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1.
BMC Plant Biol ; 19(1): 292, 2019 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-31272394

RESUMO

BACKGROUND: The oilseed Camelina sativa is grown for a range of applications, including for biofuel, biolubricants, and as a source of omega-3 fatty acids for the aquaculture feed industry. The seed meal co-product is used as a source of protein for animal feed; however, the low value of the meal hinders profitability and more widespread application of camelina. The nutritional quality of the seed meal is largely determined by the abundance of specific seed storage proteins and their amino acid composition. Manipulation of seed storage proteins has been shown to be an effective means for either adjustment of nutritional content of seeds or for enhancing accumulation of high-value recombinant proteins in seeds. RESULTS: CRISPR/Cas9 gene editing technology was used to generate deletions in the first exon of the three homoeologous genes encoding the seed storage protein CRUCIFERIN C (CsCRUC), creating an identical premature stop-codon in each and resulting in a CsCRUC knockout line. The mutant alleles were detected by applying a droplet digital PCR drop-off assay. The quantitative nature of this technique is particularly valuable when applied to polyploid species because it can accurately determine the number of mutated alleles in a gene family. Loss of CRUC protein did not alter total seed protein content; however, the abundance of other cruciferin isoforms and other seed storage proteins was altered. Consequently, seed amino acid content was significantly changed with an increase in the proportion of alanine, cysteine and proline, and decrease of isoleucine, tyrosine and valine. CsCRUC knockout seeds did not have changed total oil content, but the fatty acid profile was significantly altered with increased relative abundance of all saturated fatty acids. CONCLUSIONS: This study demonstrates the plasticity of the camelina seed proteome and establishes a CRUC-devoid line, providing a framework for modifying camelina seed protein composition. The results also illustrate a possible link between the composition of the seed proteome and fatty acid profile.


Assuntos
Brassicaceae/genética , Globulinas/genética , Proteínas de Plantas/genética , Proteínas de Armazenamento de Sementes/genética , Sequência de Bases , Brassicaceae/metabolismo , Sistemas CRISPR-Cas , Edição de Genes , Globulinas/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Armazenamento de Sementes/metabolismo , Sementes/genética
2.
Gene ; 647: 31-38, 2018 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-29320758

RESUMO

Mutations in the exonuclease domain of polymerase epsilon (POLE), an enzyme of DNA synthesis, are involved in a newly described syndrome of colorectal polyposis and cancer, and have been associated with a high mutation burden with or without microsatellite instability (MSI) phenotype. The exonuclease domain of POLE executes a proofreading function that decreases the mutation rate during DNA replication by an estimated of one to two orders. The high mutation burden resulting from its loss of function could create a load of neo-antigens that would put the neoplastic cells in severe disadvantage of an immune attack if properly presented to the immune system. This paper investigates the mutagenic effect of different POLE mutations in various cancers, in published genomic studies and the effect that these POLE mutations have in selecting for mutations of the ß2 microglobulin (B2M) gene involved in antigen presentation.


Assuntos
DNA Polimerase II/genética , Globulinas/genética , Mutação/genética , Neoplasias/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Replicação do DNA/genética , Exodesoxirribonucleases/genética , Humanos , Instabilidade de Microssatélites , Mutagênese/genética , Taxa de Mutação , Fenótipo
3.
Eur J Nutr ; 57(3): 1157-1168, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28324208

RESUMO

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 , Desmame
4.
Theor Appl Genet ; 131(3): 659-671, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29224171

RESUMO

KEY MESSAGE: Four soybean storage protein subunit QTLs were mapped using bulked segregant analysis and an F2 population, which were validated with an F5 RIL population. The storage protein globulins ß-conglycinin (7S subunit) and glycinin (11S subunits) can affect the quantity and quality of proteins found in soybean seeds and account for more than 70% of the total soybean protein. Manipulating the storage protein subunits to enhance soymeal nutrition and for desirable tofu manufacturing characteristics are two end-use quality goals in soybean breeding programs. To aid in developing soybean cultivars with desired seed composition, an F2 mapping population (n = 448) and an F5 RIL population (n = 180) were developed by crossing high protein cultivar 'Harovinton' with the breeding line SQ97-0263_3-1a, which lacks the 7S α', 11S A1, 11S A2, 11S A3 and 11S A4 subunits. The storage protein composition of each individual in the F2 and F5 populations were profiled using SDS-PAGE. Based on the presence/absence of the subunits, genomic DNA bulks were formed among the F2 plants to identify genomic regions controlling the 7S α' and 11S protein subunits. By utilizing polymorphic SNPs between the bulks characterized with Illumina SoySNP50K iSelect BeadChips at targeted genomic regions, KASP assays were designed and used to map QTLs causing the loss of the subunits. Soybean storage protein QTLs were identified on Chromosome 3 (11S A1), Chromosome 10 (7S α' and 11S A4), and Chromosome 13 (11S A3), which were also validated in the F5 RIL population. The results of this research could allow for the deployment of marker-assisted selection for desired storage protein subunits by screening breeding populations using the SNPs linked with the subunits of interest.


