Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.611
Filtrar
1.
World J Microbiol Biotechnol ; 36(1): 17, 2020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31912247

RESUMO

In this study, the effects of limited and excess nitrate on biomass, lipid production, and fatty acid profile in Messastrum gracile SE-MC4 were determined. The expression of fatty acid desaturase genes, namely stearoyl-ACP desaturase (SAD), omega-6 fatty acid desaturase (ω-6 FAD), omega-3 fatty acid desaturase isoform 1 (ω-3 FADi1), and omega-3 fatty acid desaturase isoform 2 (ω-3 FADi2) was also assessed. It was found that nitrate limitation generally increased the total oil, α-linolenic acid (C18:3n3) and total polyunsaturated fatty acid (PUFA) contents in M. gracile. The reduction of nitrate concentration from 1.76 to 0.11 mM increased the total oil content significantly from 32.5 to 41.85% (dry weight). Palmitic (C16:0) and oleic (C18:1) acids as the predominant fatty acids in this microalgae constituted between 82 and 87% of the total oil content and were relatively consistent throughout all nitrate concentrations tested. The expression of SAD, ω-6 FAD, and ω-3 FADi2 genes increased under nitrate limitation, especially at 0.11 mM nitrate. The ω-3 FADi1 demonstrated a binary up-regulation pattern of expression under both nitrate-deficient (0.11 mM) and -excess (3.55 mM) conditions. Thus, findings from this study suggested that limited or excess nitrate could be used as part of a cultivation strategy to increase oil and PUFA content following media optimisation and more efficient culture methodology. Data obtained from the expression of desaturase genes would provide valuable insights into their roles under excess and limited nitrate conditions in M. gracile, potentially paving the way for future genetic modifications.


Assuntos
Ácidos Graxos Dessaturases/genética , Ácidos Graxos/análise , Microalgas/crescimento & desenvolvimento , Nitrogênio/metabolismo , Biomassa , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos Insaturados/metabolismo , Regulação Enzimológica da Expressão Gênica , Metabolismo dos Lipídeos , Microalgas/genética , Microalgas/metabolismo , Ácido Oleico/metabolismo , Ácido Palmítico/metabolismo , Ácido alfa-Linoleico/metabolismo
2.
J Agric Food Chem ; 68(1): 426-438, 2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31855429

RESUMO

Modern peanut contains fatty acid desaturase 2 (FAD2) mutation, which is capable of producing high oleic acid for human health. However, the dynamic changes of the lipidome regarding fad2 remain elusive in peanut seed. In the present study, 547 lipid features were identified in high- and normal-oleic peanut seeds by utilizing the mass spectrometric approach. The fad2-induced differently expressed lipids (DELs) were polarly distributed at early and maturation stages during high-oleic acid (OA) seed development. Subsequently, integration of previously published proteomic data and lipidomic data revealed that 21 proteins and 149 DELs were annotated into the triacylglycerol assembly map, of which nine enzymes and 31 lipid species shared similar variation tendencies. Additionally, the variation tendencies of 17 acyl fatty acids were described in a hypothetical biosynthetic pathway. Collectively, the understanding of the lipid composition correlated with fad2 established a foundation for future high-OA peanut breeding based on lipidomic data.


Assuntos
Arachis/química , Lipídeos/química , Ácido Oleico/química , Proteínas de Plantas/química , Arachis/genética , Arachis/crescimento & desenvolvimento , Arachis/metabolismo , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Metabolismo dos Lipídeos , Mutação , Ácido Oleico/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteômica , Sementes/química , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/metabolismo
3.
Artigo em Inglês | MEDLINE | ID: mdl-31525459

RESUMO

As the first marine teleost demonstrated to biosynthesize long-chain polyunsaturated fatty acids (LC-PUFAs) from C18 precursors such as linoleic acid (LOA, 18:2n-6) and α-linolenic acid (ALA, 18:3n-3), the rabbitfish (Siganus canaliculatus) contains the complete enzymatic system for LC-PUFA biosynthesis, including Δ6/Δ5 fatty acid desaturase (Fad), Δ4 Fad, and elongase 5 (Elovl5). Previously, our group demonstrated that hepatocyte nuclear factor 4α (Hnf4α) is a transcription factor (TF) for rabbitfish Δ4 fad and elovl5, and interacts with the core promoter of Δ6/Δ5 fad. To fully clarify the role of Hnf4α in the regulation of LC-PUFA biosynthesis, the present study aimed to explore the regulatory role of Hnf4α on Δ6/Δ5 fad gene expression. First, Hnf4α overexpression and agonist assays identified the Hnf4α response region in the Δ6/Δ5 fad core promoter as -456 bp to +51 bp. Bioinformatic analysis predicted four potential Hnf4α binding elements in the core promoter, which were confirmed by site-directed mutation and functional assays in a dual luciferase assay system. Moreover, the mRNA expression levels of hnf4α, Δ6/Δ5 fad, and Δ4 fad were significantly increased in the S. canaliculatus hepatocyte line (SCHL) cells after treatment with Hnf4α agonists (Alverine and Benfluorex) or its mRNA overexpression. By contrast, the expression levels of these three genes were markedly decreased after hnf4a small interfering RNA (siRNA) transfection. The results indicated that Hnf4α has a regulatory effect on rabbitfish Δ6/Δ5 fad gene transcription, identifying Hnf4α as a TF of Δ6/Δ5 fad in vertebrates for the first time.


