Research Progress of Fatty Acid Desaturase 2 Gene in Glycolipid Metabolism / 中国医学科学院学报

The fatty acid desaturase 2 (FADS2) gene encodes delta-6 desaturase (D6D) and is a member of the fatty acid desaturase gene family.D6D is the key enzyme catalyzing the transformation of linoleic acid and α-linolenic acid to long-chain polyunsaturated fatty acid (LC-PUFA).LC-PUFA play a crucial role in regulating the glycolipid metabolism of living organisms.In recent years,the activity of D6D and the single nucleotide polymorphism (SNP) of FADS2 gene have become a hot topic in the research on glycolipid metabolism.This article reviews the role of FADS2 gene in glycolipid metabolism.

Construction of transgenic mice with Δ15 Des enzyme activity by using a PiggyBac transposon / 生物工程学报

Essential fatty acids are those that could not be synthesized by the body itself but crucial for health and life. Studies have shown that ω-3 fatty acids may facilitate human physiological functions. Mammals lack ω-3 desaturase gene, and the Δ15 fatty acid desaturase (Δ15 Des) from Caenorhabditis elegans can transform the ω-6 polyunsaturated fatty acids (PUFAs) into ω-3 PUFAs. Transgenic mice expressing Δ15 Des enzyme activity was constructed by using a PiggyBac transposon (PB). Homozygous transgenic mice with stable inheritance was bred in a short time, with a positive rate of 35.1% achieved. The mice were fed with 6% ω-6 PUFAs and the changes of fatty acids in mice were detected by gas chromatography (GC). The expression level of Δ15 Des in mice was detected by quantitative PCR (qPCR) and Western blotting (WB). qPCR and GC analysis revealed that the percentage of positive mice harboring the active gene was 61.53%. Compared with traditional methods, the transformation efficiency and activity of Δ15 Des were significantly improved, and homozygotes showed higher activity than that of heterozygotes. This further verified the efficient transduction efficiency of the PiggyBac transposon system.

Identification and functional analysis of soybean stearoyl-ACP Δ⁹ desaturase (GmSAD) gene family / 生物工程学报

Stearoyl-ACP Δ⁹ desaturase (SAD) catalyzes the synthesis of monounsaturated oleic acid or palmitoleic acid in plastids. SAD is the key enzyme to control the ratio of saturated fatty acids to unsaturated fatty acids in plant cells. In order to analyze the regulation mechanism of soybean oleic acid synthesis, soybean (Glycine max) GmSAD family members were genome-wide identified, and their conserved functional domains and physicochemical properties were also analyzed by bioinformatics tools. The spatiotemporal expression profile of each member of GmSADs was detected by qRT-PCR. The expression vectors of GmSAD5 were constructed. The enzyme activity and biological function of GmSAD5 were examined by Agrobacterium-mediated transient expression in Nicotiana tabacum leaves and genetic transformation of oleic acid-deficient yeast (Saccharomyces cerevisiae) mutant BY4389. Results show that the soybean genome contains five GmSAD family members, all encoding an enzyme protein with diiron center and two conservative histidine enrichment motifs (EENRHG and DEKRHE) specific to SAD enzymes. The active enzyme protein was predicted as a homodimer. Phylogenetic analysis indicated that five GmSADs were divided into two subgroups, which were closely related to AtSSI2 and AtSAD6, respectively. The expression profiles of GmSAD members were significantly different in soybean roots, stems, leaves, flowers, and seeds at different developmental stages. Among them, GmSAD5 expressed highly in the middle and late stages of developmental seeds, which coincided with the oil accumulation period. Transient expression of GmSAD5 in tobacco leaves increased the oleic acid and total oil content in leaf tissue by 5.56% and 2.73%, respectively, while stearic acid content was reduced by 2.46%. Functional complementation assay in defective yeast strain BY4389 demonstrated that overexpression of GmSAD5 was able to restore the synthesis of monounsaturated oleic acid, resulting in high oil accumulation. Taken together, soybean GmSAD5 has strong selectivity to stearic acid substrates and can efficiently catalyze the biosynthesis of monounsaturated oleic acid. It lays the foundation for the study of soybean seed oleic acid and total oil accumulation mechanism, providing an excellent target for genetic improvement of oil quality in soybean.

