is associated with neuronal diseases (Sirover, 1999;
Altenberg and Greulich, 2004; Kim and Dang, 2005 in
Lau J, and Robinson D, 2009).

3. Glycolysis

4. Significance
 Study important genes involved in energy
production in plants.
 Compare GAPDH genes from various plants
to study their similarities and differences.
 Analyze unique DNA sequences that could be
published in GenBank.
 Introduce important techniques in Biotechnology in
an educational setting.

5. Problem
 Do GAPDH proteins have conserved amino acids
related to the active site involved in catalytic
activity and other conserved amino acids related to
protein interactions?
 These amino acids are important in the formation
and function of the GAPDH protein.

6. Hypothesis
 GAPDH proteins will have conserved amino acids
148, 149, 150, 176, 209, and 231 related to the active
site involved in catalytic function. Other conserved
sites include the binding of the GAP substrate
(amino acids 179, 181, and 231), NAD+ (amino acids
8, 10, 11, 32, 96, and 313), and phosphate binding
(held by amino acids 148, 150, and 208).
 GAPDH proteins will have different amino acids
at some other positions not related to the active site
involved in catalytic activity and other sites
important for protein function.

7. Oxalis corniculata
Sleeping Beauty
 Juice made from
the leaves is used
as a treatment for
dysentery
 Helps relieve pain
and inflammation.
http://www.ppws.vt.edu/scott/weed_id/oxast.htm

8. Plectranthus amboinicus
Oregano brujo
 Used for cooking
 Gives flavor to
meats
http://www.bitterrootrestoration.com/medicin
al-plants/plectranthus-amboinicus.html

9. Method
 DNA was isolated.
 Primers were used to amplify GAPDH genes.
 PCR product was purified and ligated into pJET vector and
transformed into E.coli.
 DNA was purified and digested with endonuclease BglII.
 Digests were visualized using Gel electrophoresis.
 Large Scale Midi Plasmid Preps were purified.
 The cloned GAPDH amplified PCR products were
sequenced using Sanger Methodology.
 The GAPDH sequences were analyzed using Bioinformatics
(Cap 3, VecScreen, and BLAST)

10. Bacterial Cultivation and DNA Purification
Transform into E. coli Cultivate Bacteria
and extract Quantify DNA using a
plasmid DNA Spectrophotometer
BglII Restriction
Digestion Incubate for 2 hours at 37°C

11. Agarose Gel Electrophoresis
Gel Picture
Agarose Electrophoresis

12. DNA Sequencing
Automated Sequence
Add Primers to Automated Sequencing Reactions Determination
Plasmid DNAs
Chromatogram and Base Calling

13. Cloning of Plant GAPDH PCR Products
F1 F2 F3 G1 G2 G3

14. BglII Restriction F1 G1 1 Kb
Endonuclease Digestion
F1 O. corniculata 1Kb
G1 P. amboinicus 1.5Kb
1kb ladder

15. Prediction of O. corniculata mRNA
Using BLAST
Plasmid
F1

16. O. corniculata

17. Prediction of P. amboinicus
mRNA Using BLAST
Plasmid G1

18. P. amboinicus

19. Conclusion
 Midi-Prep plasmid DNAs were purified and quantified
for cloned GAPDH PCR products from O. corniculata
and P. amboinicus genomic DNAs.
 DNA samples were digested with the restriction
endonuclease BglII and visualized using agarose gel
electrophoresis to confirm that the DNAs were cloned.
 Sequences were determined for cloned GAPDH PCR
products from O.corniculata and P. amboinicus.
 Preliminary Bioinformatics analyses revealed the cloned
genes to be: GAPC (O. corniculata) and GAPDH (P.
amboinicus)

20. Future Directions
 Careful and thorough in depth Bioinformatics
analyses of the sequenced GAPDH PCR products
from O. corniculata and P. amboinicus genomic
DNAs.
 Determine intron and exon boundaries
 Phylogenetic analyses of plant GAPDH genes
 Structural modeling using novel GAPDH protein
sequences
 Submit novel GAPDH sequences from O. corniculata
and P. amboinicus to GenBank DNA database

21. Acknowledgements
 RISE Program (GM59429-13)
 Dr. Michael Rubin
 Melisa Medina
 Dr. Elena Gonzalez
 Yadira Ortiz

22. Cloning and
Bioinformatics Analyses
of Novel Plant GAPDH Genes
Valeria Rivera
Dr. Michael Rubin
RISE Program, Department of Biology,
University of Puerto Rico at Cayey