Assuntos
Antígenos de Plantas/genética , Globulinas/genética , Locos de Características Quantitativas , Proteínas de Armazenamento de Sementes/genética , Proteínas de Soja/genética , Soja/genética , Mapeamento Cromossômico , Cruzamentos Genéticos , Frequência do Gene , Genótipo , Polimorfismo de Nucleotídeo Único , Subunidades Proteicas/genética , Sementes
5.
Electrophoresis ; 38(20): 2622-2630, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28683176

RESUMO

Proteolytic cleavage or partial degradation of proteins is one of the important post-translational modifications for various biological processes, but it is difficult to analyze. Previously, we demonstrated that some subunits of the major rice (Oryza sativa L.) seed storage protein glutelin are partially degraded to produce newly identified polypeptides X1-X5 in mutants in which another major seed storage protein globulin is absent. In this study, the new polypeptides X3 and X4/X5 were immunologically confirmed to be derived from GluA3 and GluA1/GluA2 subunits, respectively. Additionally, the new polypeptides X1 and X2 were at least in part the α polypeptides of the GluB4 subunit partially degraded at the C-terminus. Simulated 2D-PAGE migration patterns of intact and partially degraded α polypeptides based on the calculation of their MWs and pIs enabled us to narrow or predict the possible locations of cleavage sites. The predicted cleavage sites were also verified by the comparison of 2D-PAGE patterns between seed-extracted and E. coli-expressed proteins of the intact and truncated α polypeptides. The results and methodologies demonstrated here would be useful for analyses of partial degradation of proteins and the structure-function relationships of rice seed protein bodies.


Assuntos
Globulinas/química , Glutens/química , Oryza/química , Simulação por Computador , Eletroforese em Gel de Poliacrilamida , Globulinas/genética , Glutens/genética , Mutação , Oryza/genética , Peptídeos/química , Conformação Proteica , Processamento de Proteína Pós-Traducional , Subunidades Proteicas/química , Proteólise , Sementes
6.
G3 (Bethesda) ; 7(7): 2345-2352, 2017 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-28592556

RESUMO

During ongoing proteomic analysis of the soybean (Glycine max (L.) Merr) germplasm collection, PI 603408 was identified as a landrace whose seeds lack accumulation of one of the major seed storage glycinin protein subunits. Whole genomic resequencing was used to identify a two-base deletion affecting glycinin 5 The newly discovered deletion was confirmed to be causative through immunological, genetic, and proteomic analysis, and no significant differences in total seed protein content were found to be due to the glycinin 5 loss-of-function mutation per se In addition to focused studies on this one specific glycinin subunit-encoding gene, a total of 1,858,185 nucleotide variants were identified, of which 39,344 were predicted to affect protein coding regions. In order to semiautomate analysis of a large number of soybean gene variants, a new SIFT 4G (Sorting Intolerant From Tolerated 4 Genomes) database was designed to predict the impact of nonsynonymous single nucleotide soybean gene variants, potentially enabling more rapid analysis of soybean resequencing data in the future.


Assuntos
Sequência de Bases , Genoma de Planta , Globulinas/genética , Polimorfismo Genético , Deleção de Sequência , Proteínas de Soja/genética , Soja/genética , Bases de Dados Genéticas , Estudo de Associação Genômica Ampla
7.
Protein Expr Purif ; 135: 78-82, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28526454

RESUMO

Chymosin is widely used in the dairy industry, and much is produced through recombinant DNA in organisms such as bacteria and tobacco. In this study, we used a new transgenic method to express caprine chymosin in corn seeds with lower cost and better storage capability. The recombinant chymosin protein was successfully expressed at an average level of 0.37 mg/g dry weight, which is 0.27% of the total soluble protein in the corn seed. Prochymosin can be activated to produce a chymosin protein with the ability to induce clotting in milk, similar to the commercial protein. The activity of the purified recombinant chymosin was as high as 178.5 U/mg. These results indicate that we have successfully established a technology for generating corn seed-derived caprine chymosin for potential use in the dairy industry.


