RESUMO
The majority of plant protein in the world's food supply is derived from soybean (Glycine max). Soybean is a key protein source for global animal feed and is incorporated into plant-based foods for people, including meat alternatives. Soybean protein content is genetically variable and is usually inversely related to seed oil content. ABI3-interacting protein 2 (AIP2) is an E3-RING ubiquitin ligase that targets the seed-specific transcription factor ABI3. Silencing both soybean AIP2 genes (AIP2a and AIP2b) by RNAi enhanced seed protein content by up to seven percentage points, with no significant decrease in seed oil content. The protein content enhancement did not alter the composition of the seed storage proteins. Inactivation of either AIP2a or AIP2b by a CRISPR-Cas9-mediated mutation increased seed protein content, and this effect was greater when both genes were inactivated. Transactivation assays in transfected soybean hypocotyl protoplasts indicated that ABI3 changes the expression of glycinin, conglycinin, 2S albumin, and oleosin genes, indicating that AIP2 depletion increased seed protein content by regulating activity of the ABI3 transcription factor protein. These results provide an example of a gene-editing prototype directed to improve global food security and protein availability in soybean that may also be applicable to other protein-source crops.
Assuntos
Sistemas CRISPR-Cas , Proteínas de Soja , Proteínas de Soja/genética , Sementes/genética , Fatores de Transcrição , Óleos de Plantas , Ubiquitina , LigasesRESUMO
KEY MESSAGE: Proteomic and lipidomics analyses of WT and GmDGAT1-2 transgenic soybeans showed that GmDGAT1-2 over-expression induced lipoxygenase down-regulatation and oleoin up-regulatation, which significantly changed the compositions and total fatty acid. The main goal of soybean breeding is to increase the oil content. Diacylglycerol acyltransferase (DGAT) is a key rate-limiting enzyme in fatty acid metabolism and may regulate oil content. Herein, 10 GmDGAT genes were isolated from soybean and transferred into wild-type (WT) Arabidopsis. The total fatty acid was 1.2 times higher in T3 GmDGAT1-2 transgenic Arabidopsis seeds than in WT. Therefore, GmDGAT1-2 was transferred into WT soybean (JACK), and four T3 transgenic soybean lines were obtained. The results of high-performance gas chromatography and Soxhlet extractor showed that, compared with those of JACK, oleic acid (18:1), and total fatty acid levels in transgenic soybean plants were much higher, but linoleic acid (18:2) was lower than WT. Palmitic acid (16:0), stearic acid (18:0), and linolenic acid (18:3) were not significantly different. For mechanistic studies, 436 differentially expressed proteins (DEPs) and 180 differentially expressed metabolites (DEMs) were identified between WT (JACK) and transgenic soybean pods using proteomic and lipidomics analyses. Four lipoxygenase proteins were down-regulated in linoleic acid metabolism while four oleosin proteins were up-regulated in the final oil formation. The results showed an increase in the total fatty acid and 18:1 composition, and a decrease in the 18:2 composition of fatty acid. Our study brings new insights into soybean genetic transformation and the deep study of molecular mechanism that changes the total fatty acid, 18:1, and 18:2 compositions in GmDGAT1-2 transgenic soybean.
Assuntos
Diacilglicerol O-Aciltransferase/genética , Glycine max/genética , Lipoxigenase/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Plantas/metabolismo , Óleo de Soja/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Diacilglicerol O-Aciltransferase/metabolismo , Regulação da Expressão Gênica de Plantas , Ácido Linoleico/genética , Ácido Linoleico/metabolismo , Lipidômica/métodos , Lipoxigenase/genética , Proteínas de Membrana/genética , Família Multigênica , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Proteômica/métodos , Sementes/genética , Sementes/metabolismo , Óleo de Soja/genética , Proteínas de Soja/genética , Proteínas de Soja/metabolismo , Glycine max/metabolismoRESUMO
The inhibitory activities of buckwheat trypsin inhibitor (rBTI) towards trypsin were compared with soybean trypsin inhibitor (SBTI) in terms of their sensitivities to temperature, pH, salt ions and organic solvents. Both rBTI and SBTI were stable over a broad pH range of 2.0-12.0. rBTI exhibited higher thermal stability than SBTI. The inhibitory activity of rBTI was decreased by Zinc ions (Zn2+), KSCN, vitamin C and urea, while that of SBTI remained unchanged. However, H2O2, Mg2+ and Cu2+ ions had no significant effects on the inhibitory activities of rBTI and SBTI. Acetonitrile enhanced the inhibitory activity of rBTI, but had no effect on SBTI, while dimethylacetamide (DMAC) increased the inhibitory effect of both rBTI and SBTI. On the contrary, the inhibitory activities of rBTI and SBTI were reduced by isopropyl alcohol and methanol. The inhibition constants Ki of rBTI and SBTI were calculated to be 7.41 × 10-9 M and 6.52 × 10-9 M, respectively.
