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1.
BMC Plant Biol ; 19(1): 333, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31370789

RESUMO

BACKGROUND: Wheat grains contain gluten proteins, which harbour immunogenic epitopes that trigger Coeliac disease in 1-2% of the human population. Wheat varieties or accessions containing only safe gluten have not been identified and conventional breeding alone struggles to achieve such a goal, as the epitopes occur in gluten proteins encoded by five multigene families, these genes are partly located in tandem arrays, and bread wheat is allohexaploid. Gluten immunogenicity can be reduced by modification or deletion of epitopes. Mutagenesis technologies, including CRISPR/Cas9, provide a route to obtain bread wheat containing gluten proteins with fewer immunogenic epitopes. RESULTS: In this study, we analysed the genetic diversity of over 600 α- and γ-gliadin gene sequences to design six sgRNA sequences on relatively conserved domains that we identified near coeliac disease epitopes. They were combined in four CRISPR/Cas9 constructs to target the α- or γ-gliadins, or both simultaneously, in the hexaploid bread wheat cultivar Fielder. We compared the results with those obtained with random mutagenesis in cultivar Paragon by γ-irradiation. For this, Acid-PAGE was used to identify T1 grains with altered gliadin protein profiles compared to the wild-type endosperm. We first optimised the interpretation of Acid-PAGE gels using Chinese Spring deletion lines. We then analysed the changes generated in 360 Paragon γ-irradiated lines and in 117 Fielder CRISPR/Cas9 lines. Similar gliadin profile alterations, with missing protein bands, could be observed in grains produced by both methods. CONCLUSIONS: The results demonstrate the feasibility and efficacy of using CRISPR/Cas9 to simultaneously edit multiple genes in the large α- and γ-gliadin gene families in polyploid bread wheat. Additional methods, generating genomics and proteomics data, will be necessary to determine the exact nature of the mutations generated with both methods.


Assuntos
Edição de Genes/métodos , Genes de Plantas/genética , Gliadina/genética , Glutens/genética , Triticum/genética , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Eletroforese em Gel de Poliacrilamida , Glutens/imunologia , Melhoramento Vegetal/métodos , Plantas Geneticamente Modificadas , Alinhamento de Sequência
2.
BMC Plant Biol ; 19(1): 336, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31370790

RESUMO

BACKGROUND: APETALA2-like genes encode plant-specific transcription factors, some of which possess one microRNA172 (miR172) binding site. The miR172 and its target euAP2 genes are involved in the process of phase transformation and flower organ development in many plants. However, the roles of miR172 and its target AP2 genes remain largely unknown in Brassica napus (B. napus). RESULTS: In this study, 19 euAP2 and four miR172 genes were identified in the B. napus genome. A sequence analysis suggested that 17 euAP2 genes were targeted by Bna-miR172 in the 3' coding region. EuAP2s were classified into five major groups in B.napus. This classification was consistent with the exon-intron structure and motif organization. An analysis of the nonsynonymous and synonymous substitution rates revealed that the euAP2 genes had gone through purifying selection. Whole genome duplication (WGD) or segmental duplication events played a major role in the expansion of the euAP2 gene family. A cis-regulatory element (CRE) analysis suggested that the euAP2s were involved in the response to light, hormones, stress, and developmental processes including circadian control, endosperm and meristem expression. Expression analysis of the miR172-targeted euAP2s in nine different tissues showed diverse spatiotemporal expression patterns. Most euAP2 genes were highly expressed in the floral organs, suggesting their specific functions in flower development. BnaAP2-1, BnaAP2-5 and BnaTOE1-2 had higher expression levels in late-flowering material than early-flowering material based on RNA-seq and qRT-PCR, indicating that they may act as floral suppressors. CONCLUSIONS: Overall, analyses of the evolution, structure, tissue specificity and expression of the euAP2 genes were peformed in B.napus. Based on the RNA-seq and experimental data, euAP2 may be involved in flower development. Three euAP2 genes (BnaAP2-1, BnaAP2-5 and BnaTOE1-2) might be regarded as floral suppressors. The results of this study provide insights for further functional characterization of the miR172 /euAP2 module in B.napus.