Assuntos
Ácidos Graxos Dessaturases/biossíntese , Proteínas de Peixes/metabolismo , Peixes/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Fator 4 Nuclear de Hepatócito/metabolismo , Linoleoil-CoA Desaturase/biossíntese , Animais , Ácidos Graxos Dessaturases/genética , Proteínas de Peixes/genética , Peixes/genética , Fator 4 Nuclear de Hepatócito/genética , Linoleoil-CoA Desaturase/genética
4.
BMC Plant Biol ; 19(1): 481, 2019 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-31703625

RESUMO

BACKGROUND: Linoleic acid is an important polyunsaturated fatty acid, required for all eukaryotes. Microsomal delta-12 (Δ12) oleate desaturase (FAD2) is a key enzyme for linoleic acid biosynthesis. Desert shrub Artemisia sphaerocephala is rich in linoleic acid, it has a large FAD2 gene family with twenty-six members. The aim of this work is to unveil the difference and potentially functionality of AsFAD2 family members. RESULTS: Full-length cDNAs of twenty-one AsFAD2 genes were obtained from A. sphaerocephala. The putative polypeptides encoded by AsFAD2 family genes showed a high level of sequence similarity and were relatively conserved during evolution. The motif composition was also relatively conservative. Quantitative real-time PCR analysis revealed that the AsFAD2-1 gene was strongly expressed in developing seeds, which may be closely associated with the high accumulating ability of linoleic acid in A. sphaerocephala seeds. Although different AsFAD2 family members showed diverse response to salt stress, the overall mRNA levels of the AsFAD2 family genes was stable. Transient expression of AsFAD2 genes in the Nicotiana benthamiana leaves revealed that the encoded proteins were all located in the endoplasmic reticulum. Heterologous expression in Saccharomyces cerevisiae suggested that only three AsFAD2 enzymes, AsFAD2-1, - 10, and - 23, were Δ12 oleate desaturases, which could convert oleic acid to linoleic acid, whereas AsFAD2-1 and AsFAD2-10 could also produce palmitolinoleic acid. CONCLUSIONS: This research reported the cloning, expression studies, subcellular localization and functional identification of the large AsFAD2 gene family. These results should be helpful in understanding fatty acid biosynthesis in A. sphaerocephala, and has the potential to be applied in the study of plant fatty acids traits.


Assuntos
Artemisia/genética , Ácidos Graxos Dessaturases/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Estresse Salino/genética , Artemisia/enzimologia , Artemisia/metabolismo , Ácidos Graxos Dessaturases/metabolismo , Perfilação da Expressão Gênica , Genes de Plantas , Família Multigênica , Filogenia , Proteínas de Plantas/metabolismo
5.
Int J Mol Sci ; 20(20)2019 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-31614732

RESUMO

The rabbitfish Siganus canaliculatus was the first marine teleost demonstrated to have the ability for the biosynthesis of long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA) from C18 PUFA precursors, and all the catalytic enzymes including two fatty acyl desaturase 2 (Δ4 Fads2 and Δ6/Δ5 Fads2) and two elongases (Elovl4 and Elovl5) have been identified, providing a good model for studying the regulatory mechanisms of LC-PUFA biosynthesis in fish. Stimulatory protein 1 (Sp1) has been speculated to be a vital transcription factor in determining the promoter activity of Fads-like genes in fish, however its regulatory effects on gene expression and LC-PUFA biosynthesis have not been demonstrated. Bioinformatic analysis predicted potential Sp1 binding sites in the promoters of the rabbitfish Δ6/Δ5 fads2 and elovl5, but not in Δ4 fads2 promoter. Here we cloned full-length cDNA of the rabbitfish sp1 gene, which encoded a putative protein of 701 amino acids, and was expressed in all tissues studied with highest levels in gill and eyes. The dual luciferase reporter assay in HepG2 line cells demonstrated the importance of the Sp1 binding site for the promoter activities of both Δ6/Δ5 fads2 and elovl5. Moreover, the electrophoretic mobility shift assay confirmed the direct interaction of Sp1 with the two promoters. Insertion of the Sp1 binding site of Δ6/Δ5 fads2 promoter into the corresponding region of the Δ4 fads2 promoter significantly increased activity of the latter. In the Siganus canaliculatus hepatocyte line (SCHL) cells, mRNA levels of Δ6/Δ5 fads2 and elovl5 were positively correlated with the expression of sp1 when sp1 was overexpressed or knocked-down by RNAi or antagonist (mithramycin) treatment. Moreover, overexpression of sp1 also led to a higher conversion of 18:2n-6 to 18:3n-6, 18:2n-6 to 20:2n-6, and 18:3n-3 to 20:3n-3, which related to the functions of Δ6/Δ5 Fads2 and Elovl5, respectively. These results indicated that Sp1 is involved in the transcriptional regulation of LC-PUFA biosynthesis by directly targeting Δ6/Δ5 fads2 and elovl5 in rabbitfish, which is the first report of Sp1 involvement in the regulation of LC-PUFA biosynthesis in vertebrates.


