Use of Q-PCR to Monitor Expression of Vegetative Lipoxygenase in Soybeans
Location
CSU 253/4/5
Start Date
5-4-2010 10:00 AM
End Date
5-4-2010 12:00 PM
Student's Major
Chemistry and Geology
Student's College
Science, Engineering and Technology
Mentor's Name
James Rife
Mentor's Department
Chemistry and Geology
Mentor's College
Science, Engineering and Technology
Description
Lipoxygenases (LOX) catalyze the addition of molecular oxygen to 1, 4-pentadiene systems in polyunsaturated fatty acids to form hydroperoxides. Soybeans have many different LOX isoforms. Three different isoforms have been found in the seeds while at least seven distinct isoforms in vegetative tissue have been reported. Several functions have been proposed for these enzymes including production of defense molecules, lipid metabolism and nitrogen storage. The focus of this project was to explore the roles of the vegetative LOX isoforms in soybeans by measuring their expression in different tissues at different developmental stages. The effects of wounding and nitrogen supplementation on LOX expression were also studied. RNA was isolated from roots, stems, cotyledons and leaves. Reverse transcription was used to make cDNA copies of the mRNAs. Quantitative Polymerase Chain Reaction (Q-PCR) was conducted to measure relative quantities of these cDNAs from different samples. Primers were designed to selectively amplify LOX 5, 6, 7 and 9 cDNAs and successfully amplified LOX 5, 7 and 9. Efficiency studies indicated that Q- PCR could quantify expression of these different LOX enzymes over a range of four orders of magnitude. Melt curves signified that the primers successfully discriminated between the different LOX mRNAs. Leaves from a control plant at ten days post germination suggested different amounts of each of the forms, with LOX 5 expression being the largest. Tissue has been harvested from plants at different developmental stages, wounding conditions and levels of nitrogen supplementation for future Q-PCR analysis.
Use of Q-PCR to Monitor Expression of Vegetative Lipoxygenase in Soybeans
CSU 253/4/5
Lipoxygenases (LOX) catalyze the addition of molecular oxygen to 1, 4-pentadiene systems in polyunsaturated fatty acids to form hydroperoxides. Soybeans have many different LOX isoforms. Three different isoforms have been found in the seeds while at least seven distinct isoforms in vegetative tissue have been reported. Several functions have been proposed for these enzymes including production of defense molecules, lipid metabolism and nitrogen storage. The focus of this project was to explore the roles of the vegetative LOX isoforms in soybeans by measuring their expression in different tissues at different developmental stages. The effects of wounding and nitrogen supplementation on LOX expression were also studied. RNA was isolated from roots, stems, cotyledons and leaves. Reverse transcription was used to make cDNA copies of the mRNAs. Quantitative Polymerase Chain Reaction (Q-PCR) was conducted to measure relative quantities of these cDNAs from different samples. Primers were designed to selectively amplify LOX 5, 6, 7 and 9 cDNAs and successfully amplified LOX 5, 7 and 9. Efficiency studies indicated that Q- PCR could quantify expression of these different LOX enzymes over a range of four orders of magnitude. Melt curves signified that the primers successfully discriminated between the different LOX mRNAs. Leaves from a control plant at ten days post germination suggested different amounts of each of the forms, with LOX 5 expression being the largest. Tissue has been harvested from plants at different developmental stages, wounding conditions and levels of nitrogen supplementation for future Q-PCR analysis.
Recommended Citation
Wageman, Sarah. "Use of Q-PCR to Monitor Expression of Vegetative Lipoxygenase in Soybeans." Undergraduate Research Symposium, Mankato, MN, April 5, 2010.
https://cornerstone.lib.mnsu.edu/urs/2010/poster-session-A/9
