DNA sequences of fatty acid elongase 1 genes FAE1.1 （EA） and FAE1.2 （Ec） were isolated and characterized for 30 com- mercialized low erucic acid rapeseed （LEAR） cultivars in China. Four types of independent muta...DNA sequences of fatty acid elongase 1 genes FAE1.1 （EA） and FAE1.2 （Ec） were isolated and characterized for 30 com- mercialized low erucic acid rapeseed （LEAR） cultivars in China. Four types of independent mutation leading to low erucic acid trait were found, i.e., a single-base transition （eAl）, a two-base deletion （ec2） and four-base deletion （eC4） as well as single-base transition with a four-base deletion （eA.）. Three genotypes, i.e., eA1eA1eC2eC2, eA1eA1eC4eC4 and eA.eA.ec4ec4 were responsible for LEA content in storage Iipids of different rapeseed cultivars. Most of the LEAR cultivars had a genotype of eA1eA1ec2ec2, which were descended from the first LEAR cultivar, Oro. Yeast expression analysis revealed that two-base-pair （AA） deletion （ec2） at the base sites of 1 422-1 423 in the C genome FAE1 gene resulted in the absence of the condensing enzyme and led to the failure to produce erucic acid. Coexpression of FAE1 and ketoacyI-CoA reductase （KCR） or enoyI-CoA reductase （ECR） was found in high erucic acid rapeseed （HEAR） but not in LEAR （eA1eA1ec2ec2oreA1eA1ec4ec4）. Moreover, KCR and ECR were still coordinately regulated in eA1eA1ec2ec2 or eA1eA1ec4ec4 genotypes, suggesting that the expression of two genes was tightly linked. In addition, specific detection methods were developed by high-resolution melting curve analysis in order to detect eA1 and ec4.展开更多
Myrmecia incisa is a green coccoid freshwater microalgae, which is rich in arachidonic acid (ArA, C20: 4?-6, Δ5, 8, 11, 14), a long chain polyunsaturated fatty acid (PUFA), especially under nitrogen starvation stress...Myrmecia incisa is a green coccoid freshwater microalgae, which is rich in arachidonic acid (ArA, C20: 4?-6, Δ5, 8, 11, 14), a long chain polyunsaturated fatty acid (PUFA), especially under nitrogen starvation stress. A cDNA library of M. incisa was constructed with λ phage vectors and a 545 nt expressed sequence tag (EST) was screened from this library as a putative elongase gene due to its 56% and 49% identity to Marchantia polymorpha L. and Ostreococcus tauri Courties et Chrétiennot-Dinet, respectively. Based upon this EST sequence, an elongase gene designated MiFAE was isolated from M. incisa via 5′/3′ rapid amplification of cDNA ends (RACE). The cDNA sequence was 1 331 bp long and included a 33 bp 5′-untranslated region (UTR) and a 431 bp 3′-UTR with a typical poly-A tail. The 867 bp ORF encoded a predicted protein of 288 amino acids. This protein was characterized by a conserved histidine-rich box and a MYxYY motif that was present in other members of the elongase family. The genomic DNA sequence of MiFAE was found to be interrupted by three introns with splicing sites of Introns I (81 bp), II (81 bp), and III (67 bp) that conformed to the GT-AG rule. Quantitative real-time PCR showed that the transcription level of MiFAE in this microalga under nitrogen starvation was higher than that under normal condition. Prior to the ArA content accumulation, the transcription of MiFAE was enhanced, suggesting that it was possibly responsible for the ArA accumulation in this microalga cultured under nitrogen starvation conditions.展开更多