Assuntos
Ácidos Graxos Dessaturases/genética , Ácidos Graxos Ômega-3/biossíntese , Proteínas de Peixes/genética , Fator de Transcrição Sp1/metabolismo , Animais , Ácidos Graxos Dessaturases/metabolismo , Proteínas de Peixes/metabolismo , Células Hep G2 , Humanos , Fígado/enzimologia , Fígado/metabolismo , Perciformes/genética , Perciformes/metabolismo , Fator de Transcrição Sp1/genética , Regulação para Cima
6.
J Agric Food Chem ; 67(39): 10984-10993, 2019 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-31525294

RESUMO

The objective of the present study was to reveal the effects of four types of nitrogen sources (soymeal, yeast extract, KNO3, and ammonium tartrate) on the lipid metabolism of the oleaginous fungus Mortierella alpina using untargeted lipidomics, targeted fatty acid, and reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis. Our results showed clear differences in the contents and compositions of lipids between four types of nitrogen sources. Soymeal and ammonium tartrate supplementation favored the accumulation of triglycerides with arachidonic acid (ARA) and C16-18 fatty acids, respectively. These results were further validated by our targeted fatty acid analysis. RT-qPCR analysis of related genes in M. alpina between the four nitrogen source conditions found that soymeal supplementation dramatically increased the expression of GPAT, ELOVL, and Δ12/Δ6 desaturase. Our findings provided new insights into the regulation of lipid biosynthesis in M. alpina and potential avenues for genetic manipulation and highlighted the importance of an optimal nitrogen source for ARA-rich oil production.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Lipídeos/biossíntese , Lipídeos/química , Espectrometria de Massas/métodos , Mortierella/metabolismo , Nitrogênio/metabolismo , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos/biossíntese , Ácidos Graxos/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Mortierella/química , Mortierella/enzimologia , Mortierella/genética
7.
Phytochemistry ; 168: 112114, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31499273

RESUMO

Idesia polycarpa is a valuable oil-producing tree and can potentially be used for edible oil and biofuel production. The fruits of I. polycarpa are unique in that they contain both saturated and unsaturated lipids. Fatty acid desaturase 2 (FAD2), also as known as omega-6 fatty acid desaturase in endoplasmic, is a key enzyme for linoleic acid and α-linolenic acid biosynthesis. However, bioinformatics and expression of FAD2 in I. polycarpa are still absent. Here, to gain insight into the lipid and linoleic synthesis of I. polycarpa, we compared the fruits from different growth stages. Lipid accumulation rates, final lipid content, linoleic accumulation rates and final linoleic content were significantly different among the different stages. In a further step, the FAD2 gene from fruits of I. polycarpa, named IpFAD2, was cloned and characterized. A partial fragment of 169 bp of IpFAD2 was amplified by degenerate PCR. Full cDNA of IpFAD2 was obtained by the RACE technique. The open-reading frame of IpFAD2 was 1149 bp in length, encoding 382 amino acids. A comparison of the deduced amino acids sequence of IpFAD2 with FAD2 from other species showed high similarities, ranging from 78.8 to 92.6%. The IpFAD2-predicted protein has a theoretical molecular mass of 44.03 kDa and an isoelectric point (pI) of 8.04. It has five transmembrane helices located on the endoplasmic reticulum. The IpFAD2-predicted protein was classified as belonging to the Membrane-FADS-like superfamily based on its conserved domain analysis. Expression analysis based on qRT-PCR indicated that IpFAD2 was expressed in different fruit growth stages, with the highest expression level at 80 DAP and the lowest at 130 DAP. The expression of IpFAD2 was positively correlated with the linoleic accumulation rates in I. polycarpa fruits. Prokaryotic expression in Escherichia. Coli BL21(DE3) indicated that IpFAD2 gene could encode a bio-functional omega-6 fatty acid desaturase. Heterologous expression in Arabidopsis thaliana confirmed that the isolated IpFAD2 proteins could catalyse linoleic synthesis. This is the first cloning and expression analysis of FAD2 from I. polycarpa, significantly contributing to our understanding of the role of IpFAD2 in linoleic synthesis, esp. in terms of genetic engineering breeding for linoleic production.


Assuntos
Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Compostos Fitoquímicos/biossíntese , Salicaceae/enzimologia , Salicaceae/genética , Sequência de Aminoácidos , Clonagem Molecular , Ácidos Graxos Dessaturases/química , Ácido Linoleico/biossíntese , Modelos Moleculares , Compostos Fitoquímicos/química , Alinhamento de Sequência
8.
BMC Plant Biol ; 19(1): 393, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31500565