Assuntos
Fagopyrum/química , Glycine max/química , Proteínas de Soja/química , Inibidores da Tripsina/química , Fagopyrum/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas de Soja/genética , Glycine max/genéticaRESUMO
Lunasin, a bioactive peptide with a variety of physiological functions, was overexpressed in soybean to generate a transgenic soybean. Polymerase chain reaction (PCR) analysis suggested that lunasin was successfully inserted into the soybean genome, and three transgenic lines, L12, L43, and L45, were selected for further study. Lunasin expression was characterized in the lines by Western blot and ultra-performance liquid chromatography with tandem mass spectrometry. Enzyme-linked immunosorbent assay showed that lunasin content in L12, L43, and L45 lines was 1.47 mg g-1, 1.32 mg g-1 and 1.98 mg g-1, respectively; these values were significantly higher than that in wild-type soybean (0.94 mg g-1). Lunasin enrichments from transgenic soybean (LET) exhibited stronger DPPH, ABTS+, and oxygen radical scavenging activity than lunasin enrichments from wild-type soybean (LEW). Further, LET presented superior anti-inflammatory activity on lipopolysaccharide-induced macrophage cells compared to LEW, and it significantly suppressed the release of nitric oxide (NO) and pro-inflammatory cytokines including interleukin-1 and -6. Moreover, LET showed higher anti-proliferation activity on MDA-MB-231 cells than LEW. Immunofluorescence staining showed that LET could internalize into NIH-3T3 cells, and localize in the nucleus. In conclusion, it is feasible and efficient to produce lunasin through a transgenic soybean expression system. Lunasin overexpressing soybean could be consumed as a functional food in the diets of patients with cancer and obesity in the future.
Assuntos
Anti-Inflamatórios , Antineoplásicos Fitogênicos , Glycine max/genética , Proteínas de Soja/genética , Proteínas de Soja/farmacologia , Animais , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacologia , Antineoplásicos Fitogênicos/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Ensaio de Imunoadsorção Enzimática , Alimento Funcional , Humanos , Camundongos , Células NIH 3T3 , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Plantas Geneticamente Modificadas , Células RAW 264.7 , Proteínas de Soja/análise , Proteínas de Soja/metabolismo , Glycine max/química , Glycine max/metabolismo , Espectrometria de Massas em TandemRESUMO
Soybean is an excellent source of vegetable protein and edible oil. Understanding the genetic basis of protein and oil content will improve the breeding programs for soybean. Linkage analysis and genome-wide association study (GWAS) tools were combined to detect quantitative trait loci (QTL) that are associated with protein and oil content in soybean. Three hundred and eight recombinant inbred lines (RILs) containing 3454 single nucleotide polymorphism (SNP) markers and 200 soybean accessions, including 94,462 SNPs and indels, were applied to identify QTL intervals and significant SNP loci. Intervals on chromosomes 1, 15, and 20 were correlated with both traits, and QTL qPro15-1, qPro20-1, and qOil5-1 reproducibly correlated with large phenotypic variations. SNP loci on chromosome 20 that overlapped with qPro20-1 were reproducibly connected to both traits by GWAS (p < 10-4). Twenty-five candidate genes with putative roles in protein and/or oil metabolisms within two regions (qPro15-1, qPro20-1) were identified, and eight of these genes showed differential expressions in parent lines during late reproductive growth stages, consistent with a role in controlling protein and oil content. The new well-defined QTL should significantly improve molecular breeding programs, and the identified candidate genes may help elucidate the mechanisms of protein and oil biosynthesis.
