Fatty Acid Desaturase Gene Of Comparative Genomics Analysis, An Important Gene Function Validation

Unsaturatedfattyacidshavemanyimportantphysiologicalfunctionsinorganism.They are necessary for membrane structure and function. They also serve as precursorsof eicosanoids, including prostaglandins and leukotrienes, and has a high antioxidantactivity, an anti-cancer activity, and some other biological activities. Fatty aciddesaturases are enzymes that introduce double bonds into the hydrocarbon chains offattyacids toproducemonounsaturated andpolyunsaturatedfattyacids. Thispaperaimsat the comparative genomics of the cyanobacterial and eukaryotic microalgal fatty aciddesaturases. In addition, CvFad2, CvFad3 and CvFad6were cloned and the function ofCvFad2 genewasstudiedfromChlorella vulgaris NJ-7.Genome-wide screening of fatty acid desaturase genes based on thegenome-sequencing project provided us a new and comprehensive insight into thecyanobacterialandeukaryoticmicroalgalPUFAs biosyntheticpathway.The filamentous or N2-fixing cyanobacteria usually possess more types of fattyacid desaturases than unicellular species. The pathway of acyl-lipid desaturation forunicellular marine cyanobacteria Synechococcus and Prochlorococcus differ from thatof other cyanobacteria, indicating different phylogenetic histories of the two generafrom othercyanobacteria isolatedfrom freshwater,soil orsymbiont.Three thermophilicunicellular strains, Thermosynechococcus elongatus BP-1 and two SynechococcusYellowstonespecies, contain only oneΔ9 desaturase incontrastwithmesophilicstrains,whichprobablyduetotheirthermichabitats.Except for the red algae Cyanidioschyzon merolae, the remaining microalgaeshare quite similar pathway to synthesize PUFAs as higher plants. There are twointerconnected pathways of fatty acid biosynthesis: the"eukaryotic"pathwayis locatedintheendoplasmicreticulum andthe"prokaryotic"pathwayislocatedinthechloroplast.Butthemajorfattyacids biosynthesizedbythesetwopathwaysforthesemicroalgaearenot the same. Cyanidioschyzon merolae possesses only three desaturase genes, two forΔ9 desaturase and one forΔ12 desaturase. The putative pathway of fatty acidbiosynthesis for Cyanidioschyzon merolae differs from that of other cyanobacteria, green algae, diatom and higher plants, which may be due to their specializedenvironmentalniche (acidichotsprings).Phylogenetic analysis of the genome sequence of Chlamydomonas reinhardtiirevealed the presence of one sequence showing significant similarity to functionallycharacterized microsomalΔ12 desaturases (59%). This gene was isolated, and itsfunctionwasverifiedbyheterologousexpression inyeast(Saccharomycescerevisiae).Threepairs of degenerate primers were designed to amplifyspecific fragments ofCvFad2, CvFad3 and CvFad6 using cDNAof Chlorella vulgaris NJ-7according to thehomologous sequences. The middle fragments of interested cDNA were obtained byRT-PCR.Moreover, the full length of the CvFad2 gene was isolated by5' RACE and 3'RACE,anditsfunctionwasverifiedbyheterologousexpressioninyeast.This is the first report on the comparative genomics of PUFAs synthetic pathwayof cyanobacteria and eukaryotic microalgae. Additonally, we isolated the genes ofCvFad2, CvFad3 and CvFad6 from Chlorella vulgaris NJ-7. This research provided afoundation for further study to analysis the expression patterns of desaturase genes andto explorethemolecularmechanismofmicroalgaladaptiontolowtemperature.