Overexpression Of Arabidopsis WIN1 Affects The Composition Of Long Fatty Acids In Saccharomyces Cerevisiae

Wax form a continuous lipid membrance covering the epidermal cells of all aerial plant organs. It mainly comprised of very-lony-chain aliphatic compounds derived from very long chain fatty acid(sC₂₀—C₆₀).The chemical and physical properties of cuticular waxes indicate that they have vital functions for plant life. The primary role of cuticular waxes is to restrict non-stomatal waterloss. Waxes also have many other functions, such as protection against UV radiation and resisting bacterial and fungal pathogenes. Many models have been put out to elucidate these processes of wax biosynthesis and wax secretion.Fatty acid of Saccharomyces cerevisiae is the important component of cell structure in yeast such as the lipid bilayer of plasma membrance and nero membrance, and the proportion of fatty acid is about 5% of the total weight. Lipid contains many kinds of fatty acid, from C₈ to C₂₆, and mainly includes saturation and unsaturation fatty acids of C₁₆ and C₁₈, in which suboleic acid and flax acid is the integrant to mammal and the content is high. The very-long-chain-fatty-acids(VLCFA,≥20C)are the components of many lipid complexes such as N-stearoy-sphingosin, sphigolipid, phospfatidylinositol and so on. Studies indicate the important role of VLCFAs to the structure, function and liquidity of cell membrane.WIN1 gene is a transcriptional activator of AP2 domain in Arabidopsis. Overexpresstion of WIN1 gene could increase plant cuticular wax load on aerial organ and change the composition of the cuticular wax in transgenic plants.Whereas, downregulation of WIN1 expression could bring reverse effect. For further study the function of WIN1, we overexpressed WIN1 gene in Saccharomyces cerevisiae and analysised the change of the composition of long fatty acids in Saccharomyces cerevisiae.Frist, we acquire the whole sequence of WIN1 cDNA from GenBank and design the special primers. Second, WIN1 gene was cloned by reverse transcriptase PCR from the flower RNA in Arabidopsis. In comparison with the sequence from GenBank, the seqence is 100% consistently. Third, the overexpression vector (p416TEF-WIN1) was constructed by linking the gene WIN1 into the yeast vector (p416TEF) between the EcoRâ… and Salâ… . The wild type yeast (K601) was transformed with p416TEF-WIN1 and p416TEF by LiAc method. The results showed that the transgenic yeast K601 (p416TEF-WIN1) grew slower than K601 (p416TEF) in SD medium for 48h at 30℃. By photics microscope, the cell of K601 (p416TEF) could form a string and the cell is smaller than the yeast which is transfered in empty vector (p416TEF). The total fatty acids were extracted and the analysis was performed by GC^MS. The content of the long chain fatty acids and VLCFAs have some change .The unsaturation fatty acid C₁₆:1 reduced 10% and C₁₈:1 increased slightly. The saturation fatty acids such as C₁₆:0 and C18:0 increased slightly. The VLCFAs have some obvious change, C₂₀ increased about 20%, C₂₂ reduced about 50% and C₂₄ reduced slightly. But no significant changes in content of the other VLCFAs such as C₂₆ were observed.In this research, we could be sure that overexpression WIN1gene would bring the abnormity of yeast growth and the change of fatty acid composition in yeast. Besides, the expressions of some genes of fatty acid metabolism are regulated by the WIN1gene. So we will hold on study the mechanism of WIN1 in the future.The main innovation point: The transgenic yeast overexpressing WIN1 was generated. It was the first time to indicate that overexpression of WIN1 in yeast affect the growth and fatty acid composition of the yeast.