RESUMO

BACKGROUND: In higher plants, the FAD2 gene encodes the microsomal oleate Δ12-desaturase, one of the key enzymes essential for the biosynthesis of the polyunsaturated lipids that serve many important functions in plant development and stress responses. FAD2 catalyzes the first step, in the biosynthesis of the polyunsaturated fatty acids (PUFAs) found in the cell membrane and cell wall, and it is thus of great importance to investigate the regulatory role of FAD2 in anther development. RESULTS: We reported the molecular characterization of the cotton (Gossypium hirsutum) GhFAD2 gene family and the essential role of GhFAD2-3 in cotton anther development. G. hirsutum contains four pairs of homoeologous FAD2 genes (GhFAD2-1 to GhFAD2-4). GhFAD2-3 is ubiquitously and relatively highly expressed in all analyzed tissues, particularly in anthers. Specific inhibition of GhFAD2-3 using the RNA interference approach resulted in male sterility due to impaired anther development at the stages from meiosis to maturation. The cellular phenotypic abnormality observed at the meiosis stage of the GhFAD2-3 silenced plant (fad2-3) coincides with the significant reduction of C18:2 in anthers at the same stage. Compared with that of the wild type (WT), the content of C18:1 was 41.48%, which increased by 5 fold in the fad2-3 anther at the pollen maturation stage. Moreover, the ratio of monounsaturated to polyunsaturated fatty acid was 5.43 in fad2-3 anther, which was much higher than that of the WT (only 0.39). Through compositional analysis of anthers cuticle and transcriptome data, we demonstrated it was unfavorable to the development of anther by regulating GhFAD2-3 expression level to increase the oleic acid content. CONCLUSIONS: Our work demonstrated the importance of C18:2 and/or C18:3 in the development of the pollen exine and anther cuticle in cotton and provided clue for further investigation of the physiological significance of the fatty acid composition for plant growth and development.


Assuntos
Ácidos Graxos Dessaturases/genética , Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Gossypium/genética , Ácido Oleico/metabolismo , Proteínas de Plantas/genética , Ácidos Graxos Dessaturases/metabolismo , Flores/genética , Gossypium/crescimento & desenvolvimento , Gossypium/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo
9.
J Agric Food Chem ; 67(35): 9851-9857, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31418561

RESUMO

Arachidonic acid (ARA, C20:4) is a typical ω-6 polyunsaturated fatty acid with special functions. Using Yarrowia lipolytica as an unconventional chassis, we previously showed the performance of the Δ-6 pathway in ARA production. However, a significant increase in the Δ-9 pathway has rarely been reported. Herein, the Δ-9 pathway from Isochrysis galbana was constructed via pathway engineering, allowing us to synthesize ARA at 91.5 mg L-1. To further improve the ARA titer, novel enzyme fusions of Δ-9 elongase and Δ-8 desaturase were redesigned in special combinations containing different linkers. Finally, with the integrated pathway engineering and synthetic enzyme fusion, a 29% increase in the ARA titer, up to 118.1 mg/L, was achieved using the reconstructed strain RH-4 that harbors the rigid linker (GGGGS). The results show that the combined pathway and protein engineering can significantly facilitate applications of Y. lipolytica.


Assuntos
Ácido Araquidônico/biossíntese , Engenharia Metabólica , Yarrowia/genética , Yarrowia/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Glucose/metabolismo , Haptófitas/enzimologia
10.
Int J Mol Sci ; 20(15)2019 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-31382584

RESUMO

Low temperature is an environmental stress factor that is always been applied in research on improving crop growth, productivity, and quality of crops. Polyunsaturated fatty acids (PUFAs) play an important role in cold tolerance, so its genetic manipulation of the PUFA contents in crops has led to the modification of cold sensitivity. In this study, we over-expressed an ω-3 fatty acid desaturase from Glycine max (GmFAD3A) drove by a maize ubiquitin promoter in rice. Compared to the wild type (ZH11), ectopic expression of GmFAD3A increased the contents of lipids and total PUFAs. Seed germination rates in GmFAD3A transgenic rice were enhanced under low temperature (15 °C). Moreover, cold tolerance and survival ratio were significantly improved in GmFAD3A transgenic seedlings. Malondialdehyde (MDA) content in GmFAD3A transgenic rice was lower than that in WT under cold stress, while proline content obviously increased. Meanwhile, the activities of superoxide dismutase (SOD), hydroperoxidase (CAT), and peroxidase (POD) increased substantially in GmFAD3A transgenic rice after 4 h of cold treatment. Taken together, our results suggest that GmFAD3A can enhances cold tolerance and the seed germination rate at a low temperature in rice through the accumulation of proline content, the synergistic increase of the antioxidant enzymes activity, which finally ameliorated the oxidative damage.


Assuntos
Ácidos Graxos Dessaturases/genética , Oryza/genética , Plântula/genética , Estresse Fisiológico/genética , Catalase/genética , Regulação da Expressão Gênica de Plantas/genética , Germinação/genética , Metabolismo dos Lipídeos/genética , Oryza/enzimologia , Oryza/crescimento & desenvolvimento , Peroxidase/genética , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plântula/crescimento & desenvolvimento , Sementes/genética , Sementes/crescimento & desenvolvimento , Soja/enzimologia , Soja/genética , Superóxido Dismutase/genética
11.
Life Sci ; 233: 116757, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31419446