Assuntos
Quimosina/biossíntese , Vetores Genéticos/química , Plantas Geneticamente Modificadas , Sementes/genética , Zea mays/genética , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Animais , Quimosina/genética , Quimosina/isolamento & purificação , Quimosina/farmacologia , Clonagem Molecular , Ensaios Enzimáticos , Floculação/efeitos dos fármacos , Tecnologia de Alimentos , Expressão Gênica , Vetores Genéticos/metabolismo , Globulinas/genética , Globulinas/metabolismo , Cabras , Cinética , Leite/química , Leite/efeitos dos fármacos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Sementes/enzimologia , Transformação Genética , Zea mays/enzimologia
8.
New Phytol ; 214(4): 1597-1613, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28322451

RESUMO

Improving nutritional seed quality is an important challenge in grain legume breeding. However, the genes controlling the differential accumulation of globulins, which are major contributors to seed nutritional value in legumes, remain largely unknown. We combined a search for protein quantity loci with genome-wide association studies on the abundance of 7S and 11S globulins in seeds of the model legume species Medicago truncatula. Identified genomic regions and genes carrying polymorphisms linked to globulin variations were then cross-compared with pea (Pisum sativum), leading to the identification of candidate genes for the regulation of globulin abundance in this crop. Key candidates identified include genes involved in transcription, chromatin remodeling, post-translational modifications, transport and targeting of proteins to storage vacuoles. Inference of a gene coexpression network of 12 candidate transcription factors and globulin genes revealed the transcription factor ABA-insensitive 5 (ABI5) as a highly connected hub. Characterization of loss-of-function abi5 mutants in pea uncovered a role for ABI5 in controlling the relative abundance of vicilin, a sulfur-poor 7S globulin, in pea seeds. This demonstrates the feasibility of using genome-wide association studies in M. truncatula to reveal genes that can be modulated to improve seed nutritional value.


Assuntos
Globulinas/metabolismo , Medicago truncatula/genética , Medicago truncatula/metabolismo , Sementes/metabolismo , Eletroforese em Gel Bidimensional , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Estudo de Associação Genômica Ampla , Globulinas/genética , Mutação , Ervilhas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transporte Proteico , Proteômica/métodos , Proteínas de Armazenamento de Sementes/genética , Proteínas de Armazenamento de Sementes/metabolismo , Sementes/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
9.
G3 (Bethesda) ; 7(1): 193-202, 2017 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-27866150

RESUMO

Double-strand breaks (DSBs) are one of the most harmful DNA lesions. Cells utilize two main pathways for DSB repair: homologous recombination (HR) and nonhomologous end-joining (NHEJ). NHEJ can be subdivided into the KU-dependent classical NHEJ (c-NHEJ) and the more error-prone KU-independent backup-NHEJ (b-NHEJ) pathways, involving the poly (ADP-ribose) polymerases (PARPs). However, in the absence of these factors, cells still seem able to adequately maintain genome integrity, suggesting the presence of other b-NHEJ repair factors or pathways independent from KU and PARPs. The outcome of DSB repair by NHEJ pathways can be investigated by using artificial sequence-specific nucleases such as CRISPR/Cas9 to induce DSBs at a target of interest. Here, we used CRISPR/Cas9 for DSB induction at the Arabidopsis cruciferin 3 (CRU3) and protoporphyrinogen oxidase (PPO) genes. DSB repair outcomes via NHEJ were analyzed using footprint analysis in wild-type plants and plants deficient in key factors of c-NHEJ (ku80), b-NHEJ (parp1 parp2), or both (ku80 parp1 parp2). We found that larger deletions of >20 bp predominated after DSB repair in ku80 and ku80 parp1 parp2 mutants, corroborating with a role of KU in preventing DSB end resection. Deletion lengths did not significantly differ between ku80 and ku80 parp1 parp2 mutants, suggesting that a KU- and PARP-independent b-NHEJ mechanism becomes active in these mutants. Furthermore, microhomologies and templated insertions were observed at the repair junctions in the wild type and all mutants. Since these characteristics are hallmarks of polymerase θ-mediated DSB repair, we suggest a possible role for this recently discovered polymerase in DSB repair in plants.


