Location: Plant Physiology and Genetics Research
Project Number: 5347-21000-012-01-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Jan 1, 2012
End Date: Dec 31, 2013
Ten omega-3 desaturase-type genes were identified in the genome of G. hirsutum. Our first objective is to characterize expression of these genes in various cotton organs of G. hirsutum including leaf, stem, root, fiber, and flowers. Our second objective is to examine expression of these genes in cotton seedlings that have been subjected to cold temperature, with the goal of identifying which genes are associated with cold temperature adaptation in cotton.
Bioinformatics analysis will be used to design gene specific primers for each of the ten omega-3 desaturase genes, and specificity of the primers will be tested using PCR and plasmids containing each of the target genes. The expression of each gene in cotton will be determined using semi-quantitative and quantitative RT-PCR with cotton RNA as a template. The ubiquitin gene will be used as an internal control. Cotton mRNA will be extracted from leaf, stem, root, flowers and fiber of G. hirsutum using standard techniques. Expression levels of each gene, relative to the internal standard, will be measured by gel electrophoresis and ethidium bromide staining (for semi-quantitative RT-PCR) or quantified using SYBR Green technology (for quantitative PCR). G. hirsutum seeds will be planted in pots and grown under normal light and temperature regimes, then a subset of the plants will be subject to cold temperature exposure while the other will remain at normal temperatures. The fatty acid composition of experimental and control plants will be determined using GC-FID, and expression levels of each omega-3 desaturase gene will be quantified by RT-PCR. Comparison of changes in lipid composition to changes in gene expression will be used to identify which omega-3 desaturases are associated with cold temperature adaptation in cotton.