AIM:To investigate the effect of MEK/ERK1/2 pathway on peroxisome proliferator-activated receptors(PPARγ)agonist-induced alterations in Δ6-desaturase(Δ6D)and stearoyl-CoA desaturase 1(SCD1)in hepatocellular carcino...AIM:To investigate the effect of MEK/ERK1/2 pathway on peroxisome proliferator-activated receptors(PPARγ)agonist-induced alterations in Δ6-desaturase(Δ6D)and stearoyl-CoA desaturase 1(SCD1)in hepatocellular carcinoma cell line HepG2.METHODS:HepG2 cells cultured in RPMI-1640 were exposed to the commonly used ERK1/2 pathway inhibitor PD98059 and PPARγ agonist,pioglitazone.Total RNA was isolated and reverse transcribed from treated cells.Changes in gene expression and metabolites ratio,as activity index for Δ6D and SCD1,were then determined using reverse transcriptionpolymerase chain reaction and gas liquid chromatography,respectively.RESULTS:The expression of both Δ6D(P = 0.03)and SCD1(P = 0.01)increased following PD98059 treatment,with a higher impact on SCD1(24.5%vs 62.5%).Although pioglitazone increased the mRNA level(1.47 ± 0.10 vs 0.88 ± 0.02,P = 0.006)and activity index(1.40 ± 0.07 vs 0.79 ± 0.11,P < 0.001)of Δ6D,no such changes have been observed for SCD1 activity index in pioglitazone-treated cells.SCD1 gene expression(+26.4%,P = 0.041)and activity index(+52.8%,P = 0.035)were significantly increased by MEK inhibition in the presence of pioglitazone,as compared with pioglitazone alone and control cells.However,the response of Δ6D expression and activity index to pioglitazone was unaffected by incubation with PD98059.CONCLUSION:PPARγ and ERK1/2 signaling pathway affect differentially and may have inhibitory crosstalk effects on the genes expression of 6D and SCD1,and subsequently on their enzymatic activities.展开更多
The imbalance between pathogenic and protective T cell subsets is a cardinal feature of autoimmune disorders such as multiple sclerosis(MS).Emerging evidence indicates that endogenous and dietary-induced changes in fa...The imbalance between pathogenic and protective T cell subsets is a cardinal feature of autoimmune disorders such as multiple sclerosis(MS).Emerging evidence indicates that endogenous and dietary-induced changes in fatty acid metabolism have a major impact on both T cell fate and autoimmunity.To date,however,the molecular mechanisms that underlie the impact of fatty acid metabolism on T cell physiology and autoimmunity remain poorly understood.Here,we report that stearoyl-CoA desaturase-1(SCD1),an enzyme essential for the desaturation of fatty acids and highly regulated by dietary factors,acts as an endogenous brake on regulatory T-cell(Treg)differentiation and augments autoimmunity in an animal model of MS in a T cell-dependent manner.Guided by RNA sequencing and lipidomics analysis,we found that the absence of Scd1 in T cells promotes the hydrolysis of triglycerides and phosphatidylcholine through adipose triglyceride lipase(ATGL).ATGL-dependent release of docosahexaenoic acid enhanced Treg differentiation by activating the nuclear receptor peroxisome proliferator-activated receptor gamma.Our findings identify fatty acid desaturation by SCD1 as an essential determinant of Treg differentiation and autoimmunity,with potentially broad implications for the development of novel therapeutic strategies and dietary interventions for autoimmune disorders such as MS.展开更多
The cDNA of the delta-12 fatty acid desaturase gene, IgFAD2, was cloned from the marine microalgae Isochrysis galbana, a species capable of producing docosahexaenoic acid. Sequence analysis indicated that the open rea...The cDNA of the delta-12 fatty acid desaturase gene, IgFAD2, was cloned from the marine microalgae Isochrysis galbana, a species capable of producing docosahexaenoic acid. Sequence analysis indicated that the open reading frame measured a length of 1 158 bp and encoded 386 amino acids with a predicted molecular weight of 42.8 kDa and an isoelectric point of 9.2. Computational analysis of the protein sequence of IgFAD2 showed typical features of membrane-bound desaturase such as three conserved histidine boxes along with four membranespanning regions that were universally present among plant desaturases. Quantitative real-time PCR results showed that the abundance of IgFAD2 transcript was significantly upregulated under different environmental stresses including low temperature(15℃), high salinity(salinity of 62 and 93), and nitrogen starvation(220 μmol/L). Heterologous expression indicated that yeast cells transformed with a plasmid construct containing IgFAD2 could convert endogenous oleic acid(18:1^(?9), OA) into linoleic acid(18:2^(?9, 12), LA). These findings confirm that I. galbana IgFAD2 plays important roles in the biosynthetic pathways of unsaturated fatty acids.展开更多
