gapdh research august 2009
TRANSCRIPT
PCR AMPLIFICATION, CLONING, SEQUENCE DETERMINATION, AND BIOINFORMATICS ANALYSES OF NOVEL PLANT GAPDH GENES FROM CYPERUS
ALTERNIFOLIUS, SCHEFFLERA ACTINOPHYLLA AND TROPICAL FLORA ENDEMIC TO PUERTO RICO
Lydia E. Cortes and Dr. Michael RubinDepartment of Biology and RISE Program
University of Puerto Rico at Cayey
GAPDH
Glyceraldehyde-3-phosphate dehydrogenase
Is a protein coding gene Serves to break down glucose for
energy and carbon molecules Has recently been implicated with
transcription activation and initiation of apoptosis, and also shuttling between Endoplasmic Reticulum and Golgi vesicle.
CYPERUS ALTERNIFOLIUS and SCHEFFLERA ACTINOPHYLLA
Cyperus alternifolius umbrella papyrus or
umbrella palm is a grass-like plant has eight genes
sequenced Schefflera actinophylla
Brassaia actinophylla is a tree five genes sequenced
PROBLEM
1. Are GAPDH genes conserved between Cyperus alternifolius and Schefflera actinophylla and other sequenced GAPDH genes?
What amino acid regions are similar and different between the two plants?
SIGNIFICANCE
By sequencing the gene it will be possible to study its sequence and mutations, information that could be used in future studies.
By finding the similar and different sequences between these two plants it will be possible to find the evolutionary relation between each plant and other plants where this gene is sequenced.
SPECIFIC AIMS
1. Transform and clone Cyperus alternifolius and Schefflera actinophylla GAPDH genes into E. Coli JM109.
2. Purify plasmid DNA containing cloned Cyperus alternifolius and Schefflera actinophylla GAPDH genes.
3. Determine the sequence of cloned Cyperus alternifolius and Schefflera actinophylla GAPDH genes.
4. Bioinformatics analysis of Cyperus alternifolius and Schefflera actinophylla GAPDH genes.
HYPOTHESIS
The sequence of the GAPDH gene should be similar in some sections, but at the same time they must have some differences.
EXPERIMENTAL DESIGN Transform and clone GAPDH genes
into E. Coli JM109
1 ul DNA
20 ul of competentcells
42°C 90 seconds
2 minutes
950 ulOf SOC media
30 minutes
CONTINUATION OF TRANSFORMATION
Amp plates- 900 and 50 ul
Overnight (both)LB Broth + Amp solution (100 ul of Amp and 100 ml of LB Broth)
PLASMID PURIFICATION
Resuspend in 200ulresuspension solution
250 ul lysis solution 250ul neutralization
solution
CONTINUATION PLASMID PURIFICATION
Move to tube With column
Add 200 ul matrix
2 minutes500 ul Wash solution
100 ul water1 minute
SEQUENCE DETERMINATION
CONTINUATION OF SEQUENCE DETERMINATION
10 ul of DNA + 1 ul of primer
Pipet 10 ul
Seal and mail
BIOINFORMATICS ANALYSIS
CAP 3
CONTINUATION BIOINFORMATICS
Predicting mRNA sequence
Predict protein sequence
No DNA
pGAP
Arabidopsis
MarkerCyperus
MarkerScheffl
era
PRELIMINARY RESULTS: CLONING GEL
PRELIMINARY RESULTS OF TRANSFORMATION
Type of DNA Number of cells in 50 microliters
Number of cells in 900 microliters
CYPERUS ALTERNIFOLIUS
17 440
SCHEFFLERA ACTINOPHYLLA
10 560
ACKNOWLEDGEMENTS Dr. Robert Ross Dr. Edgar Llera Yadira Ortiz RISE Students: Mayrim Bernard, Aixa Castro,
Sheydanis Díaz, Luis López, and Pedro Rodríguez
Melisa Medina Paola Montes Ana Velazquez RISE Program (R25GM59429)
PCR AMPLIFICATION, CLONING, SEQUENCE DETERMINATION, AND BIOINFORMATICS ANALYSES OF NOVEL PLANT GAPDH GENES FROM CYPERUS
ALTERNIFOLIUS, SCHEFFLERA ACTINOPHYLLA AND TROPICAL FLORA ENDEMIC TO PUERTO RICO
Lydia E. Cortes and Dr. Michael RubinDepartment of Biology and RISE Program
University of Puerto Rico at Cayey