Deficiency or activation of peroxisome proliferator-activated receptor α reduces the tissue concentrations of endogenously synthesized docosahexaenoic acid in C57BL/6J mice

BACKGROUND/OBJECTIVES: Docosahexaenoic acid (DHA), an n-3 long chain polyunsaturated fatty acid (LCPUFA), is acquired by dietary intake or the in vivo conversion of α-linolenic acid. Many enzymes participating in LCPUFA synthesis are regulated by peroxisome proliferator-activated receptor alpha (PPARα). Therefore, it was hypothesized that the tissue accretion of endogenously synthesized DHA could be modified by PPARα. MATERIALS/METHODS: The tissue DHA concentrations and mRNA levels of genes participating in DHA biosynthesis were compared among PPARα homozygous (KO), heterozygous (HZ), and wild type (WT) mice (Exp I), and between WT mice treated with clofibrate (PPARα agonist) or those not treated (Exp II). In ExpII, the expression levels of the proteins associated with DHA function in the brain cortex and retina were also measured. An n3-PUFA depleted/replenished regimen was applied to mitigate the confounding effects of maternal DHA. RESULTS: PPARα ablation reduced the hepatic Acox, Fads1, and Fads2 mRNA levels, as well as the DHA concentration in the liver, but not in the brain cortex. In contrast, PPARα activation increased hepatic Acox, Fads1, Fads2 and Elovl5 mRNA levels, but reduced the DHA concentrations in the liver, retina, and phospholipid of brain cortex, and decreased mRNA and protein levels of the brain-derived neurotrophic factor in brain cortex. CONCLUSIONS: LCPUFA enzyme expression was altered by PPARα. Either PPARα deficiency or activation-decreased tissue DHA concentration is a stimulus for further studies to determine the functional significance.

Development and application of KASP marker for high throughput detection of AhFAD2 mutation in peanut

Background: Cultivated peanut (Arachis hypogaea L.) is a major oilseed crop worldwide. Fatty acid composition of peanut oil may affect the flavor and shelf life of the resulting food products. Oleic acid and linoleic acid are the major fatty acids of peanut oil. The conversion from oleic acid to linoleic acid is controlled by theΔ12 fatty acid desaturase (FAD) encoded byAhFAD2AandAhFAD2B, two homoeologous genes from A and B subgenomes, respectively. One nucleotide substitution (G:C→A:T) ofAhFAD2Aand an "A" insertion ofAhFAD2Bresulted in high-oleic acid phenotype. Detection ofAhFAD2mutation had been achieved by cleaved amplified polymorphic sequence (CAPS), real-time polymerase chain reaction (qRT-PCR) and allele-specific PCR (AS-PCR). However, a low cost, high throughput and high specific method is still required to detectAhFAD2genotype of large number of seeds. Kompetitive allele specific PCR (KASP) can detect both alleles in a single reaction. The aim of this work is to develop KASP for detectionAhFAD2genotype of large number of breeding materials. Results: Here, we developed a KASP method to detect the genotypes of progenies between high oleic acid peanut and common peanut. Validation was carried out by CAPS analysis. The results from KASP assay and CAPS analysis were consistent. The genotype of 18 out of 179 BC4F2seeds was aabb. Conclusions: Due to high accuracy, time saving, high throughput feature and low cost, KASP is more suitable fordeterminingAhFAD2genotype than other methods.