The marine microalga Nannochloropsis sp.contains various elongases and desaturases that are critical for biosynthesis of polyunsaturated fatty acids.A full-length cDNA encoding a long-chain fatty acid elongase,named N...The marine microalga Nannochloropsis sp.contains various elongases and desaturases that are critical for biosynthesis of polyunsaturated fatty acids.A full-length cDNA encoding a long-chain fatty acid elongase,named Ns FAE,was cloned from Nannochloropsis sp..The open reading frame of Ns FAE(GenBank accession no.MF680548)consisted of 1068 bp and encoded a predicted protein of 355 amino acids with molecular mass 38.8 k Da.The deduced polypeptide showed 43%–44%identity to fatty acyl elongases from other algae.RT-PCR experiments indicated that the Ns FAE gene exhibited the highest expression in Nannochloropsis sp.at 72 h(i.e.,during the third growth stage)and the expression was significantly lower in the other four growth stages.Plasmid p Ns FAE-CRISPR and a recombinant DNA fragment(ADH1p-Ns FAE-CYCt)were transformed into Saccharomyces cerevisiae strain BY4742 using the CRISPR-Cas system.Yeast transformants containing Ns FAE produced three fatty acids not normally present in wild-type BY4742-linoleic acid,linolenic acid and eicosadienoic acid-indicating that Ns FAE encodes a functional elongase enzyme.展开更多
Background:In rodents,research has revealed a role of liver X receptors(LXR) in controlling lipid homeostasis and regulating the synthesis of polyunsaturated fatty acids(PUFA).Recent data suggest that LXRB is the pred...Background:In rodents,research has revealed a role of liver X receptors(LXR) in controlling lipid homeostasis and regulating the synthesis of polyunsaturated fatty acids(PUFA).Recent data suggest that LXRB is the predominant LXR subtype in ruminant mammary cells,but its role in lipid metabolism is unknown.It was hypothesized that LXRB plays a role in lipid homeostasis via altering the synthesis of PUFA in the ruminant mammary gland.We used overexpression and knockdown of LXRB in goat primary mammary epithelial cells(GMEC) to evaluate abundance of lipogenic enzymes,fatty acid profiles,content of lipid stores and activity of the stearoyl-Co A desaturase(SCD1) promoter.Results:Overexpression of LXRB markedly upregulated the protein abundance of LXRB while incubation with si RNA targeting LXRB markedly decreased abundance of LXRB protein.Overexpression of LXRB plus T0901317(T09,a ligand for LXR) dramatically upregulated SCD1 and elongation of very long chain fatty acid-like fatty acid elongases 5–7(ELOVL 5–7),which are related to PUFA synthesis.Compared with the control,cells overexpressing LXRB and stimulated with T09 had greater concentrations of C16:0,16:1,18:1n7,18:1n9 and C18:2 as well as desaturation and elongation indices of C16:0.Furthermore,LXRB-overexpressing cells incubated with T09 had greater levels of triacylglycerol and cholesterol.Knockdown of LXRB in cells incubated with T09 led to downregulation of genes encoding elongases and desaturases.Knockdown of LXRB attenuated the increase in triacylglycerol and cholesterol that was induced by T09.In cells treated with dimethylsulfoxide,knockdown of LXRB increased the concentration of C16:0 at the expense of C18:0,while a significant decrease in C18:2 was observed in cells incubated with both si LXRB and T09.The abundance of sterol regulatory element binding transcription factor 1 precursor(p SREBP1) and its mature fragment(n SREBP1) was upregulated by T09,but not LXRB overexpression.In the cells cultured with T09,knockdown of LXRB downregulated the abundance for p SREBP1 and n SREBP1.Luciferase reporter assays revealed that the activities of wild type SCD1 promoter or fragment with SREBP1 response element(SRE) mutation were decreased markedly when LXRB was knocked down.Activity of the SCD1 promoter that was induced by T09 was blocked when the SRE mutation was introduced.Conclusion:The current study provides evidence of a physiological link between the LXRB and SREBP1 in the ruminant mammary cell.An important role was revealed for the LXRB-SREBP1 network in the synthesis of PUFA via the regulation of genes encoding elongases and desaturases.Thus,targeting this network might elicit broad effects on lipid homeostasis in ruminant mammary gland.展开更多
