2013 Annual Report
1a.Objectives (from AD-416):
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.
1b.Approach (from AD-416):
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.
This subordinate project is related to Objective 3 of the parent project, "Identify genes and/or molecular process that regulate seed oil concentration and composition in oilseed crops, including under abiotic stress conditions, and determine molecular mechanisms that regulate oil production pathways in plant vegetative biomass". The goal of this subordinate project is to increase the productivity of cotton by enhancing the cold tolerance of cotton seedlings. While the principle economic driver for the cotton industry is fiber production, cotton also produces a significant amount of cottonseed oil, and improving the stress tolerance of cotton seedlings would thus benefit not only fiber production, but production of seed oil as well.
Omega-3 fatty acid desaturases play a central role in the cold temperature adaptation of plants, and the long term objectives of this project are to.
1)determine the omega-3 fatty acid desaturase gene family in two diploid cotton progenitor species, Gossypsium herbaceum and G. raimondii, as well as in modern Upland cotton, G. hirsutum, which is an allotetraploid derived from the two diploid species. Knowledge of the gene family in the diploid progenitors is important for elucidating related pairs of genes in tertraploid cotton;.
2)Examine gene expression in G. hirsutum to characterize expression patterns in various organs;.
3)Measure changes in gene expression in G. hirsutum in response to cold temperature treatment of cotton seedlings to determine which genes are associated with cold temperature adaptation; and,.
4)Develop gene-specific probes for monitoring expressing of the genes in a diverse cotton population that is subjected to cold temperature treatment to identify “high performing” alleles associated with enhanced cold tolerance. These alleles would provide molecular markers for introducing this important trait into commercial varieties.
At the beginning of this reporting period, 5 different omega-3 desaturase genes were identified in G. raimondii, 6 in G. herbaceum, and 10 in G. hirsutum. Comparison of gene sequences in the diploid progenitor species revealed that each of the 5 genes in G. raimondii was also present in G. herbaceum, indicating that this is a relative ancient gene family that was present in a common progenitor prior to the split that gave rise to these two species. One of the genes in G. herbaceum, however, underwent a gene duplication event after the split from this common progenitor, resulting in the 6 genes currently in this species. Based on this analysis, the prediction was that we should find 11 genes total in the tetraploid G. hirsutum, but we initially found only 10 genes. To help identify the last remaining gene, or to confirm that there were only 10 genes, we established a collaboration with scientists at Cornell University and Brigham Young University to analyze their recent genome sequence data from a variety of cotton species, including G. hirsutum, and these studies confirmed that there should be an 11th gene in the G. hirsutum genome. Knowledge obtained from this study allowed us to design gene-specific PCR primers that were used to amplify the remaining gene from our G. hirsutum line. Thus, in total we have now confirmed the identify of 22 omega-3 desaturases in diploid and modern tetraploid cotton.
Gene-specific PCR primers capable of amplifying coding sequences from each gene in G. hirsutum were designed for eventual use in experiments to measure gene expression in cotton. But given the high degree of DNA sequence similarity between related pairs of genes in G. hirsutum, which was often greater than 97% identity, it was necessary to first test the fidelity of each primer pair to ensure that they would amplify sequences from a single gene, and not multiple genes. To test for primer fidelity, each primer pair was used in PCR reactions containing plasmid DNA templates containing either the target gene, or the most closely related non-target gene. Several different PCR conditions were also tested to help optimize primer fidelity. Using this approach we achieved gene-specific amplification for the majority of genes, and in those cases where PCR products were obtained from both plasmid templates, new PCR primers were designed and tested.
Several different methods were evaluated for optimizing RNA extraction from various cotton organs. This RNA will subsequently be used in reverse-transcription PCR (RT-PCR) reactions to measure gene expression levels for each of the 11 omega-3 desaturase genes in G. hirsutum. PCR primers for cotton actin and ubiquitin genes were designed, based on published sequences, and preliminary RT-PCR results are being compared to results already present in the literature so that experimental conditions for gene expression analysis can be standardized in our laboratory.