RESUMO

AIMS: Previous studies have demonstrated that long non-coding RNAs (lncRNAs) were involved in tumorigenesis in various human neoplasms, including osteosarcoma (OS). However, the expression and specific role of lncRNA linc00460 in OS remain unknown. MATERIALS AND METHODS: Bioinformatics analysis, Quantitative real-time polymerase chain reaction (qRT-PCR), CCK-8 assay, Colony formation assay, Wound healing assay, Transwell assay, Dual luciferase reporter assay, RNA immunoprecipitation and Western blot were utilized to analyze or detect survival, gene expression, cell proliferation, cell migration, cell invasion and interest protein levels, respectively. KEY FINDINGS: In this study, we found high linc00460 expression predicted poor prognosis of pan-cancer patients. Linc00460 was up-regulated in OS tissues and cells. High linc00460 expression was positively correlated with distant metastasis and poor overall survival of OS patients. Knockdown of linc00460 suppressed OS cells proliferation and metastasis in vitro. In addition, an inverse correlation between linc00460/miR-1224-5p and miR-1224-5p/FADS1 was observed in OS. Mechanistically, linc00460 functioned as a competitively endogenous RNA (ceRNA) to up-regulate FADS1 expression via sponging miR-1224-5p in OS, thereby promoting OS progression. SIGNIFICANCE: In conclusion, this study recognized linc00460 as a new oncogenic lncRNA in OS and suggests that the linc00460/miR-1224-5p/FADS1 axis might be a potential therapeutic target for OS.


Assuntos
Neoplasias Ósseas/patologia , Ácidos Graxos Dessaturases/metabolismo , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Osteossarcoma/patologia , RNA Longo não Codificante/genética , Adulto , Apoptose , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/cirurgia , Movimento Celular , Proliferação de Células , Progressão da Doença , Ácidos Graxos Dessaturases/genética , Feminino , Humanos , Masculino , Osteossarcoma/genética , Osteossarcoma/metabolismo , Osteossarcoma/cirurgia , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas , Adulto Jovem
12.
Anticancer Res ; 39(7): 3651-3660, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31262891

RESUMO

BACKGROUND/AIM: Cytochrome P450 epoxygenase is a major enzyme involved in the metabolism of ω-3 polyunsaturated fatty acids (PUFAs) to produce biologically active ω-3 epoxy fatty acids (ω-3 epoxides). In general, all epoxy PUFAs including ω-3 epoxides are quickly metabolized/inactivated by soluble epoxide hydrolase (sEH) to form diol products. The aims of this study were to determine the effect and mechanism of fat-1 transgene, and ω-3 PUFA combined with sEH gene knockout or inhibitor on inhibiting pancreatic cancer and the related mechanisms involved. MATERIALS AND METHODS: PK03-mutant KrasG12D murine pancreatic carcinoma cells were inoculated into mouse models including fat-1, sEH-/- and C57BL/6J mice. The mice were fed with AIN-76A diet with or without ω-3 PUFA supplementation or treated with sEH inhibitor. In addition to tumor growth (tumor size and weight), cell proliferation, mutant Kras-mediated signaling, inflammatory reaction and angiogenesis were analyzed immunohisto-chemically and by western blot assay. ω-3 PUFA metabolism, particularly focusing on ω-3 epoxy fatty acids (ω-3 epoxides), was measured using a liquid chromatography with tandem mass spectrometry (LC-MS/MS) approach. RESULTS: Significant decreases of weight and size of the PK03 pancreatic carcinoma were observed in the fat-1 transgenic mice treated with sEH inhibitor compared to those of C57BL/6J control mice fed with AIN-76A diet (weight: 0.28±0.04 g vs. 0.58±0.06 g; size: 187.0±17.5 mm3 vs. 519.3±60.6 mm3). In a separate experiment, sEH-/- mice fed ω-3 PUFA supplement and C57BL/6J mice treated with sEH inhibitor and fed ω-3 PUFA supplement exhibited a significant reduction in the weight and size of the pancreatic carcinoma compared to C57BL/6J control mice (weight: 0.26±.26 g and 0.39±.39 g vs. 0.69±0.11 g, respectively; size: 274.2±36.2 mm3 and 296.4±99.8 mm3 vs. 612.6±117.8 mm3, respectively). Moreover, compared to the pancreatic tumors in C57BL/6J control mice, the tumors in fat-1 transgenic mice treated with sEH inhibitor showed a significant less inflammatory cell infiltrate (62.6±9.2/HPF (high power field) vs. 8.0±1.2/HPF), tumor cell proliferation (48.5±1.7% vs. 16.5±1.6%), and angiogenesis (micro-vessel density (MVD): 35.0±1.0 vs. 11.1±0.5) immunohistochemically, as well as significantly increased caspase-3 labeled apoptosis (0.44±0.06% vs. 0.69±0.06%, respectively). Using western blot approach, significant inhibition of mutant Kras-activated signals including phosphorylated Serine/threonine kinases (cRAF), Mitogen-activated protein kinase kinase (MEK), and extracellular signal-regulated kinase (ERK) were identified in pancreatic carcinoma of fat-1 transgenic mice treated with sEH inhibitor. Eicosanoic acid metabolic profiling of the serum specimens detected a significant increase of the ratios of epoxides to dihydroxy fatty acid (DiHDPE) for docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), and epoxides/dihydroxy octadecenoic acid (DiHOME) for arachidonic acid (ARA) and linoleic acid (LA), as well as a significant increase of epoxy metabolites of DHA, EPA, ARA and LA in fat-1 transgenic mice treated with a sEH inhibitor. CONCLUSION: ω-3 epoxy products from ω-3 PUFA metabolism play a crucial role in inhibiting pancreatic cancer growth, and use of ω-3 PUFAs combined with sEH inhibition is a strategy with high potential for pancreatic cancer treatment and prevention.


