gapdh research august 2009

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