Assuntos
Brassica napus/genética , Flores/crescimento & desenvolvimento , Genes de Plantas/genética , MicroRNAs/genética , Brassica napus/crescimento & desenvolvimento , Mapeamento Cromossômico , Sequência Conservada/genética , Genes de Plantas/fisiologia , Estudo de Associação Genômica Ampla , MicroRNAs/fisiologia , Filogenia , Alinhamento de Sequência
3.
Gene ; 716: 144024, 2019 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-31390541

RESUMO

The young leaves generally accumulate a certain concentration anthocyanins in the dominant species of the subtropical forest, and the changes of anthocyanin synthesis-related enzyme genes expression levels had an important effect on the study photoprotection of anthocyanins in the young leaves of subtropical forests. The determination of anthocyanin synthesis-related enzyme gene sequences and the selection of appropriate reference genes provide a basis for analyzing the functional properties of anthocyanins. In this study, four dominant subtropical forest species (i.e., Schima superba, Castanopsis fissa, Acmena acuminatissima, Cryptocarya concinna) were taken as materials. To obtain the correct nucleotide sequences of anthocyanin-related enzymes, the nucleotide sequences of CHS, DFR and ANS in each dominant species were obtained by sequencing and comparison. Then, to select the most stable reference genes for leaves at different developmental stages and different light conditions, the expression levels of six reference genes, including 18S, Actin, GAPDH, TUB, EF1 and UBQ, were studied by real-time fluorescent quantitative PCR (qRT-PCR), and reference gene stability was analyzed by GeNorm and NormFinder software. The results showed that the expression level of Actin was the most stable in S. superba, A. acuminatissima and C. concinna, and the expression level of GAPDH was the most stable in C. fissa. Finally, the expression levels of the anthocyanin synthesis genes CHS, DFR and ANS were analyzed and found to be consistent with the accumulation trend of anthocyanins in leaves. This study has important theoretical and practical significance for future research into the expression of anthocyanin synthesis-related enzyme genes in the dominant tree species in subtropical forests and reveals that anthocyanin has a photoprotective effect for young leaves in high-light environments.


Assuntos
Antocianinas/biossíntese , Árvores/genética , Aciltransferases/genética , Aciltransferases/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Antocianinas/metabolismo , Florestas , Genes de Plantas , Folhas de Planta/genética , Folhas de Planta/metabolismo , RNA de Plantas/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/normas , Padrões de Referência , Alinhamento de Sequência , Análise de Sequência , Árvores/enzimologia , Árvores/metabolismo
4.
Dokl Biochem Biophys ; 486(1): 192-196, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31367819

RESUMO

A novel CYP74 clan gene CYP443С1 of the starlet sea anemone (Nematostella vectensis, Cnidaria) has been cloned, and the properties of the corresponding recombinant protein have been studied. Depending on the substrate, CYP443С1 exhibited double function hydroperoxide lyase/epoxyalcohol synthase activity.


Assuntos
Aldeído Liases/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Anêmonas-do-Mar/enzimologia , Aldeído Liases/química , Aldeído Liases/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Anêmonas-do-Mar/genética , Alinhamento de Sequência
5.
Microbiol Res ; 227: 126296, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31421712

RESUMO

Heat shock proteins (Hsp) are important factors in the response of organisms to oscillations in environmental conditions. Although Hsp have been studied for a long time, little is known about this protein class in Trichoderma species. Here we studied the expression of Hsp genes during T. asperellum growth, and mycoparasitism against two phytopathogens: Sclerotinia sclerotiorum and Fusarium oxysporum, as well as during thermal stress. The expression levels of these genes were observed by real-time PCR and they showed to be differentially expressed under these conditions. We verified that the TaHsp26c, TaHsp70b and TaHsp70c genes were differentially expressed over time, indicating that these genes can be developmentally regulated in T. asperellum. Except for TaHsp26a, all other genes analyzed were induced in the post-contact condition when T. asperellum was cultured in a confrontation plate assay against itself. Additionally, TaHsp26b, TaHsp26c, TaHsp90, TaHsp104a and TaHsp104b were induced during initial contact between T. asperellum hyphae, suggesting that these proteins must play a role in the organism´s self-recognition mechanism. When we examined gene expression during mycoparasitism, we observed that some genes were induced both by S. sclerotiorum and F. oxysporum, while others were not induced during interaction with either of the phytopathogens. Furthermore, we observed some genes induced only during confrontation against S. sclerotiorum, indicating that the expression of Hsp genes during mycoparasitism seems to be modulated by the phytopathogen. To assess whether such genes are expressed during temperature oscillations, we analyzed their transcription levels during thermal and cold shock. We observed that except for the TaHsp70c gene, all others presented high transcript levels when T. asperellum was submitted to high temperature (38 °C), indicating their importance in the response to heat stress. The TaHsp70c gene was significantly induced only in cold shock at 4 °C. Our results show the importance of Hsp proteins during self-recognition, mycoparasitism and thermal stress in T. asperellum.