Heart Rate Variability Correlates to Functional Aerobic Impairment in Hemodialysis Patients / Variabilidade RR ao Esforço Correlaciona-se com Déficit Funcional Aeróbico em Pacientes em Hemodiálise

Background: Autonomic dysfunction (AD) is highly prevalent in hemodialysis (HD) patients and has been implicated in their increased risk of cardiovascular mortality. Objective: To correlate heart rate variability (HRV) during exercise treadmill test (ETT) with the values obtained when measuring functional aerobic impairment (FAI) in HD patients and controls. Methods: Cross-sectional study involving HD patients and a control group. Clinical examination, blood sampling, transthoracic echocardiogram, 24-hour Holter, and ETT were performed. A symptom-limited ramp treadmill protocol with active recovery was employed. Heart rate variability was evaluated in time domain at exercise and recovery periods. Results: Forty-one HD patients and 41 controls concluded the study. HD patients had higher FAI and lower HRV than controls (p<0.001 for both). A correlation was found between exercise HRV (SDNN) and FAI in both groups. This association was independent of age, sex, smoking, body mass index, diabetes, and clonidine or beta-blocker use, but not of hemoglobin levels. Conclusion: No association was found between FAI and HRV on 24-hour Holter or at the recovery period of ETT. Of note, exercise HRV was inversely correlated with FAI in HD patients and controls. (Arq Bras Cardiol. 2015; [online]. ahead print, PP.0-0) .

Fundamento: A disfunção autonômica (DA) é altamente prevalente em pacientes em hemodiálise (HD) e tem sido implicada no risco aumentado de mortalidade cardiovascular. Objetivo: Correlacionar a variabilidade RR (VRR) durante o teste ergométrico (TE) com o déficit funcional aeróbico (FAI) em pacientes em HD e em um grupo controle. Métodos: Trata-se de um estudo transversal no qual as variáveis analisadas foram obtidas através de exame clínico, coleta de sangue, ecocardiograma transtorácico, Holter de 24 horas e TE. Foi realizado TE em esteira pelo protocolo de rampa, limitado por sintomas, com recuperação ativa. A VRR foi avaliada no domínio do tempo no exercício e na recuperação separadamente. Resultados: Quarenta e um pacientes em HD e 41 controles concluíram o estudo. Pacientes em HD tinham maior FAI e menor VRR do que os controles (p <0,001 para ambos). Houve correlação entre FAI e VRR no exercício (SDNN) em ambos os grupos. Esta associação foi independente de idade, sexo, tabagismo, índice de massa corporal, diabetes, clonidina, betabloqueador, mas não dos níveis de hemoglobina. Conclusão: A VRR no exercício foi inversamente correlacionada com o FAI em pacientes em HD e controles. Não foram observadas associações do FAI com VRR no Holter ou no período de recuperação do TE. .

Reconstitution of polyunsaturated fatty acid synthesis enzymes in mammalian cells to convert LA to DHA / 生物工程学报

DHA (22:6n-3) is a Ω-3 polyunsaturated fatty acid with 22 carbon atoms and 6 double bonds, which has important biological functions in human body. Human and other mammals synthesize only limited amounts of DHA, more requirements must be satisfied from food resources. However, the natural resources of DHA (Mainly deep-sea fish and other marine products) are prone to depletion. New resources development is still insufficient to satisfy the growing market demand. Previous studies have revealed that the mammals can increase the synthesis of DHA and other long-chain polyunsaturated fatty acids after transgenic procedures. In this study, mammalian cells were transfected with Δ6, Δ5 desaturase, Δ6, Δ5 elongase, Δ15 desaturase (Isolated from nematode Caenorhabditis elegans) and Δ4 desaturase (Isolated from Euglena gracilis), simultaneously. Results show that the expression or overexpression of these 6 enzymes is capable of conversion of the o-6 linoleic acid (LA, 18:2n-6) in DHA (22:6n-3). DHA content has increased from 16.74% in the control group to 25.3% in the experimental group. The strategy and related technology in our research provided important data for future production the valuable DHA (22:6n-3) by using genetically modified animals.