Assuntos
Proteínas de Arabidopsis/genética , DNA Helicases/genética , Reparo do DNA/genética , Recombinação Homóloga/genética , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerases/genética , Arabidopsis/genética , Sistemas CRISPR-Cas/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/genética , Proteínas de Ligação a DNA/genética , Globulinas/genética , Mutação , Protoporfirinogênio Oxidase/genética , Proteínas de Armazenamento de Sementes/genética
10.
Glycobiology ; 27(1): 50-56, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27558840

RESUMO

Tarin, the Colocasia esculenta lectin from the superfamily of α-d-mannose-specific plant bulb lectins, is a tetramer of 47 kDa composed of two heterodimers. Each heterodimer possesses homologous monomers of ~11.9 (A chain) and ~12.7 (B chain) kDa. The structures of apo and carbohydrate-bound tarin were solved to 1.7 Å and 1.91 Å, respectively. Each tarin monomer forms a canonical ß-prism II fold, common to all members of Galanthus nivalis agglutinin (GNA) family, which is partially stabilized by a disulfide bond and a conserved hydrophobic core. The heterodimer is formed through domain swapping involving the C-terminal ß-strand and the ß-sheet on face I of the prism. The tetramer is assembled through the dimerization of the B chains from heterodimers involving face II of each prism. The 1.91 Å crystal structure of tarin bound to Manα(1,3)Manα(1,6)Man reveals an expanded carbohydrate-binding sequence (QxDxNxVxYx4/6WX) on face III of the ß-prism. Both monomers possess a similar fold, except for the length of the loop, which begins after the conserved tyrosine and creates the binding pocket for the α(1,6)-terminal mannose. This loop differs in size and amino-acid composition from 10 other ß-prism II domain proteins, and may confer carbohydrate-binding specificity among members of the GNA-related lectin family.


Assuntos
Colocasia/química , Globulinas/química , Lectinas de Ligação a Manose/química , Proteínas de Plantas/química , Sequência de Aminoácidos/genética , Sítios de Ligação , Cristalografia por Raios X , Globulinas/genética , Lectinas de Ligação a Manose/genética , Modelos Moleculares , Proteínas de Plantas/genética , Conformação Proteica , Homologia de Sequência de Aminoácidos
11.
Protein Pept Lett ; 24(3): 267-277, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28000570

RESUMO

Globulins are a major class of seed storage proteins which were thought to be enzymatically inactive. These proteins belong to the most ancient cupin superfamily. They can be graded into 11S legumin type and 7S vicilin type based on their sedimentation coefficients. Members from both classes share structural homology are thought to have evolved from either one-domain germin predecessor by duplication or by horizontal gene transfer of two-domain gene from bacteria to eukaryotes. Globulins are known to define the nutritional quality of the seeds, however, they are also involved in sucrose binding, desiccation, defense against microbes, hormone binding and oxidative stress etc. Major drawback with globulins is their tendency to bind to IgE. Studying structural-functional behavior of such protein can help in modifying proteins for enhanced functionality in food processing industries.


Assuntos
Glicoproteínas/química , Proteínas de Plantas/química , Plantas/química , Proteínas de Armazenamento de Sementes/química , Sementes/química , Evolução Biológica , Duplicação Gênica , Expressão Gênica , Transferência Genética Horizontal , Globulinas/genética , Globulinas/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Imunoglobulina E/genética , Imunoglobulina E/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/genética , Plantas/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas de Armazenamento de Sementes/genética , Proteínas de Armazenamento de Sementes/metabolismo , Sementes/genética , Sementes/metabolismo , Homologia de Sequência de Aminoácidos
12.
Peptides ; 85: 27-40, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27612614