Assuntos
Adenocarcinoma/terapia , Proteínas de Caenorhabditis elegans/genética , Suplementos Nutricionais , Compostos de Epóxi/farmacologia , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Ômega-3/farmacologia , Neoplasias Pancreáticas/terapia , Adenocarcinoma/patologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias Pancreáticas/patologia
13.
BMC Plant Biol ; 19(1): 311, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31307375

RESUMO

BACKGROUND: CRISPR/Cas9 gene editing is now revolutionizing the ability to effectively modify plant genomes in the absence of efficient homologous recombination mechanisms that exist in other organisms. However, soybean is allotetraploid and is commonly viewed as difficult and inefficient to transform. In this study, we demonstrate the utility of CRISPR/Cas9 gene editing in soybean at relatively high efficiency. This was shown by specifically targeting the Fatty Acid Desaturase 2 (GmFAD2) that converts the monounsaturated oleic acid (C18:1) to the polyunsaturated linoleic acid (C18:2), therefore, regulating the content of monounsaturated fats in soybean seeds. RESULTS: We designed two gRNAs to guide Cas9 to simultaneously cleave two sites, spaced 1Kb apart, within the second exons of GmFAD2-1A and GmFAD2-1B. In order to test whether the Cas9 and gRNAs would perform properly in transgenic soybean plants, we first tested the CRISPR construct we developed by transient hairy root transformation using Agrobacterium rhizogenesis strain K599. Once confirmed, we performed stable soybean transformation and characterized ten, randomly selected T0 events. Genotyping of CRISPR/Cas9 T0 transgenic lines detected a variety of mutations including large and small DNA deletions, insertions and inversions in the GmFAD2 genes. We detected CRISPR- edited DNA in all the tested T0 plants and 77.8% of the events transmitted the GmFAD2 mutant alleles to T1 progenies. More importantly, null mutants for both GmFAD2 genes were obtained in 40% of the T0 plants we genotyped. The fatty acid profile analysis of T1 seeds derived from CRISPR-edited plants homozygous for both GmFAD2 genes showed dramatic increases in oleic acid content to over 80%, whereas linoleic acid decreased to 1.3-1.7%. In addition, transgene-free high oleic soybean homozygous genotypes were created as early as the T1 generation. CONCLUSIONS: Overall, our data showed that dual gRNA CRISPR/Cas9 system offers a rapid and highly efficient method to simultaneously edit homeologous soybean genes, which can greatly facilitate breeding and gene discovery in this important crop plant.


Assuntos
Ácidos Graxos Dessaturases/genética , Edição de Genes/métodos , Genes de Plantas , RNA Guia , Soja/genética , Ácido alfa-Linoleico/genética , Agrobacterium/genética , Sistemas CRISPR-Cas , Marcadores Genéticos , Vetores Genéticos , Técnicas de Genotipagem , Padrões de Herança , Plantas Geneticamente Modificadas
14.
Nutrients ; 11(7)2019 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-31340443

RESUMO

Fatty acid desaturase 1 (FADS1) gene controls the fatty acid metabolism pathway in the human body. The lower intake of α-linolenic acid (ALA) than linoleic acid (LA) among vegetarians may disrupt the fatty acid metabolism and limit the conversion of ALA to anti-inflammatory products such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). This cross-sectional study aimed to determine the interaction of rs174547 in FADS1 gene with LA and ALA on metabolic syndrome (MetS) components. A total of 200 Chinese and Indian vegetarians in Kuala Lumpur and Selangor, Malaysia participated in the present study. The data on socio-demographic characteristics, vegetarianism practices, dietary practices, anthropometric measurements, blood pressure (BP), and overnight venous fasting blood samples were collected from the vegetarians. The rs174547 in FADS1 gene was significantly associated with MetS and its components such as waist circumference (WC) and fasting blood glucose (FBG). Multiple logistic regression analyses revealed that vegetarians with TT genotype of rs174547 in FADS1 gene had higher odds for MetS, larger WC, higher BP, and a lower level of HDL-c. Two-way ANOVA analysis showed that LA interacts with rs174547 in FADS1 gene to affect HDL-c (p < 0.05) among vegetarians. The present findings suggest the need to develop dietary guidelines for vegetarians in Malaysia. Prospective studies are also needed to affirm the interaction between LA and rs174547 in FADS1 gene on HDL-c among Malaysian vegetarians.