Fatty Acid Composition of Tissue Cultured Breast Carcinoma and the Effect of Stearoyl-CoA Desaturase 1 Inhibition / 한국유방암학회지

PURPOSE: Stearoyl-CoA desaturase 1 (SCD1) is a novel therapeutic target in various malignancies, including breast cancer. The present study was designed to investigate the effect of the pharmacologic inhibition of SCD1 on fatty acid composition in tissue explant cultures of human breast cancer and to compare these effects with those in adjacent nonneoplastic breast tissue. METHODS: Paired samples of tumor and adjacent noncancerous tissue were isolated from 12 patients with infiltrating ductal breast cancer. Samples were explant cultured in vitro, exposed to the highly selective SCD1 inhibitor CAY10566, and examined for fatty acid composition by gas liquid chromatography. The cytotoxic and antigrowth effects were evaluated by quantification of lactate dehydrogenase release and by sulforhodamine B (SRB) measurement, respectively. RESULTS: Breast cancer tissue samples were found to have higher levels of monounsaturated fatty acids (MUFA) (p<0.001) and arachidonic acid (20:4n-6, p<0.001) and a lower level of linoleic acid (18:2n-6, p=0.02) than the normal-appearing breast tissues. While exhibiting no evident cytotoxicity, treatment with the SCD1 inhibitor, CAY10566 (0.1-1 microM), for 48 hours significantly increased 18:2n-6 levels in both the tumor and adjacent normal-appearing tissue (approximately 1.2 fold, p<0.05). However, the breast cancer tissue samples showed significant increases in the levels of MUFA and 20:4n-6 compared to the normal-appearing breast tissues (p<0.05). The SRB growth assay revealed a higher rate of inhibition with the SCD1 inhibitor in breast cancer tissues than in normal-appearing tissues (p<0.01, 41% vs. 29%). The SCD1 inhibitor also elevated saturated fatty acid (1.46-fold, p=0.001) levels only in the tumor tissue explant. CONCLUSION: The fatty acid composition and response to SCD1 inhibition differed between the explant cultures from breast cancer and the adjacent normal-appearing tissue. Altered fatty acid composition induced by SCD1 inhibition may also, in addition to Delta9 desaturation, modulate other reactions in de novo fatty acid synthesis and lipogenesis, and subsequently affect the overall survival and progression of breast cancer.

Overexpression of four fatty acid synthase genes elevated the efficiency of long-chain polyunsaturated fatty acids biosynthesis in mammalian cells / 生物工程学报

Three long-chain polyunsaturated fatty acids, docosahexaenoic acid (DHA, 22:6n-3), eicosapentaenoic acid (EPA, 20:5n-3) and arachidonic acid (ARA, 20:4n-6), are the most biologically active polyunsaturated fatty acids in the body. They are important in developing and maintaining the brain function, and in preventing and treating many diseases such as cardiovascular disease, inflammation and cancer. Although mammals can biosynthesize these long-chain polyunsaturated fatty acids, the efficiency is very low and dietary intake is needed to meet the requirement. In this study, a multiple-genes expression vector carrying mammalian A6/A5 fatty acid desaturases and multiple-genes expression vector carrying mammalian Δ6/Δ5 fatty acid desaturases and Δ6/Δ5 fatty acid elongases coding genes was used to transfect HEK293T cells, then the overexpression of the target genes was detected. GC-MS analysis shows that the biosynthesis efficiency and level of DHA, EPA and ARA were significantly increased in cells transfected with the multiple-genes expression vector. Particularly, DHA level in these cells was 2.5 times higher than in the control cells. This study indicates mammal possess a certain mechanism for suppression of high level of biosynthesis of long chain polyunsaturated fatty acids, and the overexpression of Δ6/Δ5 fatty acid desaturases and Δ6/Δ5 fatty acid elongases broke this suppression mechanism so that the level of DHA, EPA and ARA was significantly increased. This study also provides a basis for potential applications of this gene construct in transgenic animal to produce high level of these long-chain polyunsaturated fatty acid.