RESUMO

Antimicrobial peptides (AMPs) inactivate microbial cells through pore formation in cell membrane. Because of their different mode of action compared to antibiotics, AMPs can be effectively used to combat drug resistant bacteria in human health. AMPs can also be used to replace antibiotics in animal feed and immobilized on food packaging films. In this research, we developed a methodology based on mechanistic evaluation of peptide-lipid bilayer interaction to identify AMPs from soy protein. Production of AMPs from soy protein is an attractive, cost-saving alternative for commercial consideration, because soy protein is an abundant and common protein resource. This methodology is also applicable for identification of AMPs from any protein. Initial screening of peptide segments from soy glycinin (11S) and soy ß-conglycinin (7S) subunits was based on their hydrophobicity, hydrophobic moment and net charge. Delicate balance between hydrophilic and hydrophobic interactions is necessary for pore formation. High hydrophobicity decreases the peptide solubility in aqueous phase whereas high hydrophilicity limits binding of the peptide to the bilayer. Out of several candidates chosen from the initial screening, two peptides satisfied the criteria for antimicrobial activity, viz. (i) lipid-peptide binding in surface state and (ii) pore formation in transmembrane state of the aggregate. This method of identification of antimicrobial activity via molecular dynamics simulation was shown to be robust in that it is insensitive to the number of peptides employed in the simulation, initial peptide structure and force field. Their antimicrobial activity against Listeria monocytogenes and Escherichia coli was further confirmed by spot-on-lawn test.


Assuntos
Antígenos de Plantas/genética , Peptídeos Catiônicos Antimicrobianos/genética , Globulinas/genética , Infecção/tratamento farmacológico , Proteínas de Armazenamento de Sementes/genética , Proteínas de Soja/genética , Sequência de Aminoácidos/genética , Animais , Antibacterianos/uso terapêutico , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Antígenos de Plantas/química , Antígenos de Plantas/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/patogenicidade , Globulinas/química , Globulinas/farmacologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Infecção/genética , Infecção/microbiologia , Bicamadas Lipídicas/química , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/patogenicidade , Simulação de Dinâmica Molecular , Proteínas de Armazenamento de Sementes/química , Proteínas de Armazenamento de Sementes/farmacologia , Proteínas de Soja/química , Proteínas de Soja/farmacologia , Soja/química , Soja/genética
13.
PLoS One ; 11(8): e0159723, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27532666

RESUMO

Crossing, backcrossing, and molecular marker-assisted background selection produced a soybean (Glycine max) near-isogenic line (cgy-2-NIL) containing the cgy-2 allele, which is responsible for the absence of the allergenic α-subunit of ß-conglycinin. To identify α-null-related transcriptional changes, the gene expressions of cgy-2-NIL and its recurrent parent DN47 were compared using Illumina high-throughput RNA-sequencing of samples at 25, 35, 50, and 55 days after flowering (DAF). Seeds at 18 DAF served as the control. Comparison of the transcript profiles identified 3,543 differentially expressed genes (DEGs) between the two genotypes, with 2,193 genes downregulated and 1,350 genes upregulated. The largest numbers of DEGs were identified at 55 DAF. The DEGs identified at 25 DAF represented a unique pattern of GO category distributions. KEGG pathway analyses identified 541 altered metabolic pathways in cgy-2-NIL. At 18DAF, 12 DEGs were involved in arginine and proline metabolism. The cgy-2 allele in the homozygous form modified the expression of several Cupin allergen genes. The cgy-2 allele is an alteration of a functional allele that is closely related to soybean protein amino acid quality, and is useful for hypoallergenic soybean breeding programs that aim to improve seed protein quality.


Assuntos
Antígenos de Plantas/genética , Regulação da Expressão Gênica de Plantas/genética , Globulinas/genética , Proteínas de Armazenamento de Sementes/genética , Sementes/crescimento & desenvolvimento , Proteínas de Soja/genética , Soja/genética , Sequência de Bases , Perfilação da Expressão Gênica , Genes de Plantas/genética , Sequenciamento de Nucleotídeos em Larga Escala , Melhoramento Vegetal , Análise de Sequência de RNA , Transcriptoma/genética
14.
Mol Biol Rep ; 43(9): 897-909, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27317377