Assuntos
Ligação Genética , Estudo de Associação Genômica Ampla , Glycine max/genética , Óleos de Plantas/metabolismo , Locos de Características Quantitativas , Sementes/genética , Proteínas de Soja/genética , Mapeamento Cromossômico , Cromossomos de Plantas , Genoma de Planta , Desequilíbrio de Ligação , Fenótipo , Polimorfismo de Nucleotídeo Único , Sementes/metabolismo , Proteínas de Soja/metabolismo , Glycine max/metabolismoRESUMO
Lipoxygenases are widespread enzymes found in virtually all eukaryotes, including fungi, and, more recently, in prokaryotes. These enzymes act on long-chain polyunsaturated fatty acid substrates (C18 to C20), raising questions regarding how the substrate threads its way from solvent to the active site. Herein, we report a comparison of the temperature dependence of isotope effects on first- and second-order rate constants among single-site variants of the prototypic plant enzyme soybean lipoxygenase-1 substituted at amino acid residues inferred to impact substrate binding. We created 10 protein variants including four amino acid positions, Val-750, Ile-552, Ile-839, and Trp-500, located within a previously proposed substrate portal. The conversion of these bulky hydrophobic side chains to smaller side chains is concluded to increase the mobility of flanking helices, giving rise to increased off rates for substrate dissociation from the enzyme. In this manner, we identified a specific "binding network" that can regulate movement of the substrate from the solvent to the active site. Taken together with our previous findings on C-H and O2 activation of soybean lipoxygenase-1, these results support the emergence of multiple complementary networks within a single protein scaffold that modulate different steps along the enzymatic reaction coordinate.
Assuntos
Glycine max/enzimologia , Lipoxigenase/química , Proteínas de Soja/química , Substituição de Aminoácidos , Domínio Catalítico , Lipoxigenase/genética , Mutação de Sentido Incorreto , Proteínas de Soja/genética , Glycine max/genéticaRESUMO
Soybean is a globally important legume crop that provides a primary source of high-quality vegetable protein and oil. Seed protein and oil content are two valuable quality traits controlled by multiple genes in soybean. In this study, the restricted two-stage multi-locus genome-wide association analysis (RTM-GWAS) procedure was performed to dissect the genetic architecture of seed protein and oil content in a diverse panel of 279 soybean accessions from the Yangtze and Huaihe River Valleys in China. We identified 26 quantitative trait loci (QTLs) for seed protein content and 23 for seed oil content, including five associated with both traits. Among these, 39 QTLs corresponded to previously reported QTLs, whereas 10 loci were novel. As reported previously, the QTL on chromosome 20 was associated with both seed protein and oil content. This QTL exhibited opposing effects on these traits and contributed the most to phenotype variation. From the detected QTLs, 55 and 51 candidate genes were identified for seed protein and oil content, respectively. Among these genes, eight may be promising candidate genes for improving soybean nutritional quality. These results will facilitate marker-assisted selective breeding for soybean protein and oil content traits.
Assuntos
Glycine max/genética , Óleos de Plantas/análise , Proteínas de Armazenamento de Sementes/genética , Sementes/genética , Proteínas de Soja/genética , Genoma de Planta , Estudo de Associação Genômica Ampla , Polimorfismo de Nucleotídeo Único , Locos de Características QuantitativasRESUMO
Soybean is globally cultivated primarily for its protein and oil. The protein and oil contents of the seeds are quantitatively inherited traits determined by the interaction of numerous genes. In order to gain a better understanding of the molecular foundation of soybean protein and oil content for the marker-assisted selection (MAS) of high quality traits, a population of 185 soybean germplasms was evaluated to identify the quantitative trait loci (QTLs) associated with the seed protein and oil contents. Using specific length amplified fragment sequencing (SLAF-seq) technology, a total of 12,072 single nucleotide polymorphisms (SNPs) with a minor allele frequency (MAF)â¯≥â¯0.05 were detected across the 20 chromosomes (Chr), with a marker density of 78.7 kbp. A total of 31 SNPs located on 12 of the 20 soybean chromosomes were correlated with seed protein and oil content. Of the 31 SNPs that were associated with the two target traits, 31 beneficial alleles were identified. Two SNP markers, namely rs15774585 and rs15783346 on Chr 07, were determined to be related to seed oil content both in 2015 and 2016. Three SNP markers, rs53140888 on Chr 01, rs19485676 on Chr 13, and rs24787338 on Chr 20 were correlated with seed protein content both in 2015 and 2016. These beneficial alleles may potentially contribute towards the MAS of favorable soybean protein and oil characteristics.
