Construction of a High-efficient Expression Vector of Δ^(12) Fatty Acid Desaturase in Peanut and Its Prokaryotical Expression

A full-length sequence coding for △^12 fatty acid desaturase gene from peanut（Arachis hypogaea L.）was cloned into the expression vector, pRSETB, to generate recombinant plasmid pRSET/HO-A, which was subsequently transformed into expression Escherichia. coli BL21（DE3）pLysS. The △^12 fatty acid desaturase was highly expressed in E. coli BL21（DE3）pLysS in the presence of isopropyl-D-thiogalactopyranoside （IPTG）. The fusion protein was purified and used to form a reaction system in vitro by adding oleic acid as substrate and incubating it at 20℃ for 6 h. Total fatty acids was extracted and methlesterified and then analyzed with gas chromatography. A novel peak corresponding to linoleic acid methyl ester standards was detected with the same retention time. GC-MS （gas chromatogram and gas chromatogram-mass spectrometry） analysis showed that the novel peak was linoleic acid methyl ester. These results exhibited △^12 fatty acid desaturase activity, which could convert oleic acid to linoleic acid specifically.

Comparison of the Δ^(12) fatty acid desaturase gene between high-oleic and normal-oleic peanut genotypes

△^12 fatty acid desaturase gene has been targeted as a logical candidate controlling the high oleate trait in peanut seeds. By RT-PCR method, the full-length cDNAs of △^12 fatty acid desaturase gene were isolated from peanut （Arachis hypogaea L.） genotypes with normal and high ratio of oleic to linoleic acid, which were designated AhFAD2B and AhFAD2B＇, respectively. Sequence alignment of their coding regions revealed that an extra A was inserted at the position ＋442 bp of AhFAD2B＇ sequence of high oleic acid genotypes, which resulted in the shift of open reading frame and a truncated protein AhFAD2B＇, with the loss of one histidine box involved in metal ion complex required for the reduction of oxygen. Analysis of transcript level showed that the expression of △^12 fatty acid desaturase gene in high oleic acid genotype was slightly lower than that in normal genotype. The enzyme activity experiment of yeast （Saccharomyces cerevisiae） cell transformed with AhFAD2B or AhFAD2B＇ proved that only AhFAD2B gene product showed significant △^12 fatty acid desaturase activity, but AhFAD2B＇ gene product did not. These results suggested that the change of AhFAD2B＇ gene sequence resulted in lower activity or deactivation of △^12 fatty acid desaturase in high oleic acid genotype.

Production of γ-linolenic acid and stearidonic acid by Synechococcus sp.PCC7002 containing cyanobacterial fatty acid desaturase genes

Genetic modifi cation is useful for improving the nutritional qualities of cyanobacteria. To increase the total unsaturated fatty acid content, along with the ratio of ω-3/ω-6 fatty acids, genetic engineering can be used to modify fatty acid metabolism. S ynechococcus sp. PCC7002, a fast-growing cyanobacterium, does not contain a Δ6 desaturase gene and is therefore unable to synthesize γ-linolenic acid(GLA) and stearidonic acid(SDA), which are important in human health. In this work, we constructed recombinant vectors Syd6 D, Syd15 D and Syd6Dd15 D to express the Δ15 desaturase and Δ6 desaturase genes from Synechocystis PCC6803 in Synechococcus sp. PCC7002, with the aim of expressing polyunsaturated fatty acids. Overexpression of the Δ15 desaturase gene in S ynechococcus resulted in 5.4 times greater accumulation of α-linolenic acid compared with the wild-type while Δ6 desaturase gene expression produced both GLA and SDA. Co-expression of the two genes resulted in low-level accumulation of GLA but much larger amounts of SDA, accounting for as much to 11.64% of the total fatty acid content.

Identification and Functional Characterization of a NovelΔ12 Fatty Acid Desaturase Gene from Haematococcus pluvialis

The freshwater microalga Haematococcus pluvialis accumulates large amounts of fatty acids in response to adverse conditions.However,the key fatty acid desaturase genes in H.pluvialis remain unknown.In this study,we cloned and functionally characterized aΔ12 fatty acid desaturase gene,and designated it as HpFAD2.The open reading frame of HpFAD2 consisted of 1137 base pairs and encoded 378 amino acids.The deduced polypeptide showed 70%identity to other endoplasmic reticulumΔ12 fatty acid desaturases,whereas it had only 44%identity to plastidΔ12 fatty acid desaturases.The PSORT algorithm and phylogenetic analysis further confirmed its affiliation to the endoplasmic reticulumΔ12 fatty acid desaturases.Heterologous expression was performed in Saccharomyces cerevisiae cells transformed with the recombinant plasmid pYES2-HpFAD2.Two additional fatty acids(C16:2 and C18:2)were detected in the yeast transformants.The results indicatedΔ12 desaturation activity and substrate preference for C18:1 over C16:1.The transcriptional levels of H.pluvialis HpFAD2 at different growth stages were measured by quantitative polymerase chain reaction(PCR),indicating that the HpFAD2 transcriptional levels were significantly higher in red cells than those in green cells.Our study brings more insight into the fatty acid biosynthetic pathway of H.pluvialis.