RESUMO

Acclimatization to stress is associated with profound changes in proteome composition. The use of plant cell and tissue culture offers a means to investigate the physiological and biochemical processes involved in the adaptation to osmotic stress. We employed a new proteomic approach to further understand the response of calli to dehydration induced by polyethylene glycol (PEG6000). Calli of three durum wheat genotypes Djenah Khetifa, Oued Zenati and Waha were treated with two concentrations of polyethylene glycol to mimic osmotic stress. Changes in protein relative abundance were analyzed using a new electrophoretic approach named diagonal two-dimensional electrophoresis (D-2DE), combined with mass spectrometry. Total proteins were extracted from 30-day-old calli from three durum wheat genotypes that showed contrasting levels of drought stress tolerance in the field. The combination of one-dimensional electrophoresis and D-2DE gave a specific imprint of the protein extracts under osmotic stress, as well as characterizing and identifying individual target proteins. Of the variously expressed proteins, three were selected (globulin, GAPDH and peroxidase) and further analyzed using qRT-PCR at the transcriptome level in order to compare the results with the proteomic data. Western blot analysis was used to further validate the differences in relative abundance pattern. The proteins identified through this technique provide new insights as to how calli respond to osmotic stress. Our method of study provides an original and relevant approach of analyzing the osmotic-responsive mechanisms at the cellular level of durum wheat with agronomic perspectives.


Assuntos
Proteínas de Plantas/metabolismo , Polietilenoglicóis/farmacologia , Triticum/metabolismo , Sequência de Aminoácidos , Eletroforese em Gel Bidimensional , Expressão Gênica , Globulinas/química , Globulinas/genética , Globulinas/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/genética , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Pressão Osmótica , Peroxidase/genética , Peroxidase/metabolismo , Estresse Fisiológico
15.
Genet Mol Res ; 15(2)2016 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-27173254

RESUMO

The objective of this study was to use RNA interference (RNAi) to improve protein quality and decrease anti-nutritional effects in soybean. Agrobacterium tumefaciens-mediated transformation was conducted using RNAi and an expression vector containing the 7S globulin ß-subunit gene. The BAR gene was used as the selective marker and cotyledonary nodes of soybean genotype Jinong 27 were chosen as explant material. Regenerated plants were detected by molecular biology techniques. Transformation of the ß-subunit gene in the 7S protein was detected by PCR, Southern blot, and q-PCR. Positive plants (10 T0, and 6 T1, and 13 T2) were tested by PCR. Hybridization bands were detected by Southern blot analysis in two of the T1 transgenic plants. RNAi expression vectors containing the soybean 7S protein ß-subunit gene were successfully integrated into the genome of transgenic plants. qRT-PCR analysis in soybean seeds showed a clear decrease in expression of the soybean ß-subunit gene. The level of 7S protein ß-subunit expression in transgenic plants decreased by 77.5% as compared to that of the wild-type plants. This study has established a basis for the application of RNAi to improve the anti-nutritional effects of soybean.


Assuntos
Agrobacterium tumefaciens/genética , Antígenos de Plantas/genética , Globulinas/genética , Interferência de RNA , Proteínas de Armazenamento de Sementes/genética , Proteínas de Soja/genética , Soja/genética , Antígenos de Plantas/metabolismo , Cotilédone/citologia , Cotilédone/genética , Cotilédone/metabolismo , Técnicas de Transferência de Genes , Genoma de Planta , Globulinas/metabolismo , Recombinação Genética , Proteínas de Armazenamento de Sementes/metabolismo , Proteínas de Soja/metabolismo , Transgenes
16.
Metabolism ; 65(6): 816-24, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27173460

RESUMO

PURPOSE: After observing variation in the expression of the housekeeping gene B2M in cells of the urinary sediment during a study of candidate genes potentially involved in diabetic kidney disease (DKD), we hypothesized that B2M mRNA expression in the urinary sediment could reflect the presence of DKD. METHODS: qPCR was used to quantify B2M mRNA expression in cells of the urinary sediment of 51 type 1 diabetes (T1D) patients (61% women, 33.5 [27.0-39.7] years old, with diabetes duration of 21.0 [15.0-28.0] years and HbA1c of 8.2% [7.3-8.9]; median [interquartile interval]) sorted according to the diabetic nephropathy (DN) stages; 8 focal segmental glomerulosclerosis (FSGS) patients and 10 healthy controls. B2M mRNA expression was also evaluated in human embryonic kidney epithelium-like (HEK-293) cells exposed to 25mM glucose and to albumin in order to mimic, respectively, a diabetic and a proteinuric milieu. RESULTS: No differences were found in B2M mRNA expression among healthy controls, FSGS and T1D patients. Nonetheless B2M mRNA expression was higher in the group composed by T1D patients with incipient or overt DN combined with FSGS patients versus T1D patients without DN combined with healthy controls (P=0.0007). B2M mRNA expression was higher in T1D patients with incipient or overt DN versus without DN (P=0.03). B2M mRNA expression positively correlated with albuminuria in the overall T1D population (r=0.43; P=0.01) and negatively correlated with estimated glomerular filtration rate in male T1D patients (r=- 0.57; P=0.01). Increased B2M expression was observed in HEK-293 cells exposed to 25mM glucose and to albumin. CONCLUSIONS: Β2M mRNA expression in cells of the urinary sediment is higher in T1D patients with DKD and in patients with FSGS in comparison to healthy subjects, maybe reflecting a tubulointerstitial injury promoted by albumin. Given the proinflammatory nature of B2M, we suggest that this protein contributes to diabetic (and possibly, to non-diabetic) tubulopathy.