Assuntos
Regulação Fúngica da Expressão Gênica/genética , Genes Fúngicos/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/fisiologia , Trichoderma/genética , Sequência de Aminoácidos , Ascomicetos/genética , Fusarium/genética , Resposta ao Choque Térmico/genética , Hifas/genética , Hifas/crescimento & desenvolvimento , Interações Microbianas , Doenças das Plantas/microbiologia , Reação em Cadeia da Polimerase em Tempo Real , Alinhamento de Sequência , Estresse Fisiológico/genética , Temperatura Ambiente , Transcriptoma , Trichoderma/crescimento & desenvolvimento
6.
BMC Plant Biol ; 19(1): 347, 2019 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-31395025

RESUMO

BACKGROUND: Flavonoid 3'-hydroxlase (F3'H) is an important enzyme in determining the B-ring hydroxylation pattern of flavonoids. In monocots, previous studies indicated the presence of two groups of F3'Hs with different enzyme activities. One F3'H in rice was found to display novel chrysoeriol-specific 5'-hydroxylase activity. However, the evolutionary history of monocot F3'Hs and the molecular basis for the observed catalytic difference remained elusive. RESULTS: We performed genome-wide survey of 12 common monocot plants, and identified a total of 44 putative F3'H genes. The results showed that F3'H gene family had underwent volatile lineage-specific gene duplication and gene loss events in monocots. The expansion of F3'H gene family was mainly attributed to dispersed gene duplication. Phylogenetic analyses showed that monocot F3'Hs have evolved into two independent lineages (Class I and Class II) after gene duplication in the common ancestor of monocot plants. Evolutionary dynamics analyses had detected positive natural selection in Class II F3'Hs, acting on 7 specific amino acid sites. Protein modelling showed these selected sites were mainly located in the catalytic cavity of F3'H. Sequence alignment revealed that Class I and Class II F3'Hs displayed amino acid substitutions at two critical sites previously found to be responsible for F3'H and flavonoid 3'5'-hydroxylase (F3'5'H) activities. In addition, transcriptional divergence was also observed for Class I and Class II F3'Hs in four monocot species. CONCLUSIONS: We concluded that monocot F3'Hs have evolved into two independent lineages (Mono_F3'H Class I and Class II), after gene duplication during the common ancestor of monocot plants. The functional divergence of monocot F3'H Class II has been affected by positive natural selection, which acted on specific amino acid sites only. Critical amino acid sites have been identified to have high possibility to affect the substrate specificity of Class II F3'Hs. Our study provided an evolutionary and protein structural explanation to the previously observed chrysoeriol-specific 5'-hydroxylation activity for CYP75B4 in rice, which may also be true for other Class II F3'Hs in monocots. Our study presented clear evidence of plant-environmental interaction at the gene evolutionary level, and would guide future functional characterization of F3'Hs in cereal plants.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Grão Comestível/genética , Proteínas de Plantas/genética , Sistema Enzimático do Citocromo P-450/química , Grão Comestível/enzimologia , Evolução Molecular , Duplicação Gênica , Modelos Moleculares , Filogenia , Proteínas de Plantas/química , Seleção Genética , Alinhamento de Sequência
7.
J Agric Food Chem ; 67(37): 10458-10469, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31469568