Assuntos
Mapeamento Cromossômico , Genoma de Planta , Estudo de Associação Genômica Ampla , Glycine max/genética , Óleo de Soja/genética , Proteínas de Soja/genética , Biomarcadores , Cromossomos de Plantas/genética , Genótipo , Herança Multifatorial , Estruturas Vegetais/genética , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Sementes/genética , Seleção GenéticaRESUMO
PURPOSE: This study examined the effect of soy proteins with depletion of different subunits of the two major storage proteins, ß-conglycinin and glycinin, on hepatic lipids and proteins involved in lipid metabolism in rats, since the bioactive component of soy responsible for lipid-lowering is unclear. METHODS: Weanling Sprague Dawley rats were fed diets containing either 20% casein protein in the absence (casein) or presence (casein + ISF) of isoflavones or 20% alcohol-washed soy protein isolate (SPI) or 20% soy protein concentrates derived from a conventional (Haro) or 2 soybean lines lacking the α' subunit of ß-conglycinin and the A1-3 (1TF) or A1-5 (1a) subunits of glycinin. After 8 weeks, the rats were necropsied and liver proteins and lipids were extracted and analysed. RESULTS: The results showed that soy protein diets reduced lipid droplet accumulation and content in the liver compared to casein diets. The soy protein diets also decreased the level of hepatic mature SREBP-1 and FAS in males, with significant decreases in diets 1TF and 1a compared to the casein diets. The effect of the soy protein diets on female hepatic mature SREBP-1, FAS, and HMGCR was confounded since casein + ISF decreased these levels compared to casein alone perhaps muting the decrease by soy protein. A reduction in both phosphorylated and total STAT3 in female livers by ISF may account for the gender difference in mechanism in the regulation and protein expression of the lipid modulators. CONCLUSIONS: Overall, soy protein deficient in the α' subunit of ß-conglycinin and A1-5 subunits of glycinin maintain similar hypolipidemic function compared to the conventional soy protein. The exact bioactive component(s) warrant identification.
Assuntos
Antígenos de Plantas/uso terapêutico , Globulinas/uso terapêutico , Hiperlipidemias/prevenção & controle , Metabolismo dos Lipídeos , Fígado/metabolismo , Proteínas de Vegetais Comestíveis/uso terapêutico , Subunidades Proteicas/uso terapêutico , Proteínas de Armazenamento de Sementes/uso terapêutico , Proteínas de Soja/uso terapêutico , Animais , Antígenos de Plantas/química , Antígenos de Plantas/genética , Antígenos de Plantas/metabolismo , Caseínas/efeitos adversos , Dieta Hiperlipídica/efeitos adversos , Feminino , Alimentos Geneticamente Modificados , Globulinas/química , Globulinas/genética , Globulinas/metabolismo , Hiperlipidemias/etiologia , Hiperlipidemias/metabolismo , Hiperlipidemias/patologia , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/patologia , Fígado/enzimologia , Fígado/patologia , Masculino , Fosforilação , Proteínas de Vegetais Comestíveis/química , Proteínas de Vegetais Comestíveis/genética , Proteínas de Vegetais Comestíveis/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Processamento de Proteína Pós-Traducional , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ratos Sprague-Dawley , Fator de Transcrição STAT3/metabolismo , Proteínas de Armazenamento de Sementes/química , Proteínas de Armazenamento de Sementes/genética , Proteínas de Armazenamento de Sementes/metabolismo , Caracteres Sexuais , Proteínas de Soja/química , Proteínas de Soja/genética , Proteínas de Soja/metabolismo , Vacúolos/patologia , DesmameRESUMO
Soybean is the world's most important leguminous crop producing high-quality protein and oil. Elevating oil accumulation in soybean seed is always many researchers' goal. WRINKLED1 (WRI1) encodes a transcription factor of the APETALA2/ethylene responsive element-binding protein (AP2/EREBP) family that plays important roles during plant seed oil accumulation. In this study, we isolated and characterized three distinct orthologues of WRI1 in soybean (Glycine max) that display different organ-specific expression patterns, among which GmWRI1a was highly expressed in maturing soybean seed. Electrophoretic mobility shift assays and yeast one-hybrid experiments demonstrated that the GmWRI1a protein was capable of binding to AW-box, a conserved sequence in the proximal upstream regions of many genes involved in various steps of oil biosynthesis. Transgenic soybean seeds overexpressing GmWRI1a under the control of the seed-specific napin promoter showed the increased total oil and fatty acid content and the changed fatty acid composition. Furthermore, basing on the activated expressions in transgenic soybean seeds and existence of AW-box element in the promoter regions, direct downstream genes of GmWRI1a were identified, and their products were responsible for fatty acid production, elongation, desaturation and export from plastid. We conclude that GmWRI1a transcription factor can positively regulate oil accumulation in soybean seed by a complex gene expression network related to fatty acid biosynthesis.