Expression of yeast acyl-delta9 desaturase for fatty acid biosynthesis in tobacco / 生物工程学报

Palmitoleic acid (16:1delta9), an unusual monounsaturated fatty acid, is highly valued for human nutrition, medication and industry. Plant oils containing large amounts of palmitoleic acid are the ideal resource for biodiesel production. To increase accumulation of palmitoleic acid in plant tissues, we used a yeast (Saccharomyees cerevisiae) acyl-CoA-delta9 desaturase (Scdelta9D) for cytosol- and plastid-targeting expression in tobacco (Nicotiana tabacum L.). By doing this, we also studied the effects of the subcellular-targeted expression of this enzyme on lipid synthesis and metabolism in plant system. Compared to the wild type and vector control plants, the contents of monounsaturated palmitoleic (16:1delta9) and cis-vaccenic (18:1delta11) were significantly enhanced in the Scdelta9D-transgenic leaves whereas the levels of saturated palmitic acid (16:0) and polyunsaturated linoleic (18:2) and linolenic (18:3) acids were reduced in the transgenics. Notably, the contents of 16:1delta9 and 18:1delta11 in the Scdelta9D plastidal-expressed leaves were 2.7 and 1.9 folds of that in the cytosolic-expressed tissues. Statistical analysis appeared a negative correlation coefficient between 16:0 and 16:1delta9 levels. Our data indicate that yeast cytosolic acyl-CoA-delta9 desaturase can convert palmitic (16:0) into palmitoleic acid (16:1delta9) in high plant cells. Moreover, this effect of the enzyme is stronger with the plastid-targeted expression than the cytosol-target expression. The present study developed a new strategy for high accumulation of omega-7 fatty acids (16:1delta9 andl8:1delta11) in plant tissues by protein engineering of acyl-CoA-delta9 desaturase. The findings would particularly benefit the metabolic assembly of the lipid biosynthesis pathway in the large-biomass vegetative organs such as tobacco leaves for the production of high-quality biodiesel.

Gene transfer of Chlorella vulgaris n-3 fatty acid desaturase optimizes the fatty acid composition of human breast cancer cells

Chlorella vulgaris has the gene of n-3 fatty acid desaturase (CvFad3), which can synthesize the precursor of n-3 polyunsaturated fatty acids (PUFAs) or convert n-6 to n-3 PUFAs. The objective of the present study was to examine whether the CvFad3 gene from C. vulgaris can be functionally and efficiently expressed in human breast cancer cells and whether its expression can exert a significant effect on cell fatty acid composition. We inserted the CvFad3 gene into the plasmid pEGFP-C3 to construct the eukaryotic expression vector pEGFP-C3-n-3 and to express the n-3 Fad gene in human breast cancer cells (MCF-7 cells). Transfection of MCF-7 cells with the recombinant vector resulted in a high expression of n-3 fatty acid desaturase. Lipid analysis indicated that the ratio of n-6/n-3 PUFAs was decreased from 6:1 in the control cells to about 1:1 in the cells expressing the n-3 fatty acid desaturase. Accordingly, the CvFad3 gene significantly decreased the ratio of n-6/n-3 PUFAs of the MCF-7 cell membrane. The expression of the CvFad3 gene can decrease cell proliferation and promote cell apoptosis. This study demonstrates that the CvFad3 gene can dramatically balance the ratio of n-6/n-3 PUFAs and may provide an effective approach to the modification of the fatty acid composition of mammalian cells, also providing a basis for potential applications of its transfer in experimental and clinical settings.