Δ12 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 Δ12 fatty acid desaturase gene were isolated from p...Δ12 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 Δ12 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 Δ12 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 Δ12 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 Δ12 fatty acid desaturase in high oleic acid genotype.展开更多
It is suggested that Δ6 fatty acid desaturase(FAD) plays a critical role in the biosynthesis of polyunsaturated fatty acids in plants and microalgae. But why does it adapt to the changed environments such as nitrogen...It is suggested that Δ6 fatty acid desaturase(FAD) plays a critical role in the biosynthesis of polyunsaturated fatty acids in plants and microalgae. But why does it adapt to the changed environments such as nitrogen starvation is seldom understood. One Δ6 FAD gene( MiD6 fad) from an arachidonic acidrich microalga M yrmecia incisa Reisigl(Chlorophyta) was first heterologously expressed in S accharomyces cerevisiae for the identification of function. The fatty acid profile of transgenic yeast detected by gas chromatography-mass spectrometry illustrated that the enzyme MiD6 FAD could convert linoleic and ?-linolenic acids to γ-linolenic and stearidonic acids, respectively, demonstrating that M iD6 fad encoded a Δ6 FAD. A 1 965-bp fragment of the cloned 2 347-bp 5′-upstream region of M iD6 fad was next subcloned and fused upstream with green fluorescent protein(GFP) gene to replace the GAL1 promoter of the vector pYES2. The generated construct was transformed into S. cerevisiae for function determination. Confocal microscopic images of the transformed line illustrated that this inserted fragment could drive GFP expression, which was further verified by fluorescence intensity quantification and Western blot analysis using antiGFP antibody. The conversion efficiency(approximately 2%-3%) of MiD6 FAD was much lower than the reported ? 3 FAD and Δ6 elongase in this microalga, suggesting that MiD6 FAD catalysed the possible ratelimiting step for ArA biosynthesis. The presence of several putative c is-acting regulatory elements in this identified promoter sheds new light on the regulation mechanism research of Δ6 FAD transcription for the ArA production in M. incisa in changing environmental factors.展开更多
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 cl...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.展开更多
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...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.展开更多
A gene(NANOC-D6D) encoding a desaturase that removes two hydrogen atoms from fatty acids at delta 6 position was isolated from a cDNA library of Nannochloropsis oculata(Droop) D.J.Hibberd(Eustigmatophyceae).The unicel...A gene(NANOC-D6D) encoding a desaturase that removes two hydrogen atoms from fatty acids at delta 6 position was isolated from a cDNA library of Nannochloropsis oculata(Droop) D.J.Hibberd(Eustigmatophyceae).The unicellular marine microalga N.oculata synthesizes rich long chain polyunsaturated fatty acids(LCPUFAs),including eicosapentaenoic acid(20:5n-3,EPA).The deduced protein contains 474 amino acids that fold into 4 trans-membrane domains.The neighbor-joining phylogenetic tree indicates that NANOC-D6D is phylogenetically close to the delta-6 fatty acid desaturase of marine microalgae such as Glossomastix chrysoplasta,Thalassiosira pseudonana,and Phaeodactylum tricornutum.The gene was expressed in Saccharomyces cerevisiae INVSc1 to verify the substrate specificity of NANOC-D6D.Our results suggest that the recombinant NANOC-D6D simultaneously desaturates linoleic acid(LA) and a-linolenic acid(ALA).展开更多
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 ...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.展开更多
The unicellular green alga Dunaliella is outstanding for its ability of massive accumulation of carotenoids. To elucidate the carotenoids synthesis pathway in this alga,phytoene desaturase(pds) gene cDNA together with...The unicellular green alga Dunaliella is outstanding for its ability of massive accumulation of carotenoids. To elucidate the carotenoids synthesis pathway in this alga,phytoene desaturase(pds) gene cDNA together with its DNA sequences were isolated and their structures and functions analyzed. The full-length pds cDNA of 2290 bp(GenBank Accession No. DQ243892) was de-duced from RACE results,including untranslated 21 bp 5'-and 520 bp 3'-flanking regions and an open reading frame of 582 amino acids,coding a protein of 64.196 kDa. The DNA sequence of 2908 bp(GenBank Accession No. DQ845248) including five introns was obtained. The fifth intron was uncompleted and complex,including two bases' perfect repeats(GT) 10 and large different-sized repeats within the last 400 bp. The Southern blot hybridization result demonstrated that this gene occurred as a single copy in this species,and the quantitative RT-PCR result showed that the transcription of this gene was constitutive. The evolutional significance of pds was discussed.展开更多
Arachidonic acid(AA or ARA), an essential ω-6 polyunsaturated fatty acid(PUFA), can be produced by Mortierella isabellina. Mutagenesis on Mortierella isabellina As3.3410 was induced to raise ARA production. The mutan...Arachidonic acid(AA or ARA), an essential ω-6 polyunsaturated fatty acid(PUFA), can be produced by Mortierella isabellina. Mutagenesis on Mortierella isabellina As3.3410 was induced to raise ARA production. The mutant strain of YZ-124 had the highest ARA of 4.72 g L-1, which was 5.5 times higher than that of the original strain 3.3410. mRNA expression level of Δ6-desaturase was determined in five different kinds of ARA-producing Mortierella isabellina after cultured for 7 days, and in the mutant strain YZ-124 over a 3-8 day time-course. In addition, the desaturase activity and ARA content were measured at the selected time points. The lowest expression of Δ6-desaturase was observed in the original strain and the highest expression in the mutant strain YZ-124, which increased with increasing time in culture. Furthermore, a positive correlation was observed between the expression levels of Δ6-desaturase and ARA content. Based on this, Δ6-desaturase played a significant role in ARA synthesis pathway in Mortierella isabellina.展开更多
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 resp...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.展开更多
The performance of lactating dairy cows and composition of the fatty acids in milk fat were measured to investigate the effect of parity on conjugated linoleic acid(CLA)in milk fat and△~9-desaturase indices from Hols...The performance of lactating dairy cows and composition of the fatty acids in milk fat were measured to investigate the effect of parity on conjugated linoleic acid(CLA)in milk fat and△~9-desaturase indices from Holstein dairy cows.Milk samples of two hundred and thirty-five lactating Holstein dairy cows in north China were tested.In order to avoid possible confounding effects of diet and season,Holstein cows were fed a single diet and milk was sampled on the same day.The average content of cis-9,trans-11 CLA was 5.14 mg/g fatty acids in milk fat,varying between 1.5 and 10.5 mg/g fatty acids.The average content of cis-9,trans-11 CLA of primiparous cattle was 5.19 mg/g fatty acids in milk fat,while it was 5.11 mg/g fatty acids in multiparous animals,with no significant difference between them(P】0.05).There was a significant(P【0.05)difference between the△~9-desaturase index of cis-9 C14:l and cis-9 C16:l between primiparous and multiparous cattle but not(P】0.05)in the△~9-desaturase index of cis-9 C18:l and cis-9,trans-11 CLA.展开更多
A 2 149 bp full length phytoene desaturase (PDS) cDNA was first cloned from saffron (Crocus sativus L.) stigma using RT-PCR technique and a rapid amplification of cDNA end (RACE) strategy. The cDNA has an open reading...A 2 149 bp full length phytoene desaturase (PDS) cDNA was first cloned from saffron (Crocus sativus L.) stigma using RT-PCR technique and a rapid amplification of cDNA end (RACE) strategy. The cDNA has an open reading frame of 1 697 bp, which encodes a polypeptide of 565 amino acids. The coding region of the cDNA was inserted into a prokaryotic expression vector pET -21a(+) and overexpressed in E. coli BL21 (DE3). The fusion proteins were found largely in an insoluble inclusion bodies. The purified fusion protein was used to immunize rabbits to obtain polyclonal antiserum with titer of 1×10 5. Western blot analysis by using this particular antiserum showed that the higher expression level of PDS in mature stigma than in leaves and stamen, and the higher expression level of PDS in mature stigma than in young stigma.展开更多
基金Supported by A Grant from the Drug Applied Research Center of Tabriz University of Medical Sciences,to Darabi M,Research Projects numbers 89/102 and 90/73
文摘AIM:To investigate the effect of MEK/ERK1/2 pathway on peroxisome proliferator-activated receptors(PPARγ)agonist-induced alterations in Δ6-desaturase(Δ6D)and stearoyl-CoA desaturase 1(SCD1)in hepatocellular carcinoma cell line HepG2.METHODS:HepG2 cells cultured in RPMI-1640 were exposed to the commonly used ERK1/2 pathway inhibitor PD98059 and PPARγ agonist,pioglitazone.Total RNA was isolated and reverse transcribed from treated cells.Changes in gene expression and metabolites ratio,as activity index for Δ6D and SCD1,were then determined using reverse transcriptionpolymerase chain reaction and gas liquid chromatography,respectively.RESULTS:The expression of both Δ6D(P = 0.03)and SCD1(P = 0.01)increased following PD98059 treatment,with a higher impact on SCD1(24.5%vs 62.5%).Although pioglitazone increased the mRNA level(1.47 ± 0.10 vs 0.88 ± 0.02,P = 0.006)and activity index(1.40 ± 0.07 vs 0.79 ± 0.11,P < 0.001)of Δ6D,no such changes have been observed for SCD1 activity index in pioglitazone-treated cells.SCD1 gene expression(+26.4%,P = 0.041)and activity index(+52.8%,P = 0.035)were significantly increased by MEK inhibition in the presence of pioglitazone,as compared with pioglitazone alone and control cells.However,the response of Δ6D expression and activity index to pioglitazone was unaffected by incubation with PD98059.CONCLUSION:PPARγ and ERK1/2 signaling pathway affect differentially and may have inhibitory crosstalk effects on the genes expression of 6D and SCD1,and subsequently on their enzymatic activities.
基金supported by the Flemish Fund for Scientific Research(FWO Vlaanderen,12J9116N,12JG119N,12U7718N,1S15519N,and G099618N)the Belgian Charcot Foundation(FCS-2016-EG7,R-8676,and R-6832)+4 种基金the Interreg V‐A EMR program(EURLIPIDS,EMR23)the special research fund UHasselt(BOF)JMN is supported by a National Institutes of Health Grant(R01 DK062388)supported by the European Research Council(ERC)under the European Union’s Horizon 2020 research and innovation program(640116)by a SALK grant from the government of Flanders and by an Odysseus grant of the Research Foundation Flanders,Belgium(FWO).
文摘The imbalance between pathogenic and protective T cell subsets is a cardinal feature of autoimmune disorders such as multiple sclerosis(MS).Emerging evidence indicates that endogenous and dietary-induced changes in fatty acid metabolism have a major impact on both T cell fate and autoimmunity.To date,however,the molecular mechanisms that underlie the impact of fatty acid metabolism on T cell physiology and autoimmunity remain poorly understood.Here,we report that stearoyl-CoA desaturase-1(SCD1),an enzyme essential for the desaturation of fatty acids and highly regulated by dietary factors,acts as an endogenous brake on regulatory T-cell(Treg)differentiation and augments autoimmunity in an animal model of MS in a T cell-dependent manner.Guided by RNA sequencing and lipidomics analysis,we found that the absence of Scd1 in T cells promotes the hydrolysis of triglycerides and phosphatidylcholine through adipose triglyceride lipase(ATGL).ATGL-dependent release of docosahexaenoic acid enhanced Treg differentiation by activating the nuclear receptor peroxisome proliferator-activated receptor gamma.Our findings identify fatty acid desaturation by SCD1 as an essential determinant of Treg differentiation and autoimmunity,with potentially broad implications for the development of novel therapeutic strategies and dietary interventions for autoimmune disorders such as MS.
基金The Basic Scientific Fund for National Public Research Institutes of China under contract No.2017Q09the Aoshan Science and Technology Innovation Project of Pilot National Laboratory for Marine Science and Technology(Qingdao)under contract No.2016ASKJ02+3 种基金the National Natural Science Foundation of China-Shandong Joint Funded Project under contract No.U1606404the National Natural Science Foundation of China under contract Nos 41776176 and 41806201the 973 Project from Chinese Ministry of Science and Technology under contract No.2015CB755904the Shandong Provincial Natural Science Foundation under contract No.ZR2015PD003
文摘The cDNA of the delta-12 fatty acid desaturase gene, IgFAD2, was cloned from the marine microalgae Isochrysis galbana, a species capable of producing docosahexaenoic acid. Sequence analysis indicated that the open reading frame measured a length of 1 158 bp and encoded 386 amino acids with a predicted molecular weight of 42.8 kDa and an isoelectric point of 9.2. Computational analysis of the protein sequence of IgFAD2 showed typical features of membrane-bound desaturase such as three conserved histidine boxes along with four membranespanning regions that were universally present among plant desaturases. Quantitative real-time PCR results showed that the abundance of IgFAD2 transcript was significantly upregulated under different environmental stresses including low temperature(15℃), high salinity(salinity of 62 and 93), and nitrogen starvation(220 μmol/L). Heterologous expression indicated that yeast cells transformed with a plasmid construct containing IgFAD2 could convert endogenous oleic acid(18:1^(?9), OA) into linoleic acid(18:2^(?9, 12), LA). These findings confirm that I. galbana IgFAD2 plays important roles in the biosynthetic pathways of unsaturated fatty acids.
文摘Δ12 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 Δ12 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 Δ12 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 Δ12 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 Δ12 fatty acid desaturase in high oleic acid genotype.
基金Supported by the National Natural Science Foundation of China(No.31172389)the Special Project of Marine Renewable Energy from the State Oceanic Administration(No.SHME2011SW02)the Shanghai Universities Peak Discipline Project of Aquaculture
文摘It is suggested that Δ6 fatty acid desaturase(FAD) plays a critical role in the biosynthesis of polyunsaturated fatty acids in plants and microalgae. But why does it adapt to the changed environments such as nitrogen starvation is seldom understood. One Δ6 FAD gene( MiD6 fad) from an arachidonic acidrich microalga M yrmecia incisa Reisigl(Chlorophyta) was first heterologously expressed in S accharomyces cerevisiae for the identification of function. The fatty acid profile of transgenic yeast detected by gas chromatography-mass spectrometry illustrated that the enzyme MiD6 FAD could convert linoleic and ?-linolenic acids to γ-linolenic and stearidonic acids, respectively, demonstrating that M iD6 fad encoded a Δ6 FAD. A 1 965-bp fragment of the cloned 2 347-bp 5′-upstream region of M iD6 fad was next subcloned and fused upstream with green fluorescent protein(GFP) gene to replace the GAL1 promoter of the vector pYES2. The generated construct was transformed into S. cerevisiae for function determination. Confocal microscopic images of the transformed line illustrated that this inserted fragment could drive GFP expression, which was further verified by fluorescence intensity quantification and Western blot analysis using antiGFP antibody. The conversion efficiency(approximately 2%-3%) of MiD6 FAD was much lower than the reported ? 3 FAD and Δ6 elongase in this microalga, suggesting that MiD6 FAD catalysed the possible ratelimiting step for ArA biosynthesis. The presence of several putative c is-acting regulatory elements in this identified promoter sheds new light on the regulation mechanism research of Δ6 FAD transcription for the ArA production in M. incisa in changing environmental factors.
基金This study was supported by the Zhejiang Provincial Natural Science Foundation of China(No.LQ16D060001)the National Natural Science Foundation of China(No.41606163)+3 种基金the Natural Science Foundation of the Ningbo Government(No.2017A610288)the Ningbo Science and Technology Research Projects,China(No.2019B10006)the Zhejiang Major Science Project,China(No.2019C02057)the Earmarked Fund for Modern Agro-Industry Technology Research System,China(No.CARS-49)and partly sponsored by K.C.Wong Magna Fund at Ningbo University.
文摘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.
基金Supported by the International S&T Cooperation Program of China(No.2012DFA30450)the National Natural Science Foundation of China(No.30871541)+1 种基金the Taishan Scholar Foundation of Shandong Province(No.tshw20091014)the Innovation Program of the University Institutes of Jinan,Shandong Province(No.201004044)
文摘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.
基金Supported by the National Key Technology R&D Program in the 11th Five-Year Plan of China (No. 2006BAD09A03-2)the National High-tech Research and Development Program of China(863 Program) (No. 2007AA09Z427)
文摘A gene(NANOC-D6D) encoding a desaturase that removes two hydrogen atoms from fatty acids at delta 6 position was isolated from a cDNA library of Nannochloropsis oculata(Droop) D.J.Hibberd(Eustigmatophyceae).The unicellular marine microalga N.oculata synthesizes rich long chain polyunsaturated fatty acids(LCPUFAs),including eicosapentaenoic acid(20:5n-3,EPA).The deduced protein contains 474 amino acids that fold into 4 trans-membrane domains.The neighbor-joining phylogenetic tree indicates that NANOC-D6D is phylogenetically close to the delta-6 fatty acid desaturase of marine microalgae such as Glossomastix chrysoplasta,Thalassiosira pseudonana,and Phaeodactylum tricornutum.The gene was expressed in Saccharomyces cerevisiae INVSc1 to verify the substrate specificity of NANOC-D6D.Our results suggest that the recombinant NANOC-D6D simultaneously desaturates linoleic acid(LA) and a-linolenic acid(ALA).
