The Cloning And Molecular Characterization Of Safflower FAD2Gene Family With Particular Reference On High Oleic Genotype

Safflower is an important oilseed crop that is currently grown for its high quality edibleoil used in cooking, salad dressings and margarines, and to a lesser degree as a bird seed. Theapplication and nutritional value of vegetable oil is highly dependent on its fatty acidcomposition, especially the relative proportion of its two major fatty acids, i.e oleic acid andlinoleic acid. Microsomal oleoyl phosphatidylcholine desaturase encoded by FAD2gene isknown to introduce a double bond at the Δ12position of an oleic acid on phosphatidylcholineand convert it to linoleic acid. The known plant FAD2enzymes are encoded by small genefamilies consisting of1-4members. In addition to the classic oleate Δ12-desaturation activity,functional variants of FAD2that are capable of undertaking additional or alternative acylmodifications have also been reported in a limited number of plant species. In this study, Ihave isolated an exceptionally large FAD2gene family from safflower and analysed thedifferential expression profile and diversified functionality of these genes.This thesis embodies the molecular cloning and functional characterization of the largestFAD2gene family ever reported from a higher plant. The temporal and spatial expressionprofiles of11FAD2genes were revealed through Real-Time quantitative PCR. Thediversified functionalities of the safflower FAD2gene family members were demonstrated byectopic expression in yeast and transient expression in Nicotiana benthamiana leaves.CtFAD2-1and CtFAD2-10were demonstrated to be oleate desaturases specifically expressedin developing seeds and flower head, respectively, while CtFAD2-2appears to have relativelylow oleate desaturation activity throughout the plant. CtFAD2-5and CtFAD2-8arespecifically expressed in root tissues, while CtFAD2-3,4,6,7are mostly expressed in thecotyledons and hypocotyls in young safflower seedlings. CtFAD2-9was found to encode anovel desaturase operating on C16:1substrate. CtFAD2-11is a tri-functional enzyme able tointroduce a carbon double bond in either cis or trans configuration, or a carbon triple(acetylenic) bond at the Δ12position. The seed expressed FAD2oleate Δ12desaturase genesidentified in this study will provide candidate targets to manipulate the oleic acid level insafflower seed oil. Further, the divergent FAD2enzymes with novel functionality could beused to produce rare fatty acids, such as crepenynic acid, in genetically engineered crop plantsthat are precursors for economically important phytoalexins and oleochemical products.As a part of our investigation into the molecular features of the high oleic (HO) trait insafflower we have profiled the microRNA (miRNA) populations from the developingsafflower seeds expressing the ol allele in comparison to the wild type high linoleic (HL)safflower using deep sequencing technology. The small RNA populations of the mid-maturity developing embryos of HO (olol) and wild type HL safflower (OlOl) had a very similar sizedistribution pattern but only~16.5%of the unique small RNAs were overlapping in these twogenotypes. From these two small RNA populations，we have found55known miRNAs andidentified two candidate novel miRNA families that are likely unique to the developingsafflower seeds. Target genes with conserved as well as novel functions were predicted for theconserved miRNAs. We have also identified miRNAs differentially expressed between theHO and HL safflower genotypes. This study comparatively characterised the miRNApopulations in the mid-maturity developing safflower embryos between the HO and HLgenotypes. The results may lay a foundation for unravelling the miRNA-mediated molecularprocesses that regulate oleic acid accumulation in the HO safflower mutant.To explore the temperature regulation of safflower FAD2gene family, I measured thetranscript accumulation of FAD2genes and corresponding fatty acid changes in both the wildtype and high oleic mutant at three different temperature regimes. In addition to developingseeds, roots and leaves are also used in this study. CtFAD2-1did not respond at the lowtemperature in the developing embryo of both HL and HO,they expressed lower than26℃,and exprssion increased when temperature went to30℃. It seemed that CtFAD2-11responded to the low temperature. At12℃,the expression of this gene was higher than othertwo temperature. The expression mode of CtFAD2-1,CtFAD2-2and CtFAD2-11were similar,as higher temperature higher levels.