Assuntos
Diabetes Mellitus Tipo 1/urina , Nefropatias Diabéticas/urina , Globulinas/urina , Glomerulosclerose Segmentar e Focal/urina , Adulto , Albuminas/farmacologia , Albuminúria/genética , Albuminúria/urina , Biomarcadores , Diabetes Mellitus Tipo 1/genética , Nefropatias Diabéticas/genética , Feminino , Globulinas/genética , Glomerulosclerose Segmentar e Focal/genética , Glucose/farmacologia , Células HEK293 , Humanos , Rim/efeitos dos fármacos , Masculino , RNA Mensageiro/genética , RNA Mensageiro/urina
17.
Food Chem ; 210: 148-55, 2016 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-27211633

RESUMO

Protein content and globulin subunit composition of soybean seeds affect the quality of soy foods. In this proteomic study, the protein profile of soybean seeds with high (∼45.5%) or low (∼38.6%) protein content and with or without the glycinin (11S) subunit 11SA4 was examined. 44 unique proteins and their homologues were identified and showed that both protein content and 11SA4 influenced the abundance of a number of proteins. The absence of 11SA4 exerted a greater impact than the protein content, and led to a decreased abundance of glycinin G2/A2B1 and G5/A5A4B3 subunits, which resulted in lower total 11S with a concomitant higher total ß-conglycinin (7S). Low protein content was associated with higher glycinin G3/A1aB1b and lower glycinin G4/A5A4B3. Using the proteomic approach, it was demonstrated that 11SA4 deficiency induced compensatory accumulation of 7S globulins and led to a similar total abundance for 7S+11S irrespective of protein content or 11SA4.


Assuntos
Globulinas/química , Proteínas de Plantas/química , Soja/metabolismo , Globulinas/genética , Globulinas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteômica , Sementes/química , Sementes/genética , Sementes/metabolismo , Alimentos de Soja/análise , Proteínas de Soja/metabolismo , Soja/química , Soja/genética
18.
J Agric Food Chem ; 64(17): 3473-83, 2016 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-27070305

RESUMO

Compared to ß-conglycinin, glycinin contains 3-4 times the methionine and cysteine (sulfur-containing amino acids), accounting for approximately 40 and 30%, respectively, of the total storage protein in soybean. Increasing the soybean storage protein content while improving the ratio of glycinin to ß-conglycinin is of great significance for soybean breeding and soy food products. The objective of this study is to analyze the genetic mechanism regulating the glycinin and ß-conglycinin contents of soybean by using a recombinant inbred line (RIL) population derived from a cross between Kefeng No. 1 and Nannong 1138-2. Two hundred and twenty-one markers were used to map quantitative trait loci (QTLs) for glycinin (11S) and ß-conglycinin (7S) contents, the ratio of glycinin to ß-conglycinin (RGC), and the sum of glycinin and ß-conglycinin (SGC). A total of 35 QTLs, 3 pairs of epistatic QTLs, and 5 major regions encompassing multiple QTLs were detected. Genes encoding the subunits of ß-conglycinin were localized to marker intervals sat_418-satt650 and sat_196-sat_303, which are linked to RGC and SGC; marker sat_318, associated with 11S, 7S, and SGC, was located near Glyma10g04280 (Gy4), which encodes a subunit of glycinin. These results, which take epistatic interactions into account, will improve our understanding of the genetic basis of 11S and 7S contents and will lay a foundation for marker-assisted selection (MAS) breeding of soybean and improving the quality of soybean products.