Assuntos
Glycine max/metabolismo , Óleos de Plantas/metabolismo , Proteínas de Plantas/metabolismo , Sementes/metabolismo , Proteínas de Soja/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ácidos Graxos/biossíntese , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Ligação Proteica , Sementes/genética , Homologia de Sequência de Aminoácidos , Proteínas de Soja/classificação , Proteínas de Soja/genética , Glycine max/genética , Fatores de Transcrição/classificação , Fatores de Transcrição/genéticaRESUMO
As one of the major oil crops, soybean might be seriously affected by phosphorus deficiency on both yield and quality. Understanding the molecular basis of phosphorus uptake and utilization in soybean may help to develop phosphorus (P) efficient cultivars. On this purpose, we conducted a comparative proteomic analysis on a high P acquisition soybean cultivar BX10 under low and high P conditions. A total of 61 unique proteins were identified as putative P deficiency responsive proteins. These proteins were involved in carbohydrate metabolism, protein biosynthesis/processing, energy metabolism, cellular processes, environmental defense/interaction, nucleotide metabolism, signal transduction, secondary metabolism and other metabolism related processes. Several proteins involved in energy metabolism, cellular processes, and protein biosynthesis and processing were found to be up-regulated in both shoots and roots, whereas, proteins involved in carbohydrate metabolism appeared to be down-regulated. These proteins are potential candidates for improving P acquisition. These findings provide a useful starting point for further research that will provide a more comprehensive understanding of molecular mechanisms through which soybeans adapt to P deficiency condition.
Assuntos
Fósforo/metabolismo , Raízes de Plantas/genética , Proteômica , Proteínas de Soja/biossíntese , Metabolismo Energético/genética , Regulação da Expressão Gênica de Plantas , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Proteínas de Soja/genética , Glycine maxRESUMO
The phytotoxic effect of the allelochemical cyanamide has been well-documented yet the underlying mechanism for this phenomenon has not been fully characterized. Cognizant of the putative inhibitory effect of cyanamide on aldehyde dehydrogenases (ALDHs), we hereby show that the capacity of mitochondrial preparations from cyanamide-treated soybean seedlings to oxidize acetaldehyde and succinic-semialdehyde was dose-dependently reduced to at most 55% and 70%, respectively. Cyanamide-treated plants exhibited oxidative stress (i.e. increased lipid peroxidation and H2O2 accumulation) that was exacerbated upon exposure to UV-A--symptoms reminiscent of ALDH and succinic-semialdehyde dehydrogenase (SSADH) knock-out Arabidopsis mutants. We suggest that the inhibition of mitochondrial ALDH and SSADH may be a contributory mechanism to the burst in oxidative stress mediated by cyanamide.
Assuntos
Aldeído Desidrogenase/antagonistas & inibidores , Cianamida/toxicidade , Inibidores Enzimáticos/farmacologia , Glycine max/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Proteínas de Soja/antagonistas & inibidores , Aldeído Desidrogenase/genética , Aldeído Desidrogenase/metabolismo , Peróxido de Hidrogênio/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Feromônios/toxicidade , Plântula/efeitos dos fármacos , Plântula/enzimologia , Plântula/genética , Plântula/metabolismo , Proteínas de Soja/genética , Proteínas de Soja/metabolismo , Glycine max/enzimologia , Glycine max/genética , Glycine max/metabolismoRESUMO
Molybdenum cofactor (Moco) is required for the activities of Moco-dependant enzymes. Cofactor for nitrate reductase and xanthine dehydrogenase (Cnx1) is known to be involved in the biosynthesis of Moco in plants. In this work, a soybean (Glycine max L.) Cnx1 gene (GmCnx1) was transferred into soybean using Agrobacterium tumefaciens-mediated transformation method. Twenty seven positive transgenic soybean plants were identified by coating leaves with phosphinothricin, bar protein quick dip stick and PCR analysis. Moreover, Southern blot analysis was carried out to confirm the insertion of GmCnx1 gene. Furthermore, expression of GmCnx1 gene in leaf and root of all transgenic lines increased 1.04-2.12 and 1.55-3.89 folds, respectively, as compared to wild type with GmCnx1 gene and in line 10 , 22 showing the highest expression. The activities of Moco-related enzymes viz nitrate reductase (NR) and aldehydeoxidase (AO) of T1 generation plants revealed that the best line among the GmCnx1 transgenic plants accumulated 4.25 µg g(-1) h(-1) and 30 pmol L(-1), respectively (approximately 2.6-fold and 3.9-fold higher than non-transgenic control plants).In addition, overexpression ofGmCnx1boosted the resistance to various strains of soybean mosaic virus (SMV). DAS-ELISA analysis further revealed that infection rate of GmCnx1 transgenic plants were generally lower than those of non-transgenic plants among two different virus strains tested. Taken together, this study showed that overexpression of a GmCnx1 gene enhanced NR and AO activities and SMV resistance, suggesting its important role in soybean genetic improvement.