Dietary macronutrients and micronutrients play important roles in human health.On the other hand,the excessive energy derived from food is stored in the form of triacylglycerol.A variety of dietary and hormonal factor...Dietary macronutrients and micronutrients play important roles in human health.On the other hand,the excessive energy derived from food is stored in the form of triacylglycerol.A variety of dietary and hormonal factors affect this process through the regulation of the activities and expression levels of those key player enzymes involved in fatty acid biosynthesis such as acetyl-CoA carboxylase,fatty acid synthase,fatty acid elongases,and desaturases.As a micronutrient,vitamin A is essential for the health of humans.Recently,vitamin A has been shown to play a role in the regulation of glucose and lipid metabolism.This review summarizes recent research progresses about the roles of vitamin A in fatty acid synthesis.It focuses on the effects of vitamin A on the activities and expression levels of mRNA and proteins of key enzymes for fatty acid synthesis in vitro and in vivo.It appears that vitamin A status and its signaling pathway regulate the expression levels of enzymes involved in fatty acid synthesis.Future research directions are also discussed.展开更多
The fatty acid elongase 1 (FAE1) genes of Brassic napus were cloned from two cultivars, i.e. Zhong- shuan No. 9 with low erucic acid content, and Zhongyou 821 with high erucic acid content, using the degenerate PCR pr...The fatty acid elongase 1 (FAE1) genes of Brassic napus were cloned from two cultivars, i.e. Zhong- shuan No. 9 with low erucic acid content, and Zhongyou 821 with high erucic acid content, using the degenerate PCR primers. The sequence analysis showed that there was no intron within the FAE1 genes. The FAE1 genes from Zhongyou 821 contained a coding sequence of 1521 nucleotides, and those cloned from Zhongshuan No. 9 contained a 1517 bp coding sequence. Alignment of the FAE1 sequences from Brassica rapa, B. oleracea and B. napus detected 31 single nucleotide polymorphic sites (2.03%), which resulted in 7 amino-acid substitutions. Further analysis indicated that 19 SNPs were genome-specific, of which, 95% were synonymous mutations. The nucleotide substitution at po- sition 1217 in the FAE1 genes led to a specific site of restricted cleavage. An AvrII cleavage site was present only in the C genome genes and absent in the A genome FAE1 genes. Digestion profile of the FAE1 sequences from B. rapa, B. oleracea and B. napus produced with AvrII confirmed that the FAE1 genes of B. oleracea origin was recognized and digested, while that of B. rapa origin could not. The results indicated that by AvrII cleavage it was possible to distinguish B. rapa from B. oleracea and be- tween the A and C genome of B. napus. In addition, the FAE1 genes could be used as marker genes to detect the pollen flow of B. napus, thus providing an alternative method for risk assessment of gene flow.展开更多
超长链脂肪酸(very long chain fatty acids,VLCFAs)在生物体中具有广泛的生理功能,它们参与种子甘油酯、生物膜膜脂及鞘脂的合成,并为角质层蜡质的生物合成提供前体物质。角质层是覆盖在植物地上部分最表层的保护层,由角质和蜡质组成,...超长链脂肪酸(very long chain fatty acids,VLCFAs)在生物体中具有广泛的生理功能,它们参与种子甘油酯、生物膜膜脂及鞘脂的合成,并为角质层蜡质的生物合成提供前体物质。角质层是覆盖在植物地上部分最表层的保护层,由角质和蜡质组成,其中蜡质又分为角质层表皮蜡和内部蜡,在植物生长发育、适应外界环境方面起重要作用。VLCFAs的合成由脂肪酰-CoA延长酶催化,该酶是由β-酮脂酰-CoA合酶、β-酮脂酰-CoA还原酶、β-羟脂酰-CoA脱水酶和反式烯脂酰-CoA还原酶组成的多酶体系。合成后的VLCFAs通过脱羰基与酰基还原作用进入角质层蜡质合成途径,形成各种蜡质组分。文章就VLCFAs及角质层蜡质合成代谢途径中相关酶基因研究进展方面做了综述,并对植物蜡质基因研究中存在的问题提出一些看法。展开更多