Assuntos
Dieta Vegetariana , Ácidos Graxos Dessaturases/genética , Ácido Linoleico/administração & dosagem , Síndrome Metabólica/genética , Polimorfismo de Nucleotídeo Único , Vegetarianos , Ácido alfa-Linoleico/administração & dosagem , Adulto , Estudos Transversais , Ácidos Graxos Dessaturases/metabolismo , Feminino , Humanos , Ácido Linoleico/metabolismo , Malásia , Masculino , Síndrome Metabólica/diagnóstico , Síndrome Metabólica/enzimologia , Pessoa de Meia-Idade , Medição de Risco , Fatores de Risco , Ácido alfa-Linoleico/metabolismo
15.
Food Funct ; 10(8): 4966-4974, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31343008

RESUMO

trans-Trismethoxy resveratrol (TMR) is a methyl analog of resveratrol. It is found to exhibit enhanced biological effects compared to resveratrol, such as inhibition of cancer cell growth and pro-apoptotic activities. However, the role of TMR in lipid metabolism is not fully understood. In this study, we used Caenorhabditis elegans, an in vivo nematode model which has been widely applied in disease research, including research on obesity, to investigate the effect of TMR on lipid metabolism. Treatment with TMR (100 and 200 µM) for 4 days significantly reduced triglyceride accumulation (14% and 20% reduction over the control, respectively) of C. elegans, without affecting nematode growth, food intake and reproduction. Treatment with TMR significantly downregulated stearoyl-CoA desaturase genes, fat-6 and fat-7, accompanied by a decrease in the desaturation index of fatty acids, the ratio of oleic acid to stearic acid. These results suggest that TMR inhibits fat accumulation by downregulating stearoyl-CoA desaturase in C. elegans.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/metabolismo , Gorduras/metabolismo , Ácidos Graxos Dessaturases/metabolismo , Resveratrol/farmacologia , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Ácidos Graxos Dessaturases/genética , Resveratrol/química , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismo , Triglicerídeos/metabolismo
16.
Plant Physiol Biochem ; 141: 423-430, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31233983

RESUMO

The effect of salinity on physiological traits, fatty acid composition and desaturase genes expression in fruit mesocarp of olive cultivar Leccino was investigated. Significant reduction of shoot elongation (-12%) during salt treatments (80 mM NaCl) was associated with the translocation of Na in the aerial part. After 75 days of treatment, fruits from each plant were subdivided into four maturation groups (MG0, MG1, MG2, MG3) according to ripening degrees. Na accumulation increased in each MG under salinity, reaching the highest values in MG1 fruits (2654 mg kg-1 DW). Salinity caused an acceleration of the ripening process, increased fruit number and decreased total fatty acids content in MG3. An increase in oleic acid at MG1 (53%) was detected, with consequent increase in the oleic/linoleic (41%) and decrease in the polyunsaturated/monounsaturated ratios (30%). Those variations could be explained by the synergic up-regulation of OeSAD1, together with the down-regulation of OeFAD6 transcript levels.


Assuntos
Ácidos Graxos Dessaturases/genética , Ácidos Graxos/química , Frutas/enzimologia , Olea/enzimologia , Sais/química , Irrigação Agrícola , Expressão Gênica , Ácido Linoleico/química , Ácido Oleico/química , Fenótipo , Fotossíntese , Óleos Vegetais/química , Sódio/química , Regulação para Cima
17.
Int J Mol Sci ; 20(12)2019 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-31242553

RESUMO

Peanuts with high oleic acid content are usually considered to be beneficial for human health and edible oil storage. In breeding practice, peanut lines with high monounsaturated fatty acids are selected using fatty acid desaturase 2 (FAD2), which is responsible for the conversion of oleic acid (C18:1) to linoleic acid (C18:2). Here, comparative transcriptomics were used to analyze the global gene expression profile of high- and normal-oleic peanut cultivars at six time points during seed development. First, the mutant type of FAD2 was determined in the high-oleic peanut (H176). The result suggested that early translation termination occurred simultaneously in the coding sequence of FAD2-A and FAD2-B, and the cultivar H176 is capable of utilizing a potential germplasm resource for future high-oleic peanut breeding. Furthermore, transcriptomic analysis identified 74 differentially expressed genes (DEGs) involved in lipid metabolism in high-oleic peanut seed, of which five DEGs encoded the fatty acid desaturase. Aradu.XM2MR belonged to the homologous gene of stearoyl-ACP (acyl carrier protein) desaturase 2 (SAD2) that converted the C18:0 into C18:1. Further subcellular localization studies indicated that FAD2 was located at the endoplasmic reticulum (ER), and Aradu.XM2MR was targeted to the plastid in Arabidopsis protoplast cells. To examine the dynamic mechanism of this finding, we focused on the peroxidase (POD)-mediated fatty acid (FA) degradation pathway. The fad2 mutant significantly increased the POD activity and H2O2 concentration at the early stage of seed development, implying that redox signaling likely acted as a messenger to connect the signaling transduction between the high-oleic content and Aradu.XM2MR transcription level. Taken together, transcriptome analysis revealed the feedback mechanism of SAD2 (Aradu.XM2MR) associated with FAD2 mutation during the seed developmental stage, which could provide a potential peanut breeding strategy based on identified candidate genes to improve the content of oleic acid.