Assuntos
Aldeído Oxidase/metabolismo , Glycine max/metabolismo , Vírus do Mosaico/fisiologia , Nitrato Redutase/metabolismo , Doenças das Plantas/genética , Proteínas de Plantas/fisiologia , Proteínas de Soja/fisiologia , Xantina Desidrogenase/fisiologia , Agrobacterium tumefaciens , Coenzimas/biossíntese , Sequência Conservada , DNA Complementar/genética , DNA de Plantas/genética , Resistência à Doença , Vetores Genéticos , Metaloproteínas/biossíntese , Dados de Sequência Molecular , Cofatores de Molibdênio , Filogenia , Doenças das Plantas/virologia , Folhas de Planta/metabolismo , Folhas de Planta/virologia , Proteínas de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Estrutura Terciária de Proteína , Pteridinas , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas de Soja/genética , Glycine max/genética , Glycine max/virologia , Regulação para Cima , Xantina Desidrogenase/genéticaRESUMO
KEY MESSAGE: Both in vitro and in vivo hairy root transformation systems could not replace whole plant transformation for promoter analysis of root-specific and low-P induced genes in soybean. An efficient genetic transformation system is crucial for promoter analysis in plants. Agrobacterium-mediated transformation is the most popular method to produce transgenic hairy roots or plants. In the present study, first, we compared the two different Agrobacterium rhizogenes-mediated hairy root transformation methods using either constitutive CaMV35S or the promoters of root-preferential genes, GmEXPB2 and GmPAP21, in soybean, and found the efficiency of in vitro hairy root transformation was significantly higher than that of in vivo transformation. We compared Agrobacterium rhizogenes-mediated hairy root and Agrobacterium tumefaciens-mediated whole plant transformation systems. The results showed that low-phosphorous (P) inducible GmEXPB2 and GmPAP21 promoters could not induce the increased expression of the GUS reporter gene under low P stress in both in vivo and in vitro transgenic hairy roots. Conversely, GUS activity of GmPAP21 promoter was significantly higher at low P than high P in whole plant transformation. Therefore, both in vitro and in vivo hairy root transformation systems could not replace whole plant transformation for promoter analysis of root-specific and low-P induced genes in soybean.
Assuntos
Agrobacterium/genética , Glycine max/genética , Raízes de Plantas/genética , Regiões Promotoras Genéticas/genética , Transformação Genética , Agrobacterium tumefaciens/genética , Caulimovirus/genética , Flores/genética , Flores/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucuronidase/genética , Glucuronidase/metabolismo , Histocitoquímica , Fósforo/metabolismo , Fósforo/farmacologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Caules de Planta/genética , Caules de Planta/metabolismo , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas de Soja/genética , Glycine max/metabolismoRESUMO
Basic 7S globulin (Bg7S), which accumulates in mature soybean (Glycine max) seeds, is an extracellular matrix protein. A large amount of Bg7S is synthesized de novo and is eluted from soybean seeds when immersed in 50-60°C water (hot water treatment, HWT). However, the Bg7S elution mechanism remains unclear. Under HWT, the seeds probably undergo heat stress and flooding stress. To obtain fundamental knowledge related to how Bg7S is eluted from hot-water-treated seeds, this study compared Bg7S elution among soybean cultivars having different flooding tolerance during pre-germination. The amounts of Bg7S eluted from seeds varied significantly among cultivars. Elution was suppressed by seed coats regarded as preventing the leakage of seed contents by rapid water imbibition. Furthermore, Bg7S expression levels differed among cultivars, although the difference did not result from any variation in Bg7S promoter sequences. However, the expression levels of Bg7S under HWT were not associated with the flooding tolerance level. Immunoelectron microscopy revealed that the Bg7S accumulated in the intercellular space of hot-water-treated seeds. Plasma membrane shrinkage was observed. The main proteins eluted from seeds under HWT were located in the extracellular space. This study clarified the mechanism of Bg7S elution from seeds under HWT.
Assuntos
Antígenos de Plantas/biossíntese , Globulinas/biossíntese , Glycine max/metabolismo , Proteínas de Armazenamento de Sementes/biossíntese , Sementes/metabolismo , Proteínas de Soja/biossíntese , Sequência de Aminoácidos , Antígenos de Plantas/genética , Sequência de Bases , Globulinas/genética , Globulinas/metabolismo , Resposta ao Choque Térmico , Regiões Promotoras Genéticas , Transporte Proteico , Proteínas de Armazenamento de Sementes/genética , Proteínas de Armazenamento de Sementes/metabolismo , Sementes/ultraestrutura , Análise de Sequência de Proteína , Proteínas de Soja/genética , Proteínas de Soja/metabolismo , Glycine max/ultraestruturaRESUMO
In order to detect presence and quantity of Roundup Ready (RR) soybean in crude oil extracted from soybean seed with a different percentage of GMO seed two extraction methods were used, CTAB and DNeasy Plant Mini Kit. The amplifications of lectin gene, used to check the presence of soybean DNA, were not achieved in all CTAB extracts of DNA, while commercial kit gave satisfactory results. Comparing actual and estimated GMO content between two extraction methods, root mean square deviation for kit is 0.208 and for CTAB is 2.127, clearly demonstrated superiority of kit over CTAB extraction. The results of quantification evidently showed that if the oil samples originate from soybean seed with varying percentage of RR, it is possible to monitor the GMO content at the first stage of processing crude oil.