Association of Bovine Fatty Acid Desaturase 2 Gene Single-Nucleotide Polymorphisms with Intramuscular Fatty Acid Composition in Japanese Black Steers

Beef from Japanese Black cattle (JBK), is popular in Japan and valued for its highly marbled fat content. In JBK, genes affecting oleic acid content in meat have been studied mainly to lower the fat melting point and improve tenderness;however, there has been no direct correlation demonstrated between beef taste and oleic acid. To investigate genes affecting other fatty acids other than oleic acid, polymorphisms of the fatty acid desaturase 2 (FADS2) gene were genotyped and associations with fatty acid profile in JBK beef were investigated. Amplifications of 5’-flanking regions, 12 exons, and 3’-untranslated regions of the FADS2 gene in three Japanese and five Western cattle breeds via PCR, were amplified, sequenced and SNPs were identified using specific TaqMan genotyping assay. Fatty acid composition of intramuscular adipose tissue of the Trapezius muscle was analyzed in JBK steers. Six of the 15 identified SNPs are novel and have never been registered in any public bovine SNP database. A non-synonymous SNP (rs211580559;C > T;294 Ala > Val) in exon 7 was examined in order to evaluate its association with fatty acid profiles. The data showed that highly significant association existed between rs211580559 and C18:2 (n-6) composition, and accounted for 22.3% of the variation. There were no significant relationships between rs2115-80559 and the other fatty acids. It was concluded that rs211580559 of the FADS2 gene may be a useful selection marker for reducing unfavorable volatiles generated from linoleic acid in JBK beef during the cooking process.

Origin and evolution of fatty acid desaturase genes in oil crop Brassica napus

Fatty acid(FA)desaturases,as the key enzymes in lipid metabolism,are responsible for biosynthesis of the unsaturated fatty FAs,which play important roles in maintaining cell membrane integrity and multiple stress responses.Although attention has been drawn to some plant FA desaturase genes,their global landscape in oil crops is still lacking.Here,we performed systematic characterization and phylogenomic synteny network analyses of the FA desaturase gene family in polyploid oil crop B.napus and other 54 species covering major streptophyte lineages.A total of 1653 FA desaturase genes were identified from these plant genomes.Based on the broad-scale family phylogeny and functional domains,we proposed a unified eight-group classification system for angiosperm FA desaturases,and found that the origin of genes responsible for FA desaturation evolved early and some genes were absent in different species.Phylogenomic analyses revealed deeply conserved syntenic relationships within each of the eight FA desaturase groups.B.napus contains up to 93 FA desaturase genes from the eight groups.Recurrent duplication events in Brassicaceae contributed to the expansion of FA desaturase genes in B.napus,leading to further functional diversification.These FA desaturase genes exhibited spatio-temporal specific expression patterns in different tissues of B.napus,and a set of FA desaturase genes seem to be orchestrated by key transcriptional factors during seed development,such as zf-HD,B3,GATA3,PEI1,NFYA7,YAB1 and YAB2.Altogether,our data have inferred the evolutionary trajectory of this important gene family across distinct plant lineages,providing theoretical basis for future manipulation of FA desaturase genes to improve the seed oil quality of B.napus.

Antisense-Mediated Depletion of Tomato Chloroplast Omega-3 Fatty Acid Desaturase Enhances Thermal Tolerance

A chloroplast-localized tomato （Lycopersicon esculentum Mill.） ω-3 fatty acid desaturase gene （LeFADT） was isolated and characterized with regard to its sequence, response to various temperatures, and function in antisense transgenic tomato plants. The deduced amino acid sequence had four histidine-rich regions, of which three regions were highly conserved throughout the whole ω-3 fatty acid desaturasegene family. Southern blotting analysis showed that LeFAD7was encoded by a single copy gene and had two homologous genes in the tomato genome. Northern blot showed that LeFAD7 was expressed in all organs and was especially abundant in leaf tissue. Meanwhile, expression of LeFAD7 was induced by chilling stress （4 ℃）, but was inhibited by high temperature （45 ℃）, in leaves. Transgenic tomato plants were produced by integration of the antisense LeFAD7DNA under the control of a CaMV35S promoter into the genome. Antisense transgenic plants with lower 18 ： 3 content could maintain a higher maximal photochemical efficiency （Fv/Fm） and O2 evolution rate than wild-type plants. These results suggested that silence of the LeFAD7 gene alleviated high-temperature stress. There was also a correlation between the low content of 18 ： 3 resulting from silence of the LeFAD7 gene and tolerance to high-temperature stress.

Fatty acid desaturase 1 polymorphisms are associated with coronary heart disease in a Chinese population

Background A recent genome-wide association study in Caucasians revealed that three loci （rs174547 in fatty acid desaturase 1 （FADS1）, rs2338104 near mevalonate kinase/methylmalonic aciduria, cobalamin deficiency, cblB type （MVK/MMAB） and rs10468017 near hepatic lipase （LIPC）） influence the plasma concentrations of high-density lipoprotein-cholesterol （HDL-C） and triglycerides （TG）. However, there are few reports on the associations between these polymorphisms and plasma lipid concentrations in Chinese individuals. This study aimed to evaluate the associations between these three polymorphisms with HDL-C and TG concentrations, as well as coronary heart disease （CHD） susceptibility in Chinese individuals.