基金financially supported by the National Natural Science Foundation of China (30471099)the National High Technology and Development Program of China (2006AA10A113)
文摘DNA sequences of fatty acid elongase 1 genes FAE1.1 （EA） and FAE1.2 （Ec） were isolated and characterized for 30 com- mercialized low erucic acid rapeseed （LEAR） cultivars in China. Four types of independent mutation leading to low erucic acid trait were found, i.e., a single-base transition （eAl）, a two-base deletion （ec2） and four-base deletion （eC4） as well as single-base transition with a four-base deletion （eA.）. Three genotypes, i.e., eA1eA1eC2eC2, eA1eA1eC4eC4 and eA.eA.ec4ec4 were responsible for LEA content in storage Iipids of different rapeseed cultivars. Most of the LEAR cultivars had a genotype of eA1eA1ec2ec2, which were descended from the first LEAR cultivar, Oro. Yeast expression analysis revealed that two-base-pair （AA） deletion （ec2） at the base sites of 1 422-1 423 in the C genome FAE1 gene resulted in the absence of the condensing enzyme and led to the failure to produce erucic acid. Coexpression of FAE1 and ketoacyI-CoA reductase （KCR） or enoyI-CoA reductase （ECR） was found in high erucic acid rapeseed （HEAR） but not in LEAR （eA1eA1ec2ec2oreA1eA1ec4ec4）. Moreover, KCR and ECR were still coordinately regulated in eA1eA1ec2ec2 or eA1eA1ec4ec4 genotypes, suggesting that the expression of two genes was tightly linked. In addition, specific detection methods were developed by high-resolution melting curve analysis in order to detect eA1 and ec4.
基金Supported by the National Natural Science Foundation of China (No.30972243)the Creative Project of the Shanghai Municipal Education Committee (No.09ZZ167)+1 种基金the Scientific Research Foundation for the Returned Overseas Chinese Scholars,State Education Ministry,the Key Discipline Project of the Shanghai Municipal Education Committee (No.J50701)the Excellent Thesis Grant from Shanghai Ocean University
文摘Myrmecia incisa is a green coccoid freshwater microalgae, which is rich in arachidonic acid (ArA, C20: 4?-6, Δ5, 8, 11, 14), a long chain polyunsaturated fatty acid (PUFA), especially under nitrogen starvation stress. A cDNA library of M. incisa was constructed with λ phage vectors and a 545 nt expressed sequence tag (EST) was screened from this library as a putative elongase gene due to its 56% and 49% identity to Marchantia polymorpha L. and Ostreococcus tauri Courties et Chrétiennot-Dinet, respectively. Based upon this EST sequence, an elongase gene designated MiFAE was isolated from M. incisa via 5′/3′ rapid amplification of cDNA ends (RACE). The cDNA sequence was 1 331 bp long and included a 33 bp 5′-untranslated region (UTR) and a 431 bp 3′-UTR with a typical poly-A tail. The 867 bp ORF encoded a predicted protein of 288 amino acids. This protein was characterized by a conserved histidine-rich box and a MYxYY motif that was present in other members of the elongase family. The genomic DNA sequence of MiFAE was found to be interrupted by three introns with splicing sites of Introns I (81 bp), II (81 bp), and III (67 bp) that conformed to the GT-AG rule. Quantitative real-time PCR showed that the transcription level of MiFAE in this microalga under nitrogen starvation was higher than that under normal condition. Prior to the ArA content accumulation, the transcription of MiFAE was enhanced, suggesting that it was possibly responsible for the ArA accumulation in this microalga cultured under nitrogen starvation conditions.