Assuntos
Produtos Agrícolas/química , Contaminação de Alimentos , Glycine max/química , Plantas Geneticamente Modificadas/química , Sementes/química , Óleo de Soja/química , Produtos Agrícolas/efeitos dos fármacos , Produtos Agrícolas/genética , Produtos Agrícolas/metabolismo , DNA de Plantas/isolamento & purificação , DNA de Plantas/metabolismo , Resistência a Medicamentos , Manipulação de Alimentos , Inspeção de Alimentos/métodos , Rotulagem de Alimentos/legislação & jurisprudência , Glicina/análogos & derivados , Glicina/farmacologia , Herbicidas/farmacologia , Lectinas de Plantas/genética , Lectinas de Plantas/metabolismo , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Sementes/efeitos dos fármacos , Sementes/genética , Sementes/metabolismo , Sérvia , Proteínas de Soja/genética , Proteínas de Soja/metabolismo , Glycine max/efeitos dos fármacos , Glycine max/genética , Glycine max/metabolismo , GlifosatoRESUMO
Oleosin is the most abundant protein in the oil bodies of plant seeds, playing an important role in regulating oil body formation and lipid accumulation. To investigate whether lipid accumulation in transgenic rice seeds depends on the expression level of oleosin, we introduced two soybean oleosin genes encoding 24 kDa proteins into rice under the control of an embryo-specific rice promoter REG-2. Overexpression of soybean oleosin in transgenic rice leads to an increase of seed lipid content up to 36.93 and 46.06 % higher than that of the non-transgenic control, respectively, while the overall fatty acid profiles of triacylglycerols remained unchanged. The overexpression of soybean oleosin in transgenic rice seeds resulted in more numerous and smaller oil bodies compared with wild type, suggesting that an inverse relationship exists between oil body size and the total oleosin level. The increase in lipid content is accompanied by a reduction in the accumulation of total seed protein. Our results suggest that it is possible to increase rice seed oil content for food use and for use as a low-cost feedstock for biodiesel by overexpressing oleosin in rice seeds.
Assuntos
Regulação da Expressão Gênica de Plantas , Glycine max/genética , Oryza/química , Oryza/genética , Proteínas de Soja/genética , DNA de Plantas/genética , Genes de Plantas , Morfogênese/genética , Óleos de Plantas/análise , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/genética , Sementes/química , Sementes/genética , Proteínas de Soja/metabolismoRESUMO
UNLABELLED: With the widespread use of Roundup Ready soy (event 40-3-2) (RRS), the comprehensive detection of genetically modified component in foodstuffs is of significant interest, but few protein-based approaches have been found useful in processed foods. In this report, the combination of quantitative PCR (qPCR) and western blot was used to detect cp4-epsps gene and its protein product in different RRS plant tissues and commercial soy-containing foodstuffs. The foods included those of plant origin produced by different processing procedures and also some products containing both meat and plant protein concentrates. The validity of the 2 methods was confirmed first. We also showed that the CP4-EPSPS protein existed in different RRS plant tissues. In certain cases, the results from the western blot and the qPCR were not consistent. To be specific, at least 2 degraded fragments of CP4-EPSPS protein (35.5 and 24.6 kDa) were observed. For dried bean curd crust and deep-fried bean curd, a degraded protein fragment with the size of 24.6 kDa appeared, while cp4-epsps gene could not be traced by qPCR. In contrast, we found a signal of cp4-epsps DNA in 3 foodstuffs, including soy-containing ham cutlet product, meat ball, and sausage by qPCR, while CP4-EPSPS protein could not be detected by western blot in such samples. Our study therefore concluded that the combination of DNA- and protein-based methods would compensate each other, thus resulting in a more comprehensive detection from nucleic acid and protein levels. PRACTICAL APPLICATION: The combination of quantitative PCR (qPCR) and western blot was used to detect cp4-epsps gene and its protein product in different Roundup Ready soy (event 40-3-2) plant tissues and commercial soy-containing foodstuffs. The foods included those of plant origin produced by different processing procedures and also some products containing a combination of both meat and plant protein concentrates. This study indicated that the combination of DNA- and protein-based methods would supplement each other for genetically modified detection from nucleic acid and protein levels. Accordingly, qPCR and western blot could be used in CP4-EPSPS detection in a wide variety of soy-related foodstuffs.
