Cloning and Sequencing a Portion of Glyceraldehyde-3-phospate Dehydrogenase Gene from the
Leaves of Soleirolia Soleirolii
December 13, 2013
Without energy, plants cease to exist. Energy is a vital part in the process known as photosynthesis. In this pathway of photosynthesis, glycolysis helps yields ATP and NADH. The ATP released through glycolysis is energy that is reused to go through another production of photosynthesis. The Glyceraldehyde 3-phsophate dehydrogenase (GAPDH) gene is important in the process of glycolysis. Glycolysis is involved in energy production in carbohydrate metabolism. It helps breakdown glucose into pyruvate, thus releasing energy. GAPDH is located in the genome of plants and codes for an enzyme that catalyzes the process of glycolysis. The enzyme catalyzes NAD+, which produces an intermediate of 1,3-biphosphoglycerate (Niu et al, 1994). GAPDH is also involved in endocytosis in the cytoplasm of a plant cell. In the nucleus, it not only takes part in tRNA export but it is also involved in the translational level of gene expression through DNA replication and repair (Sirover, 1997).
In these series of lab experiments preformed throughout this project, the main objective was to isolate, sequence and clone GAPDH. The GAPDH will be extracted from a plant called soleirolia soleirolii, commonly known by it’s household name, baby tears. Another plant that will be looked at closely is Arabidopsis. This plant will act as the control throughout the experiment. The first step of this experiment is to extract the GAPDH gene from the plant. Column chromatography helps in separation and determination of compounds such as amino acids, sugars, phosphate esters and nucleotides (Thomas, 1972). Compounds that are seperated in chromatrography require elution through the use of organic buffers (Thomas, 1972). Lysis buffer and ethanol will be the two aqueous solutions that will aid in the extraction of DNA. Lysis buffer will help breakdown the cell wall of the plant cells, helping to separate the contents of the cell. Another important aqueous solution is the wash buffer. Centrifuging the DNA solution with the wash buffer eludes the DNA, which should result in a successful extraction of the GAPDH gene.
Once the DNA is extracted, Polymerase chain reaction (PCR) will be conducted. This procedure consists of repeated cycles of heating and cooling to make a copy of a specific region of the DNA from the plant. During the PCR process, the temperature is raised which causes the DNA to denature into two strands. Once the temperature is lowered, DNA primers bind to the complementary DNA sequence. After 30 cycles of epeated denaturing, annealing and extending of DNA, a billion copies of the target sequence are made just from that one molecule (Polymerase Chain Reaction, 2012). After the initial PCR, the nested PCR procedure comes next. This helps amplify the DNA that was copied in the initial PCR.