基金supported by the Basic Scientific Fund for National Public Research Institutes of China (No. 2016Q07)
文摘The marine microalga Nannochloropsis sp.contains various elongases and desaturases that are critical for biosynthesis of polyunsaturated fatty acids.A full-length cDNA encoding a long-chain fatty acid elongase,named Ns FAE,was cloned from Nannochloropsis sp..The open reading frame of Ns FAE(GenBank accession no.MF680548)consisted of 1068 bp and encoded a predicted protein of 355 amino acids with molecular mass 38.8 k Da.The deduced polypeptide showed 43%–44%identity to fatty acyl elongases from other algae.RT-PCR experiments indicated that the Ns FAE gene exhibited the highest expression in Nannochloropsis sp.at 72 h(i.e.,during the third growth stage)and the expression was significantly lower in the other four growth stages.Plasmid p Ns FAE-CRISPR and a recombinant DNA fragment(ADH1p-Ns FAE-CYCt)were transformed into Saccharomyces cerevisiae strain BY4742 using the CRISPR-Cas system.Yeast transformants containing Ns FAE produced three fatty acids not normally present in wild-type BY4742-linoleic acid,linolenic acid and eicosadienoic acid-indicating that Ns FAE encodes a functional elongase enzyme.
基金supported by the National Natural Science Foundation of China(31702090)Key R&D program of Zhejiang Province(2022C04017)+1 种基金Zhejiang Provincial Major Science and Technology Projects on Agricultural New Varieties Selection and Breeding(2021C02068-6)Opening fund in Key Laboratory of Molecular Animal Nutrition(Zhejiang University,KLMAN202103).
文摘Background:In rodents,research has revealed a role of liver X receptors(LXR) in controlling lipid homeostasis and regulating the synthesis of polyunsaturated fatty acids(PUFA).Recent data suggest that LXRB is the predominant LXR subtype in ruminant mammary cells,but its role in lipid metabolism is unknown.It was hypothesized that LXRB plays a role in lipid homeostasis via altering the synthesis of PUFA in the ruminant mammary gland.We used overexpression and knockdown of LXRB in goat primary mammary epithelial cells(GMEC) to evaluate abundance of lipogenic enzymes,fatty acid profiles,content of lipid stores and activity of the stearoyl-Co A desaturase(SCD1) promoter.Results:Overexpression of LXRB markedly upregulated the protein abundance of LXRB while incubation with si RNA targeting LXRB markedly decreased abundance of LXRB protein.Overexpression of LXRB plus T0901317(T09,a ligand for LXR) dramatically upregulated SCD1 and elongation of very long chain fatty acid-like fatty acid elongases 5–7(ELOVL 5–7),which are related to PUFA synthesis.Compared with the control,cells overexpressing LXRB and stimulated with T09 had greater concentrations of C16:0,16:1,18:1n7,18:1n9 and C18:2 as well as desaturation and elongation indices of C16:0.Furthermore,LXRB-overexpressing cells incubated with T09 had greater levels of triacylglycerol and cholesterol.Knockdown of LXRB in cells incubated with T09 led to downregulation of genes encoding elongases and desaturases.Knockdown of LXRB attenuated the increase in triacylglycerol and cholesterol that was induced by T09.In cells treated with dimethylsulfoxide,knockdown of LXRB increased the concentration of C16:0 at the expense of C18:0,while a significant decrease in C18:2 was observed in cells incubated with both si LXRB and T09.The abundance of sterol regulatory element binding transcription factor 1 precursor(p SREBP1) and its mature fragment(n SREBP1) was upregulated by T09,but not LXRB overexpression.In the cells cultured with T09,knockdown of LXRB downregulated the abundance for p SREBP1 and n SREBP1.Luciferase reporter assays revealed that the activities of wild type SCD1 promoter or fragment with SREBP1 response element(SRE) mutation were decreased markedly when LXRB was knocked down.Activity of the SCD1 promoter that was induced by T09 was blocked when the SRE mutation was introduced.Conclusion:The current study provides evidence of a physiological link between the LXRB and SREBP1 in the ruminant mammary cell.An important role was revealed for the LXRB-SREBP1 network in the synthesis of PUFA via the regulation of genes encoding elongases and desaturases.Thus,targeting this network might elicit broad effects on lipid homeostasis in ruminant mammary gland.