RESUMO

Mud crab (Scylla paramamosain) is a commonly consumed seafood as a result of its high nutritional value; however, it is associated with food allergy. The current understanding of crab allergens remains insufficient. In the present study, an 18 kDa protein was purified from crab muscle and confirmed to be myosin light chain 1 (MLC1) by matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry. Total RNA was isolated and amplified to obtain a MLC1 open reading frame of 462 bp, encoding 154 amino acids. A structural analysis revealed that recombinant MLC1 (rMLC1) expressed in Escherichia coli contained α-helix and random coil. Moreover, rMLC1 displayed strong immunoactivity by dot blot and a basophil activation test. Furthermore, seven allergenic epitopes of MLC1 were predicted, and five critical epitope regions were identified by an inhibition enzyme-linked immunosorbent assay and human mast cell degranulation assay. This comprehensive research of an allergen helps to conduct component-resolved diagnoses and immunotherapies related to crab allergies.


Assuntos
Alérgenos/imunologia , Proteínas de Artrópodes/imunologia , Braquiúros/genética , Clonagem Molecular , Epitopos/imunologia , Cadeias Leves de Miosina/imunologia , Alérgenos/química , Alérgenos/genética , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Braquiúros/química , Braquiúros/imunologia , Degranulação Celular , Epitopos/química , Epitopos/genética , Humanos , Mastócitos/imunologia , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/genética , Fases de Leitura Aberta , Alinhamento de Sequência
8.
Exp Parasitol ; 205: 107734, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31394093

RESUMO

Parasitism by Haemonchus contortus is one of the main limiting factors in small ruminant production around the globe. Although several studies suggest the use of integrated management practices, these parasites have been controlled essentially with synthetic anthelmintic drugs. The resistance mechanism against the imidazothiazole derivative levamisole in Haemonchus contortus has not been fully described. Recently, resistance was associated with a 63bp deletion in the Hco-acr-8b gene that encodes a subunit for a nicotinic acetylcholine receptor. This study aimed to standardize a real time PCR (qPCR) protocol for levamisole resistance diagnosis in H. contortus populations based on this polymorphism and use it to characterize 23 field H. contortus populations obtained from different localities of Ceará State, Northeast Brazil. In addition, two populations of H. contortus were used as a standard of susceptibility and resistance, Inbred Strain Edinburgh (ISE) and Kokstad, respectively. Larval development tests (LDT) were performed on five field isolates and both EC50 and EC95 were estimated. LDT EC95 values provided a wider interval between susceptible and resistant populations than EC50 values (EC95 = 1.96-57.93 µM; EC50 = 0.05-0.39 µM), and were found to be more appropriate for differentiating them. Real time PCR results showed resistance allele frequencies ranged from 20.9 to 76.7%. Our results suggest that levamisole resistance may be present in field populations but it is not as widespread as benzimidazole resistance. This methodology may be useful to monitor levamisole resistance in field populations of H. contortus.


Assuntos
Antinematódeos/farmacologia , Resistência a Medicamentos/genética , Haemonchus/efeitos dos fármacos , Levamisol/farmacologia , Animais , Benzimidazóis/farmacologia , DNA de Helmintos/isolamento & purificação , Fezes/parasitologia , Frequência do Gene/genética , Hemoncose/tratamento farmacológico , Hemoncose/parasitologia , Hemoncose/veterinária , Haemonchus/genética , Haemonchus/crescimento & desenvolvimento , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Receptores Colinérgicos/efeitos dos fármacos , Receptores Nicotínicos/efeitos dos fármacos , Alinhamento de Sequência/veterinária , Ovinos , Doenças dos Ovinos/tratamento farmacológico , Doenças dos Ovinos/parasitologia , Tetramizol/farmacologia
9.
Arch Virol ; 164(11): 2725-2733, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31468140