Assuntos
Antígenos de Plantas/genética , Globulinas/genética , Locos de Características Quantitativas , Proteínas de Armazenamento de Sementes/genética , Proteínas de Soja/genética , Soja/genética
19.
Mol Cells ; 39(2): 111-8, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26674968

RESUMO

MiR399f plays a crucial role in maintaining phosphate homeostasis in Arabidopsis thaliana. Under phosphate starvation conditions, AtMYB2, which plays a role in plant salt and drought stress responses, directly regulates the expression of miR399f. In this study, we found that miR399f also participates in plant responses to abscisic acid (ABA), and to abiotic stresses including salt and drought. Salt and ABA treatment induced the expression of miR399f, as confirmed by histochemical analysis of promoter-GUS fusions. Transgenic Arabidopsis plants overexpressing miR399f (miR399f-OE) exhibited enhanced tolerance to salt stress and exogenous ABA, but hypersensitivity to drought. Our in silico analysis identified ABF3 and CSP41b as putative target genes of miR399f, and expression analysis revealed that mRNA levels of ABF3 and CSP41b decreased remarkably in miR399f-OE plants under salt stress and in response to treatment with ABA. Moreover, we showed that activation of stress-responsive gene expression in response to salt stress and ABA treatment was impaired in miR399f-OE plants. Thus, these results suggested that in addition to phosphate starvation signaling, miR399f might also modulates plant responses to salt, ABA, and drought, by regulating the expression of newly discovered target genes such as ABF3 and CSP41b.


Assuntos
Ácido Abscísico/farmacologia , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Regulação da Expressão Gênica de Plantas , Globulinas/genética , MicroRNAs/genética , Ácido Abscísico/metabolismo , Adaptação Fisiológica , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Secas , Genes Reporter , Globulinas/metabolismo , MicroRNAs/metabolismo , Fosfatos/deficiência , Plantas Geneticamente Modificadas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Salinidade , Transdução de Sinais , Cloreto de Sódio/farmacologia , Estresse Fisiológico , Transativadores/genética , Transativadores/metabolismo , beta-Glucosidase/genética , beta-Glucosidase/metabolismo
20.
Protein Eng Des Sel ; 28(9): 281-91, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26243884

RESUMO

ß-Conglycinin and glycinin are soybean major seed storage proteins. Previous studies have shown that adding the extension region of ß-conglycinin α subunit improves the emulsifying properties of proglycinin and confers more favourable characteristics than fusing the extension region of ß-conglycinin α' subunit or the hypervariable regions (A4IV) of glycinin A1aB1b subunit. To evaluate the polypeptide properties, we designed mutants of A1aB1b subunits fused with truncated versions of A4IV (A4IVcut), α (αcut) or α' (α'cut) extension regions lacking the C-terminus 25 or 31 residues (A4IVC25, αC25 or α'C31), and also A4IVcut and α'cut with αC25 residues added (A4IVcut-αC25 and α'cut-αC25). All the modified proteins displayed conformations similar to the wild type. With good solubilities, the emulsion properties of the modified proteins were much better at ionic strength µ = 0.08 than at µ = 0.5. The modified A1aB1bαcut and A1aB1bα'cut showed poorer emulsion properties than those of A1aB1bα and A1aB1bα'. Replacing the hydrophobic A4IVC25 region of A1aB1bA4IV with hydrophilic αC25 created A1aB1bA4IVcut-αC25, which had the best emulsion stability among these proglycinin mutants. We found that addition of αC25 improves the emulsifying properties of two C-terminally truncated proglycinin variants, thereby illustrating its potential general utility. Our investigation showed that in order to improve the emulsifying ability and emulsion stability of a globular protein, the introduced polypeptide should (i) be highly hydrophilic, (ii) consist of multiple hydrophobic-strong hydrophilic regions comprising at least two alpha helixes, (iii) harbour a terminal α-helix at the end of the C-terminus and (iv) have properties similar to those of αC25.


Assuntos
Antígenos de Plantas/genética , Globulinas/genética , Peptídeos/química , Proteínas de Armazenamento de Sementes/genética , Proteínas de Soja/genética , Sequência de Aminoácidos/genética , Antígenos de Plantas/química , Emulsões , Globulinas/química , Interações Hidrofóbicas e Hidrofílicas , Mutação , Peptídeos/genética , Estrutura Secundária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Proteínas de Armazenamento de Sementes/química , Proteínas de Soja/química , Soja/química , Soja/genética
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