Fatty acid desaturase 1 polymorphisms are associated with coronary heart disease in a Chinese population / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>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.</p><p><b>METHODS</b>We conducted a population-based case-control study in Chinese individuals to evaluate the associations between these three polymorphisms and HDL-C and TG concentrations, and also evaluated their associations with susceptibility to CHD. Genotypes were determined using polymerase chain reaction-restriction fragment length polymorphism assays and TaqMan genotyping assays.</p><p><b>RESULTS</b>We found significant differences in TG and HDL-C concentrations among the TT, TC and CC genotypes of FADS1 rs174547 (P=0.017 and 0.003, respectively, multiple linear regression). The CC variant of rs174547 was significantly associated with hyperlipidemia compared with the TT variant (adjusted odds ratio (OR)=1.71, 95% confidence intervals (CI): 1.16-2.54). The FADS1 rs174547 CC variant was also associated with significantly increased CHD risk compared with the TT and TC variant (adjusted OR=1.53, 95%CI: 1.01-2.31), and the effect was more evident among nonsmokers and females. The polymorphisms rs2338104 and rs10468017 did not significantly influence HDL-C or TG concentrations in this Chinese population.</p><p><b>CONCLUSION</b>rs174547 in FADS1 may contribute to the susceptibility of CHD by altering HDL-C and TG levels in Chinese individuals.</p>

Sodium azide mutagenesis resulted in a peanut plant with elevated oleate content

Screening of peanut seeds resulting from 0.39 percent sodium azide treatment with NIRS calibration equation for bulk seed samples identified a plant with more than 60 percent oleate. Oleate content in individual seeds of the plant, as predicted by NIRS calibration equation for intact single peanut seeds, ranged from 50.05 percent ~ 68.69 percent. Three seeds with >60 percent oleate thus identified were further confirmed by gas chromatography. Multiple sequence alignments of the FAD2B gene from Huayu 22 (wild type) and peanut seeds with elevated oleate (mutant type) revealed a C281T transition in the coding region causing an I94T substitution in the oleoyl-PC desaturase, which may be responsible for reduction in the enzyme activity.

A real-time PCR genotyping assay to detect FAD2A SNPs in peanuts (Arachis hypogaea L)

The high oleic (C18:1) phenotype in peanuts has been previously demonstrated to result from a homozygous recessive genotype (ol1ol1ol2ol2) in two homeologous fatty acid desaturase genes (FAD2A and FAD2B) with two key SNPs. These mutant SNPs, specifically G448A in FAD2A and 442insA in FAD2B, significantly limit the normal function of the desaturase enzyme activity which converts oleic acid into linoleic acid by the addition of a second double bond in the hydrocarbon chain. Previously, a genotyping assay was developed to detect wild type and mutant alleles in FAD2B. A real-time PCR assay has now been developed to detect wild type and mutant alleles (G448A) in FAD2A using either seed or leaf tissue. This assay was demonstrated to be applicable for the detection of homozygous and heterozygous samples. The FAD2A genotyping assay was validated by employing gas chromatography (GC) to determine total fatty acid composition and by genotyping peanut lines that have been well characterized. Overall, development of rapid assays such as real-time PCR which can identify key genotypes associated with important agronomic traits such as oleic acid, will improve breeding efficiency by targeting desirable genotypes at early stages of development.

Cloning of delta8-fatty acid desaturase gene from Euglena gracilis and its expression in Saccharomyces cerevisiae / 生物工程学报