RESUMO

Porcine reproductive and respiratory syndrome (PRRS) is one of the most highly infectious diseases in the pig industry, resulting in enormous economic losses worldwide. In this study, a PRRS virus (PRRSV) strain was isolated from primary porcine alveolar macrophage cells in Xinjiang in northwest China. This new strain was sequenced and designated as XJzx1-2015, and its sequence was then compared to those of other representative PRRSV strains from around the world. Complete genomic characterisation showed that the full-length nucleotide sequence of XJzx1-2015 exhibited low-level similarity to NB/04 (91.6%), JXA1 (90.5%), CH-1a (90.2%), VR-2332 (86.9%), QYYZ (85.7%), and JL580 (82.2%), with the highest similarity to HK13 (91.7%) sequence identity. Nonstructural protein 2 (NSP2) and glycosylated protein (GP) 2 of XJzx1-2015 had deletions of five and two amino acids, respectively, corresponding to strain VR-2332 positions 475-479 and 173-174. Phylogenetic analysis based on complete genome sequences showed that XJzx1-2015 and four other strains from China formed a new subgenotype closely related to other sublineage 8.7 (JXA1-like) strains belonging to the North American genotype. However, phylogenetic analysis based on NSP2 and GP5 showed that XJzx1-2015 clustered with sublineage 8.7 (JXA1-like, CH-1a-like) and lineage 3 (QYYZ-like) strains, respectively. Recombination analysis indicated that XJzx1-2015 is an intersubgenotype recombinant of CH-1a-like and QYYZ-like strains. Overall, our findings demonstrate that XJzx1-2015 is a novel PRRSV strain with a significantly high frequency of mutation and a recombinant between lineage 3 and sublineage 8.7 identified in northwest China. These results provide important insights into PRRSV evolution.


Assuntos
Genoma Viral/genética , Síndrome Respiratória e Reprodutiva Suína/epidemiologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Doenças dos Suínos/epidemiologia , Sequência de Aminoácidos , Animais , China/epidemiologia , Macrófagos Alveolares/virologia , Filogenia , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/classificação , Vírus da Síndrome Respiratória e Reprodutiva Suína/isolamento & purificação , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Suínos , Doenças dos Suínos/virologia
10.
J Agric Food Chem ; 67(35): 9868-9876, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31389242

RESUMO

Amylosucrase (EC 2.4.1.4, ASase), a typical carbohydrate-active enzyme, can catalyze 5 types of reactions and recognize more than 50 types of glycosyl acceptors. However, most ASases are unstable even at 50 °C, which limits their practical industrial applications. In this study, an extremely thermostable ASase was discovered from Calidithermus timidus DSM 17022 (CT-ASase) with an optimal activity temperature of 55 °C, half-life of 1.09 h at 70 °C, and melting temperature of 74.47 °C. The recombinant CT-ASase was characterized as the first tetrameric ASase, and a structure-based truncation mutation was conducted to confirm the effect of tetrameric conformation on its thermostability. In addition, α-1,4-glucan was found to be the predominant product of CT-ASase at pH 6.0-8.0 and 30-60 °C.


Assuntos
Proteínas de Bactérias/química , Glucosiltransferases/química , Thermus/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Estabilidade Enzimática , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , Conformação Proteica , Alinhamento de Sequência , Thermus/química , Thermus/genética
11.
BMC Bioinformatics ; 20(1): 419, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31409275

RESUMO

BACKGROUND: Alignment of sequence families described by profiles provides a sensitive means for establishing homology between proteins and is important in protein evolutionary, structural, and functional studies. In the context of a steadily growing amount of sequence data, estimating the statistical significance of alignments, including profile-profile alignments, plays a key role in alignment-based homology search algorithms. Still, it is an open question as to what and whether one type of distribution governs profile-profile alignment score, especially when profile-profile substitution scores involve such terms as secondary structure predictions. RESULTS: This study presents a methodology for estimating the statistical significance of this type of alignments. The methodology rests on a new algorithm developed for generating random profiles such that their alignment scores are distributed similarly to those obtained for real unrelated profiles. We show that improvements in statistical accuracy and sensitivity and high-quality alignment rate result from statistically characterizing alignments by establishing the dependence of statistical parameters on various measures associated with both individual and pairwise profile characteristics. Implemented in the COMER software, the proposed methodology yielded an increase of up to 34.2% in the number of true positives and up to 61.8% in the number of high-quality alignments with respect to the previous version of the COMER method. CONCLUSIONS: The more accurate estimation of statistical significance is implemented in the COMER method, which is now more sensitive and provides an increased rate of high-quality profile-profile alignments. The results of the present study also suggest directions for future research.