基金Supported by the Financial Support of the Overseas Training Program for Outstanding Young and Middle-Aged Teachers in Universities in Jiangsu Province,China(to Yang FC).
文摘Dietary macronutrients and micronutrients play important roles in human health.On the other hand,the excessive energy derived from food is stored in the form of triacylglycerol.A variety of dietary and hormonal factors affect this process through the regulation of the activities and expression levels of those key player enzymes involved in fatty acid biosynthesis such as acetyl-CoA carboxylase,fatty acid synthase,fatty acid elongases,and desaturases.As a micronutrient,vitamin A is essential for the health of humans.Recently,vitamin A has been shown to play a role in the regulation of glucose and lipid metabolism.This review summarizes recent research progresses about the roles of vitamin A in fatty acid synthesis.It focuses on the effects of vitamin A on the activities and expression levels of mRNA and proteins of key enzymes for fatty acid synthesis in vitro and in vivo.It appears that vitamin A status and its signaling pathway regulate the expression levels of enzymes involved in fatty acid synthesis.Future research directions are also discussed.
基金the National Natural Science Foundation of China (Grant No. 30471099)Development Plan of the State Key Fundamental Research of China (Grant No. 2006CB101600)the National High Technology and Development Program of China (Grant No. 2006AA10A113)
文摘The fatty acid elongase 1 (FAE1) genes of Brassic napus were cloned from two cultivars, i.e. Zhong- shuan No. 9 with low erucic acid content, and Zhongyou 821 with high erucic acid content, using the degenerate PCR primers. The sequence analysis showed that there was no intron within the FAE1 genes. The FAE1 genes from Zhongyou 821 contained a coding sequence of 1521 nucleotides, and those cloned from Zhongshuan No. 9 contained a 1517 bp coding sequence. Alignment of the FAE1 sequences from Brassica rapa, B. oleracea and B. napus detected 31 single nucleotide polymorphic sites (2.03%), which resulted in 7 amino-acid substitutions. Further analysis indicated that 19 SNPs were genome-specific, of which, 95% were synonymous mutations. The nucleotide substitution at po- sition 1217 in the FAE1 genes led to a specific site of restricted cleavage. An AvrII cleavage site was present only in the C genome genes and absent in the A genome FAE1 genes. Digestion profile of the FAE1 sequences from B. rapa, B. oleracea and B. napus produced with AvrII confirmed that the FAE1 genes of B. oleracea origin was recognized and digested, while that of B. rapa origin could not. The results indicated that by AvrII cleavage it was possible to distinguish B. rapa from B. oleracea and be- tween the A and C genome of B. napus. In addition, the FAE1 genes could be used as marker genes to detect the pollen flow of B. napus, thus providing an alternative method for risk assessment of gene flow.
文摘超长链脂肪酸(very long chain fatty acids,VLCFAs)在生物体中具有广泛的生理功能,它们参与种子甘油酯、生物膜膜脂及鞘脂的合成,并为角质层蜡质的生物合成提供前体物质。角质层是覆盖在植物地上部分最表层的保护层,由角质和蜡质组成,其中蜡质又分为角质层表皮蜡和内部蜡,在植物生长发育、适应外界环境方面起重要作用。VLCFAs的合成由脂肪酰-CoA延长酶催化,该酶是由β-酮脂酰-CoA合酶、β-酮脂酰-CoA还原酶、β-羟脂酰-CoA脱水酶和反式烯脂酰-CoA还原酶组成的多酶体系。合成后的VLCFAs通过脱羰基与酰基还原作用进入角质层蜡质合成途径,形成各种蜡质组分。文章就VLCFAs及角质层蜡质合成代谢途径中相关酶基因研究进展方面做了综述,并对植物蜡质基因研究中存在的问题提出一些看法。