Delta8 desaturase pathway, different from common delta6 desaturase pathway, is an alternate pathway of polyunsaturated fatty acids biosynthesis. Delta8-fatty acid desaturase is one of the key enzymes in delta8 desaturase pathway. Two specific fragments were separately cloned from genomic DNA and cDNA of Euglena gracilis by PCR with the primers designed according to the reported sequence. Comparison of the genomic and cDNA sequences revealed that there wasn't intron in this delta8-fatty acid desaturase gene. This gene has an open reading frame of 1 266 bp that encodes 421 amino acids. It is 6 bp longer than the reported gene sequence, and also showed certain difference from the reported sequence in the N-terminal. The recombinant expression plasmid pYEFD by subcloning delta8-fatty acid desaturase gene into the yeast-E. coli shuttle vector pYES2.0 was constructed and was transformed into the defective mutant INVSc1 of Saccharomyces cerevisiae by electrotransformation. The resulting strain YD8 harboring plasmid pYEFD was selected and was cultured in the induction medium with exogenous substrates omega6-eicosadienoic acid and omega3-eicosatrienoic acid for the expression of delta8-fatty acid desaturase gene. The results indicated that high level expressed As-fatty acid desaturase could convert omega6-eicosadienoic acid and omega3-eicosatrienoic acid to dihomo-gamma-linolenic acid and eicosatetraenoic acid with substrate conversion ratio 31.2% and 46.3%, respectively.

Cloning and expression in Saccharomyces cerevisiae of delta5-fatty acid desaturase gene from Phaeodactylum tricornutum / 生物工程学报

Delta5-fatty acid desaturase is the key enzyme in synthesis of arachidonic acid. Two specific fragment was cloned from genomic DNA and total cDNA of Phaeodactylum tricornutum through PCR with primer designed according to the reported sequences, respectively 1520 bp and 1410 bp. Comparison of the genomic and cDNA sequences revealed that the delta5-fatty Acid Desaturase gene from genomic DNA had an 110 bp intron. The 1.4 kb was subcloned into the yeast-E. coli shuttle vector pYES2.0, then an expression recombinant plasmid pYPTD5 containerizing target gene was constructed. The plasmid pYPTD5 was transformed into defective mutant INCSc 1 of Saccharomyces cerevisiae for expression by electrotransformation method. Dihomo-gamma-linolenic acid was provided as an exogenous substrate to the yeast cultures, with galactose as inducer. By GC detecting, the recombinant S. cerecisiae had arachidonic acid. The results indicated that high level expression of delta5-fatty acid desaturase, and the substrate conversion reached 45.9%.

Isolation and characterization of microsomal omega-6-desaturase gene (fad2-1) from soybean.

A genomic DNA sequence (fad2-1) encoding seed specific microsomal 0-6 desaturase was isolated from soybean (Glycine max. L cv. Pusa-9702). A positive genomic clone of 1852 nucleotides containing a single uninterrupted 3' end exonic region with an ORF of 1140 bp encoding a peptide of 379 amino acids, a complete 3' UTR of 206 bp and 86 bp of 5' UTR interrupted by a single intron of 420 bp was obtained on screening the sub-genomic library of soybean. Southern blots revealed at least two copies of the gene per haploid genome. Analysis of the translated product showed the presence of three histidine boxes, with the general sequence HXXXH and five probable transmembrane segments reported to be involved in substrate specificity.

Functional expression of an omega-3 fatty acid desaturase gene from Glycine max in Saccharomyces cerevisiae / 生物工程学报

Alpha-linolenic acid(ALA, C18:3delta9,12,15 ) is an essential fatty acid which has many sanitary functions to human. However, its contents in diets are often not enough. In plants, omega-3 fatty acid desaturases(FAD) catalyze linoleic acid(LA, C18:2delta9,12) into ALA. The seed oil of Glycine max contains high level of ALA. To investigate the functions of Glycine max omega-3FAD, the cDNA of GmFAD3 C was amplified by RT-PCR from immature seeds, then cloned into the shuttle expression vector p416 to generate the recombinant vector p4GFAD3C. The resulting vector was transformed into Saccharomyces cerevisiae K601 throuth LiAc method. The positive clones were screened on the CM(Ura-) medium and identified by PCR, and then cultured in CM (Ura-) liquid medium with exogenous LA in 20 degrees C for three days. The intracellular fatty acid composition of the engineering strain Kp416 and Kp4GFAD3C was analyzed by gas chromatography (GC). A novel peak in strain Kp4GFAD3C was detected,which was not detectable in control, Comparison of the retention times of the newly yielded peak with that of authentic standard indicated that the fatty acid is ALA. The content of ALA reached to 3.1% of the total fatty acid in recombinant strain, the content of LA correspondingly decreased from 22% to 16.2% by contrast. It was suggested that the protein encoded by GmFAD3 C can specifically catalyze 18 carbon PUFA substrate of LA into ALA by taking off hydrogen atoms at delta15 location. In this study, we expressed a Glycine max omega-3 fatty acid desaturase gene in S. cerevisiae; An efficient and economical yeast expressing system(K601-p416 system) which is suitable for the expression of FAD was built.

The synthesis and function analysis of omega-3 fatty acid desaturase gene from Caenorhabditis briggssae / 生物工程学报

Omega-3 polyunsaturated fatty acids (PUFAs) have been broadly investigated and shown to exert many preventive and therapeutic actions besides their important role in maintenances human health and normal development. In mammals, the level of omega-3 PUFAs is relatively too low compared with omega-6 PUFAs, which metabolically and functionally distinct from omega-3 PUFAs and often have important opposing physiological functions. Either the inefficiency of omega-3 PUFAs or the excess of omega-6 PUFAs will cause many healthy problems. So methods have been sought to increase the amount of omega-3 PUFAs and to improve the omega-6/omega-3 ratio in body. In this study, the sFat-1 gene, which putatively encodes a omega-3 fatty acid desaturase, was chemically synthesized according to the sequence from Caenorhabditis briggssae (with codon usage modified), and constructed into a mammal expression vector pcDNA3. 1-sFat1-EGFP. This vector was introduced into CHO cells by lipid-mediated transfection, and it's expression quickly and effectively elevated the cellular omega-3 PUFAs (from 18-carbon to 22-carbon) contents and dramatically improved the ratio of omega-6/omega-3 PUFAs. Cellular lipids extracts from stably selected cells were analyzed with GC-MS and the results showed that amount of total omega-6 PUFAs dropped from 48.97% (in GFP cells)to 35.29% (in sFat-1 cells), whereas the amount of total omega-3 PUFAs increased from 7.86% to 24.02%, respectively. The omega-6/omega-3 ratio also dropped from 6.23 to 1.47. These data demonstrates the Caenorhabditis briggssae omega-3 Fatty Acid Desaturase gene, sFat-1, was synthesized successfully and can produce omega-3 PUFAs by using the corresponding omega-6 PUFAs as substrates, which shows its potential for use in the production of omega-3 PUFAs in transgenic animals.

Progress on molecular biology of delta6-fatty acid desaturases / 生物工程学报

Polyunsaturated fatty acids (PUFAs) including gamma-linolenic acid are valuable products because of their involvement in several aspects of human health care. GLA has been claimed to play a crucial role in development and prevention of some skin diseases, diabetes, reproductive disorder and others. At present, market demand for most gamma-linolenic acid is growing continually and current sources are inadequate for satisfying this demand due to the significant problems of low productivity, complex and expensive downstream process and unstable quality. Therefore, seeking for alternative sources are demanding. delta6-fatty acid desaturase is the rate-limiting enzyme for the biosynthesis of PUFAs, which catalyses the conversion of linoleic acid and alpha-linolenic acid to gamma-linolenic acid and stearidonic acid respectively. Unfortunately, the structure information on membrane desaturases is scarce because of the technical limitations in obtaining quantities of purified protein and the intrinsic difficulties in obtaining crystals from membrane proteins. With the isolation of the genes coding for delta6-fatty acid desaturase from various organisms, its characteristics will be elucidated gradually. Here we concisely reviewed the recent progress on studies of molecular biology including the cloning of delta6-fatty acid desaturase gene, structure and function, phylogeny and prospects of gene engineering application.