Assuntos
Arachis/genética , Arachis/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Oxigenases de Função Mista/genética , Ácido Oleico/metabolismo , Transcriptoma , Sequência de Aminoácidos , Arachis/classificação , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Genoma de Planta , Metabolismo dos Lipídeos , Oxigenases de Função Mista/metabolismo , Modelos Biológicos , Filogenia , Sementes/genética , Sementes/metabolismo
18.
Genes (Basel) ; 10(6)2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31210171

RESUMO

Plant fatty acid desaturases (FADs) catalyze the desaturation of fatty acids in various forms and play important roles in regulating fatty acid composition and maintaining membrane fluidity under temperature stress. A total of 30 FADs were identified from a maize genome, including 13 soluble and 17 membrane-bound FADs, which were further classified into two and five sub-groups, respectively, via phylogenetic analysis. Although there is no evolutionary relationship between the soluble and the membrane-bound FADs, they all harbor a highly conserved FA_desaturase domain, and the types and the distributions of conserved motifs are similar within each sub-group. The transcriptome analysis revealed that genes encoding FADs exhibited different expression profiles under cold and heat stresses. The expression of ZmFAD2.1&2.2, ZmFAD7, and ZmSLD1&3 were significantly up-regulated under cold stress; moreover, the expression of ZmFAD2.1&2.3 and ZmSLD1&3 were obviously down-regulated under heat stress. The co-expression analysis demonstrated close correlation among the transcription factors and the significant responsive FAD genes under cold or heat stress. This study helps to understand the roles of plant FADs in temperature stress responses.


Assuntos
Resposta ao Choque Frio/genética , Ácidos Graxos Dessaturases/genética , Resposta ao Choque Térmico/genética , Zea mays/genética , Cromossomos de Plantas , Temperatura Baixa , Sequência Conservada/genética , Ácidos Graxos Dessaturases/classificação , Duplicação Gênica , Regulação da Expressão Gênica de Plantas , Temperatura Alta , Filogenia , Transcriptoma/genética , Zea mays/crescimento & desenvolvimento
19.
Science ; 364(6443): 886-889, 2019 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-31147520

RESUMO

Colonization of new ecological niches has triggered large adaptive radiations. Although some lineages have made use of such opportunities, not all do so. The factors causing this variation among lineages are largely unknown. Here, we show that deficiency in docosahexaenoic acid (DHA), an essential ω-3 fatty acid, can constrain freshwater colonization by marine fishes. Our genomic analyses revealed multiple independent duplications of the fatty acid desaturase gene Fads2 in stickleback lineages that subsequently colonized and radiated in freshwater habitats, but not in close relatives that failed to colonize. Transgenic manipulation of Fads2 in marine stickleback increased their ability to synthesize DHA and survive on DHA-deficient diets. Multiple freshwater ray-finned fishes also show a convergent increase in Fads2 copies, indicating its key role in freshwater colonization.


Assuntos
Adaptação Biológica/genética , Ácidos Docosa-Hexaenoicos/metabolismo , Ácidos Graxos Dessaturases/genética , Água Doce , Duplicação Gênica , Smegmamorpha/fisiologia , Animais , Dosagem de Genes , Água do Mar , Smegmamorpha/genética , Smegmamorpha/metabolismo
20.
BMC Biotechnol ; 19(1): 24, 2019 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-31035982

RESUMO

BACKGROUND: Increasing the content of oleic acid in peanut seeds is one of the major goals in peanut breeding due to consumer and industry benefits, such as anti-oxidation and long shelf-life. Homeologous ahFAD2A and ahFAD2B genes encode fatty acid desaturases, which are the key enzymes for converting oleic acid to linoleic acid that oxidizes readily. To date, all high oleic acid peanut varieties result from natural mutations occurred in both genes. A method to induce mutations in the genes of other elite cultivars could speed introgression of this valuable trait. The gene-editing approach utilizing CRISPR/Cas9 technology was employed to induce de novo mutations in the ahFAD2 genes using peanut protoplasts and hairy root cultures as models. RESULTS: The hot spot of natural mutation in these genes was selected as the target region. Appropriate sgRNAs were designed and cloned into a CRISPR/Cas9 expression plasmid. As a result of CRISPR/Cas9 activity, three mutations were identified - G448A in ahFAD2A, and 441_442insA and G451T in ahFAD2B. The G448A and 441_442insA mutations are the same as those seen in existing high oleate varieties and the G451T is new mutation. Because natural mutations appear more often in the ahFAD2A gene than in the ahFAD2B gene in subspecies A. hypogaea var. hypogaea, the mutations induced in ahFAD2B by gene editing may be useful in developing high oleate lines with many genetic backgrounds after validation of oleic acid content in the transformed lines. The appearance of the G448A mutation in ahFAD2A is a further benefit for high oleic acid oil content. CONCLUSIONS: Overall, these results showed that mutations were, for the first time, induced by CRISPR-based gene editing approach in peanut. This research demonstrated the potential application of gene editing for mutagenesis in peanut and suggested that CRISPR/Cas9 technology may be useful in the peanut breeding programs.


Assuntos
Arachis/genética , Sistemas CRISPR-Cas , Ácidos Graxos Dessaturases/genética , Edição de Genes/métodos , Mutagênese , Proteínas de Plantas/genética , Arachis/enzimologia , Sequência de Bases , Ácidos Graxos Dessaturases/metabolismo , Ácido Linoleico/metabolismo , Ácido Oleico/metabolismo , Melhoramento Vegetal/métodos , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Sementes/enzimologia , Sementes/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA