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
△^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 fr...△^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.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
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 tr...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.展开更多
Delta-12 oleate desaturase gene （FAD2-1） which converts oleic acid into linoleic acid, is the key enzyme determining the fatty acid composition of cottonseed oil. By employing RT-PCR method, full length cDNA of cott...Delta-12 oleate desaturase gene （FAD2-1） which converts oleic acid into linoleic acid, is the key enzyme determining the fatty acid composition of cottonseed oil. By employing RT-PCR method, full length cDNA of cotton delta-12 oleate desat- urase gene GhFAD2-1 containing an open reading frame of 1 158 bp was cloned for constructing RNAi vector. A 515 bp long specific fragment of this gene was se- lected for constructing ihpRNA vector under the control of a seed-specific promoter NAPIN, named pFGC1008-NAPIN-FAD2-1; meanwhile miRNA gene-silencing vector pCAMBIA1302-amiRNA-FAD2-1 targeting GhFAD2-1 was also constructed.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
Using degenerate primers and RT-PCR, RACE techniques, a 1491 bp cDNA segment of stearoyl-acyl carrier protein desaturase （SAD） is cloned from developing seeds of Jatropha curcas L. The segment contains a 1191 bp of ...Using degenerate primers and RT-PCR, RACE techniques, a 1491 bp cDNA segment of stearoyl-acyl carrier protein desaturase （SAD） is cloned from developing seeds of Jatropha curcas L. The segment contains a 1191 bp of complete open reading frame （ORF）. Analysis in the BLAST on NCBI shows that Jatropha curcas SAD （JSAD） gene encodes a protein precursor composed of a signal peptide of 33 amino acids and a mature peptide of 363 amino acids. The homological analysis shows that JSAD has high level of homology both in nucleotide sequence and in amino acid sequence to other plants SADs. The nucleotide and peptide identity of JSAD to Ricinus communis SAD （RSAD） is up to 89% and 96.2% respectively. Molecular modeling of JSAD indicates that its three-dimensional structure strongly resembled the crystal structure of RSAD.展开更多
This study investigated the effect of dietary lipid on the growth, fatty acid composition and Δ5 fatty acyl desaturase genes(Fads) expression of juvenile abalone(Haliotis discus hannai Ino) hepatopancreas. Six purifi...This study investigated the effect of dietary lipid on the growth, fatty acid composition and Δ5 fatty acyl desaturase genes(Fads) expression of juvenile abalone(Haliotis discus hannai Ino) hepatopancreas. Six purified diets were formulated to contain tripalmitin(TP), olive oil(OO, 72.87% 18:1n-9), grape seed oil(GO, 68.67% 18:2n-6), linseed oil(LO, 70.48% 18:3n-3), ARA oil(AO, 41.81% ARA) or EPA oil(EO, 44.09% EPA and 23.67% DAH). No significant difference in survival rate was observed among abalone fed with different diets. Weight gain rate(WGR) and daily growth rate of shell length(DGRSL) were significantly increased in abalone fed with diets containing OO, AO and EO, but decreased in abalone fed with LO diet(P < 0.05) in comparison with those fed with TP. High level of dietary 18:2n-6 resulted in higher content of n-6 polyunsaturated fatty acids(PUFAs) in abalone fed with GO than those fed with TP, OO, LO and EO(P < 0.05). n-3 PUFAs in abalone fed with LO was significantly higher than those in abalone fed with TP, OO, GO and AO(P < 0.05). The highest contents of 20:1n-9 and 22:1n-9 were observed in abalone fed with OO. The expression of Δ5 Fads in hepatopancreas of abalone was enhanced by high concentration of 18:3n-3 and suppressed by dietary LC-PUFAs; however it was not affected by dietary high concentration of 18:1n-9 or 18:2n-6. These results provided valuable information for understanding the synthesis of LC-PUFAs and nutritional regulation of Δ5 Fads expression in abalone.展开更多
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...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.展开更多
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△^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.
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This work was supported by Chinese National Programs for High Technology Research and Development (No. 2002AA207004).
文摘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.
文摘Delta-12 oleate desaturase gene （FAD2-1） which converts oleic acid into linoleic acid, is the key enzyme determining the fatty acid composition of cottonseed oil. By employing RT-PCR method, full length cDNA of cotton delta-12 oleate desat- urase gene GhFAD2-1 containing an open reading frame of 1 158 bp was cloned for constructing RNAi vector. A 515 bp long specific fragment of this gene was se- lected for constructing ihpRNA vector under the control of a seed-specific promoter NAPIN, named pFGC1008-NAPIN-FAD2-1; meanwhile miRNA gene-silencing vector pCAMBIA1302-amiRNA-FAD2-1 targeting GhFAD2-1 was also constructed.
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.
基金Project supported by"Tenth Five Years"Key Program of the State Science and Technology Commission in China(Grant Nos.2002BA901A15,2004BA411B01)
文摘Using degenerate primers and RT-PCR, RACE techniques, a 1491 bp cDNA segment of stearoyl-acyl carrier protein desaturase （SAD） is cloned from developing seeds of Jatropha curcas L. The segment contains a 1191 bp of complete open reading frame （ORF）. Analysis in the BLAST on NCBI shows that Jatropha curcas SAD （JSAD） gene encodes a protein precursor composed of a signal peptide of 33 amino acids and a mature peptide of 363 amino acids. The homological analysis shows that JSAD has high level of homology both in nucleotide sequence and in amino acid sequence to other plants SADs. The nucleotide and peptide identity of JSAD to Ricinus communis SAD （RSAD） is up to 89% and 96.2% respectively. Molecular modeling of JSAD indicates that its three-dimensional structure strongly resembled the crystal structure of RSAD.
基金supported by the National Natural Science Foundation of China (No. 30871930)
文摘This study investigated the effect of dietary lipid on the growth, fatty acid composition and Δ5 fatty acyl desaturase genes(Fads) expression of juvenile abalone(Haliotis discus hannai Ino) hepatopancreas. Six purified diets were formulated to contain tripalmitin(TP), olive oil(OO, 72.87% 18:1n-9), grape seed oil(GO, 68.67% 18:2n-6), linseed oil(LO, 70.48% 18:3n-3), ARA oil(AO, 41.81% ARA) or EPA oil(EO, 44.09% EPA and 23.67% DAH). No significant difference in survival rate was observed among abalone fed with different diets. Weight gain rate(WGR) and daily growth rate of shell length(DGRSL) were significantly increased in abalone fed with diets containing OO, AO and EO, but decreased in abalone fed with LO diet(P < 0.05) in comparison with those fed with TP. High level of dietary 18:2n-6 resulted in higher content of n-6 polyunsaturated fatty acids(PUFAs) in abalone fed with GO than those fed with TP, OO, LO and EO(P < 0.05). n-3 PUFAs in abalone fed with LO was significantly higher than those in abalone fed with TP, OO, GO and AO(P < 0.05). The highest contents of 20:1n-9 and 22:1n-9 were observed in abalone fed with OO. The expression of Δ5 Fads in hepatopancreas of abalone was enhanced by high concentration of 18:3n-3 and suppressed by dietary LC-PUFAs; however it was not affected by dietary high concentration of 18:1n-9 or 18:2n-6. These results provided valuable information for understanding the synthesis of LC-PUFAs and nutritional regulation of Δ5 Fads expression in abalone.
基金Supported by the State Key Basic Research and Development Plan of China （G1998010100） and the National Natural Science Foundation of China （30471053）.
文摘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.