文摘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.
文摘The unicellular green alga Dunaliella is outstanding for its ability of massive accumulation of carotenoids. To elucidate the carotenoids synthesis pathway in this alga,phytoene desaturase(pds) gene cDNA together with its DNA sequences were isolated and their structures and functions analyzed. The full-length pds cDNA of 2290 bp(GenBank Accession No. DQ243892) was de-duced from RACE results,including untranslated 21 bp 5'-and 520 bp 3'-flanking regions and an open reading frame of 582 amino acids,coding a protein of 64.196 kDa. The DNA sequence of 2908 bp(GenBank Accession No. DQ845248) including five introns was obtained. The fifth intron was uncompleted and complex,including two bases' perfect repeats(GT) 10 and large different-sized repeats within the last 400 bp. The Southern blot hybridization result demonstrated that this gene occurred as a single copy in this species,and the quantitative RT-PCR result showed that the transcription of this gene was constitutive. The evolutional significance of pds was discussed.
基金Supported by the National Natural Science Foundation of China(31171657)Heilongjiang Youth Science Foundation(QC2010093)+1 种基金Heilongjiang Natural Science Foundation(20100481037)Key Projects on Ministry of Education in Heilongjiang(12511Z022)
文摘Arachidonic acid(AA or ARA), an essential ω-6 polyunsaturated fatty acid(PUFA), can be produced by Mortierella isabellina. Mutagenesis on Mortierella isabellina As3.3410 was induced to raise ARA production. The mutant strain of YZ-124 had the highest ARA of 4.72 g L-1, which was 5.5 times higher than that of the original strain 3.3410. mRNA expression level of Δ6-desaturase was determined in five different kinds of ARA-producing Mortierella isabellina after cultured for 7 days, and in the mutant strain YZ-124 over a 3-8 day time-course. In addition, the desaturase activity and ARA content were measured at the selected time points. The lowest expression of Δ6-desaturase was observed in the original strain and the highest expression in the mutant strain YZ-124, which increased with increasing time in culture. Furthermore, a positive correlation was observed between the expression levels of Δ6-desaturase and ARA content. Based on this, Δ6-desaturase played a significant role in ARA synthesis pathway in Mortierella isabellina.
基金funded by the Agricultural Science and Technology Innovation Project of the Chinese Academy of Agricultural Sciences(CAAS-ASTIP-OCRI),the National Natural Science Foundation of China(grant number 31801399)the Research Foundation of Education Bureau of Hunan Province,China(grant number 21A0135)。
文摘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.
文摘The performance of lactating dairy cows and composition of the fatty acids in milk fat were measured to investigate the effect of parity on conjugated linoleic acid(CLA)in milk fat and△~9-desaturase indices from Holstein dairy cows.Milk samples of two hundred and thirty-five lactating Holstein dairy cows in north China were tested.In order to avoid possible confounding effects of diet and season,Holstein cows were fed a single diet and milk was sampled on the same day.The average content of cis-9,trans-11 CLA was 5.14 mg/g fatty acids in milk fat,varying between 1.5 and 10.5 mg/g fatty acids.The average content of cis-9,trans-11 CLA of primiparous cattle was 5.19 mg/g fatty acids in milk fat,while it was 5.11 mg/g fatty acids in multiparous animals,with no significant difference between them(P】0.05).There was a significant(P【0.05)difference between the△~9-desaturase index of cis-9 C14:l and cis-9 C16:l between primiparous and multiparous cattle but not(P】0.05)in the△~9-desaturase index of cis-9 C18:l and cis-9,trans-11 CLA.
文摘A 2 149 bp full length phytoene desaturase (PDS) cDNA was first cloned from saffron (Crocus sativus L.) stigma using RT-PCR technique and a rapid amplification of cDNA end (RACE) strategy. The cDNA has an open reading frame of 1 697 bp, which encodes a polypeptide of 565 amino acids. The coding region of the cDNA was inserted into a prokaryotic expression vector pET -21a(+) and overexpressed in E. coli BL21 (DE3). The fusion proteins were found largely in an insoluble inclusion bodies. The purified fusion protein was used to immunize rabbits to obtain polyclonal antiserum with titer of 1×10 5. Western blot analysis by using this particular antiserum showed that the higher expression level of PDS in mature stigma than in leaves and stamen, and the higher expression level of PDS in mature stigma than in young stigma.