Assuntos
3-Fosfoshikimato 1-Carboxiviniltransferase/metabolismo , Inspeção de Alimentos/métodos , Alimentos Geneticamente Modificados , Glycine max/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Alimentos de Soja/análise , Proteínas de Soja/metabolismo , 3-Fosfoshikimato 1-Carboxiviniltransferase/antagonistas & inibidores , 3-Fosfoshikimato 1-Carboxiviniltransferase/química , 3-Fosfoshikimato 1-Carboxiviniltransferase/genética , Agrobacterium/genética , Agrobacterium/metabolismo , Western Blotting , China , DNA de Plantas/isolamento & purificação , DNA de Plantas/metabolismo , Resistência a Medicamentos/genética , Inibidores Enzimáticos/farmacologia , Genes Bacterianos , Glicina/análogos & derivados , Glicina/farmacologia , Herbicidas/farmacologia , Produtos da Carne/análise , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Plantas Geneticamente Modificadas/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas de Soja/antagonistas & inibidores , Proteínas de Soja/química , Proteínas de Soja/genética , Glycine max/efeitos dos fármacos , Glycine max/genética , GlifosatoRESUMO
Symbiotic nitrogen fixation is a high-phosphorus demand process. Proteomic analysis was performed to identify the differentially expressed proteins in soybean nodules under phosphate starvation, and qRT-PCR was subsequently conducted to examine the expression patterns of the genes encoding the identified proteins. There were 44 phosphate-starvation responsive proteins identified from soybean nodules. Among them, 14 plant and 3 rhizobial proteins were up-regulated, whereas 13 plant and 14 rhizobial proteins were down-regulated by phosphate starvation. The qRT-PCR assays verified that gene expression correlated with 11 of the 14 up-regulated proteins from plants, but only 4 of 13 down-regulated proteins were correlated to the expression of the corresponding genes, suggesting that most up-regulated proteins may be controlled at the transcriptional level, whereas down-regulated proteins were controlled at the post-transcriptional level. Furthermore, a group of genes exhibited differential responses to phosphate starvation in nodules versus roots, suggesting that different adaptive responses might occur between roots and nodules. To our best knowledge, this is the first study to reveal differential protein profiles of nodules responding to phosphate starvation through proteomic analysis, which could result in a relatively comprehensive understanding of molecular mechanisms through which soybean nodules adapt to phosphorus stress.
Assuntos
Glycine max/genética , Fósforo/deficiência , Nódulos Radiculares de Plantas/metabolismo , Proteínas de Soja/análise , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Fixação de Nitrogênio/genética , Proteômica/métodos , Nódulos Radiculares de Plantas/genética , Proteínas de Soja/genética , Proteínas de Soja/metabolismo , Glycine max/crescimento & desenvolvimento , Glycine max/metabolismo , Estresse Fisiológico/genéticaRESUMO
Plants have attracted increasing attention as an expression platform for the production of pharmaceutical proteins due to its unlimited scalability and low cost potential. However, compared to other expression systems, plants accumulate relatively low levels of foreign proteins, thus necessitating the development of efficient systems for purification of foreign proteins from plant tissues. We have developed a novel strategy for purification of recombinant proteins expressed in plants, based on genetic fusion to soybean agglutinin (SBA), a homotetrameric lectin that binds to N-acetyl-D-galactosamine. Previously it was shown that high purity SBA could be recovered from soybean with an efficiency of greater than 90% following one-step purification using N-acetyl-D-galactosamine-agar columns. We constructed an SBA fusion protein containing the reporter green fluorescent protein (GFP) and transiently expressed it in N. benthamiana plants. We achieved over 2.5% of TSP accumulation in leaves of N. benthamiana. Confocal microscopic analysis demonstrated in vivo activity of the fused GFP partner. Importantly, high purity rSBA-GFP was recovered from crude leaf extract with ~90% yield via one-step purification on N-acetyl-D-galactosamine-agar columns, and the purified fusion protein was able to induce the agglutination of rabbit red blood cells. Combined with this, tetrameric assembly of the fusion protein was demonstrated via western blotting. In addition, rSBA-GFP retained its GFP signal on agglutinated red blood cells, demonstrating the feasibility of using rSBA-GFP for discrimination of cells that bear the ligand glycan on their surface. This work validates SBA as an effective affinity tag for simple and rapid purification of genetically fused proteins.