Assuntos
Modelos Teóricos , Proteínas/química , Algoritmos , Sequência de Aminoácidos , Conformação Proteica , Alinhamento de Sequência
12.
BMC Plant Biol ; 19(1): 325, 2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324146

RESUMO

BACKGROUND: The heteroside floridoside is a primary photosynthetic product that is known to contribute to osmotic acclimation in almost all orders of Rhodophyta. However, the encoding genes and enzymes responsible for the synthesis of floridoside and its isomeric form, L- or D-isofloridoside, are poorly studied. RESULTS: Here, four putative trehalose-6-phosphate synthase (TPS) genes, designated as PhTPS1, PhTPS2, PhTPS3, and PhTPS4, were cloned and characterized from the red alga Pyropia haitanensis (Bangiophyceae). The deduced amino acid sequence is similar to the annotated TPS proteins of other organisms, especially the UDP-galactose substrate binding sites of PhTPS1, 2, which are highly conserved. Of these, PhTPS1, 4 are involved in the biosynthesis of floridoside and isofloridoside, with isofloridoside being the main product. PhTPS3 is an isofloridoside phosphate synthase, while PhTPS2 exhibits no activity. When challenged by desiccation, high temperature, and salt stress, PhTPS members were expressed to different degrees, but the responses to thermal stress and desiccation were stronger. CONCLUSIONS: Thus, in P. haitanensis, PhTPSs encode the enzymatical activity of floridoside and isofloridoside phosphate synthase and are crucial for the abiotic stress defense response.


Assuntos
Proteínas de Algas/metabolismo , Glucosiltransferases/metabolismo , Glicerol/análogos & derivados , Rodófitas/fisiologia , Trealose/biossíntese , Proteínas de Algas/genética , Proteínas de Algas/fisiologia , Glucosiltransferases/genética , Glicerol/metabolismo , Filogenia , Rodófitas/enzimologia , Rodófitas/genética , Rodófitas/metabolismo , Alinhamento de Sequência , Estresse Fisiológico
13.
BMC Plant Biol ; 19(1): 329, 2019 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-31337346

RESUMO

BACKGROUND: Zinc finger proteins (ZFPs) containing only a single zinc finger domain play important roles in the regulation of plant growth and development, as well as in biotic and abiotic stress responses. To date, the evolutionary history and functions of the ZFP gene family have not been identified in cotton. RESULTS: In this paper, we identified 29 ZFP genes in Gossypium hirsutum. This gene family was divided into seven subfamilies, 22 of which were distributed over 17 chromosomes. Bioinformatic analysis revealed that 20 GhZFP genes originated from whole genome duplications and two originated from dispersed duplication events, indicating that whole genome duplication is the main force in the expansion of the GhZFP gene family. Most GhZFP8 subfamily genes, except for GhZFP8-3, were highly expressed during fiber cell growth, and were induced by brassinosteroids in vitro. Furthermore, we found that a large number of GhZFP genes contained gibberellic acid responsive elements, auxin responsive elements, and E-box elements in their promoter regions. Exogenous application of these hormones significantly stimulated the expression of these genes. CONCLUSIONS: Our findings reveal that GhZFP8 genes are involved in cotton fiber development and widely induced by auxin, gibberellin and BR, which provides a foundation for the identification of more downstream genes with potential roles in phytohormone stimuli, and a basis for breeding better cotton varieties in the future.


Assuntos
Gossypium/genética , Reguladores de Crescimento de Planta/fisiologia , Proteínas de Plantas/genética , Dedos de Zinco/genética , Brassinosteroides/metabolismo , Mapeamento Cromossômico , Sequência Conservada/genética , Giberelinas/fisiologia , Gossypium/fisiologia , Ácidos Indolacéticos/metabolismo , Filogenia , Proteínas de Plantas/fisiologia , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Transcriptoma , Dedos de Zinco/fisiologia
14.
BMC Plant Biol ; 19(1): 287, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31262258

RESUMO

BACKGROUND: The majority of apricot (Prunus armeniaca L.) cultivars display orange or yellow background skin, whereas some cultivars are particularly preferred by consumers because of their red blushed skin on the background. RESULTS: In this study, two blushed ('Jianali' and 'Hongyu') and two nonblushed ('Baixing' and 'Luntaixiaobaixing') cultivars were used to investigate the formation mechanism of blushed skin in apricots. High-performance liquid chromatography (HPLC) analysis showed that the blushed cultivars accumulated higher cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside and peonidin-3-O-rutinoside levels during fruit ripening than the nonblushed cultivars. Based on coexpression network analysis (WGCNA), a putative anthocyanin-related R2R3-MYB, PaMYB10, and seven structural genes were identified from transcriptome data. The phylogenetic analysis indicated that PaMYB10 clustered in the anthocyanin-related MYB clade. Sequence alignments revealed that PaMYB10 contained a bHLH-interaction motif ([DE]Lx2[RK]x3Lx6Lx3R) and an ANDV motif. Subcellular localization analysis showed that PaMYB10 was a nuclear protein. Real-time qRT-PCR analysis demonstrated that the transcript levels of PaMYB10 and seven genes responsible for anthocyanin synthesis were significantly higher in blushed than in nonblushed apricots, which was consistent with the accumulation of anthocyanin. In addition, bagging significantly inhibited the transcript levels of PaMYB10 and the structural genes in 'Jianali' and blocked the red coloration and anthocyanin accumulation. Transient PaMYB10 overexpression in 'Luntaixiaobaixing' fruits resulted in the red blushed skin at the maturation stage. CONCLUSIONS: Taken together, these data reveal that three anthocyanins are responsible for the blushed skin of apricots, identify PaMYB10 as a positive regulator of anthocyanin biosynthesis in apricots, and demonstrate that blush formation depends on light.


Assuntos
Antocianinas/biossíntese , Regulação da Expressão Gênica de Plantas , Pigmentos Biológicos/biossíntese , Proteínas de Plantas/genética , Prunus armeniaca/fisiologia , Fatores de Transcrição/genética , Sequência de Aminoácidos , Antocianinas/genética , Cromatografia Líquida de Alta Pressão , Cor , Frutas/genética , Frutas/fisiologia , Glucosídeos/biossíntese , Glucosídeos/genética , Filogenia , Pigmentos Biológicos/genética , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Prunus armeniaca/genética , Alinhamento de Sequência , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
15.
J Agric Food Chem ; 67(31): 8548-8558, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31266305

RESUMO

Herein, we report a double enzyme system to degrade 12 phthalate esters (PAEs), particularly bulky PAEs, such as the widely used bis(2-ethylhexyl) phthalate (DEHP), in a one-pot cascade process. A PAE-degrading bacterium, Gordonia sp. strain 5F, was isolated from soil polluted with plastic waste. From this strain, a novel esterase (GoEst15) and a mono(2-ethylhexyl) phthalate hydrolase (GoEstM1) were identified by homology-based cloning. GoEst15 showed broad substrate specificity, hydrolyzing DEHP and 10 other PAEs to monoalkyl phthalates, which were further degraded by GoEstM1 to phthalic acid. GoEst15 and GoEstM1 were heterologously coexpressed in Escherichia coli BL21 (DE3), which could then completely degrade 12 PAEs (5 mM), within 1 and 24 h for small and bulky substrates, respectively. To our knowledge, GoEst15 is the first DEHP hydrolase with a known protein sequence, which will enable protein engineering to enhance its catalytic performance in the future.


Assuntos
Proteínas de Bactérias/química , Esterases/química , Ésteres/química , Gordonia (Bactéria)/enzimologia , Ácidos Ftálicos/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Biodegradação Ambiental , Dietilexilftalato/química , Dietilexilftalato/metabolismo , Esterases/genética , Esterases/metabolismo , Ésteres/metabolismo , Gordonia (Bactéria)/genética , Gordonia (Bactéria)/isolamento & purificação , Gordonia (Bactéria)/metabolismo , Hidrólise , Ácidos Ftálicos/metabolismo , Alinhamento de Sequência , Microbiologia do Solo
16.
Arch Virol ; 164(9): 2297-2307, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31267215

RESUMO

The influence of the high genetic variability of hepatitis B virus (HBV) on the sensitivity of serological assays has received little attention so far. A major source of variability is related to viral genotypes and subgenotypes. Their possible influence on diagnosis and prophylaxis is poorly known and has mostly been evaluated for genotypes A, B, C and D. Robust data showing the detection efficiency of HBsAg from genotype F is lacking. This study examined the effect of virus-like particles containing HBsAg from genotypes A and F (particularly, F1b and F4) produced in Pichia pastoris in relation to the anti-HBs antibodies used in the immunoassays for in vitro diagnosis and compared it with that exerted by the G145R S-escape mutant. The results showed that HBsAg detection rates for subgenotypes F1b and F4 differed significantly from those obtained for genotype A and that subgenotype F1b had a major impact on the sensitivity of the immunoassays tested. Prediction of the tertiary structure of subgenotypes F1b and F4 revealed changes inside and outside the major hydrophilic region (aa 101-160) of the HBsAg compared to genotype A and the G145R variant. A phosphorylation site (target for protein kinase C) produced by the G145R substitution might prevent recognition by anti-HBs antibodies. In conclusion, the use of different genotypes or variants for diagnosis could improve the rate of detection of HBV infection. The incorporation of a genotype-F-derived HBsAg vaccine in areas where this genotype is endemic should be evaluated, since this might also affect vaccination efficacy.


Assuntos
Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/isolamento & purificação , Hepatite B/virologia , Sequência de Aminoácidos , Hepatite B/diagnóstico , Antígenos de Superfície da Hepatite B/química , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/química , Vírus da Hepatite B/classificação , Vírus da Hepatite B/genética , Humanos , Dados de Sequência Molecular , Mutação , Filogenia , Conformação Proteica , Alinhamento de Sequência
17.
J Agric Food Chem ; 67(31): 8626-8631, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31287307

RESUMO

An almond allergen with two known short peptide sequences was reported as the almond 2S albumin but was later suspected to be almond vicilin. However, this allergen was not designated by the World Health Organization/International Union of Immunological Societies. This study aimed to determine the true identity of this elusive almond allergen. cDNAs were synthesized from total RNA of the Nonpareil almond. The complete sequence of the previously reported almond allergen was determined from its coding sequence. The deduced protein was produced recombinantly and was confirmed to be a food allergen by testing with 18 almond-allergic sera. The allergen is a potential cysteine-rich antimicrobial protein with characteristic C[X]3C-[X]10-12-C[X]3C motifs of the hairpinin antimicrobial protein. This first member of a novel family of food allergens was named Pru du 8. The signature motif of the hairpinin antimicrobial protein can be found in the N-terminal region of some vicilin allergens (e.g., Ara h 1). It can also be found in the signal peptide of other vicilin allergens (e.g., Car i 2). In many species, however, vicilins do not contain such a motif, indicating that the presence of the signature motifs of the hairpinin antimicrobial protein in vicilins might be a result of translocation during evolution.


Assuntos
Alérgenos/imunologia , Antígenos de Plantas/imunologia , Prunus dulcis/imunologia , Alérgenos/química , Alérgenos/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Antígenos de Plantas/química , Antígenos de Plantas/genética , DNA Complementar/genética , Hipersensibilidade Alimentar/imunologia , Humanos , Prunus dulcis/química , Prunus dulcis/genética , Proteínas de Armazenamento de Sementes/química , Proteínas de Armazenamento de Sementes/genética , Proteínas de Armazenamento de Sementes/imunologia , Alinhamento de Sequência , Análise de Sequência de DNA
18.
Plant Sci ; 286: 68-77, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31300143

RESUMO

Apple (Malus domestica) is an important fruit worldwide; however, the development of the apple industry is limited by fungal disease. Apple bitter rot caused by the pathogen Colletotrichum gloeosporioides is one of the most devastating apple diseases, leading to large-scale losses in apple quality and production. WRKY transcription factors have important functions in the regulation of biotic and abiotic stresses. However, their biological and molecular functions in non-model plants, including apple, remain poorly understood. Here, we isolated MdWRKY100 from 'Hanfu' apple. The MdWRKY100 protein fused to green fluorescent protein localized to the nucleus, and MdWRKY100 in yeast cells displayed transcriptional activation activity, which is consistent with the function of a transcription factor. Additionally, several putative cis-acting elements involved in abiotic stress responsiveness were also identified in the MdWRKY100 promoter. Transcriptional analysis revealed that MdWRKY100 was expressed ubiquitously in all examined apple organs. Overexpression in apple increased resistance to Colletotrichum gloeosporioides, while RNAi silencing transgenic plants were more sensitive to Colletotrichum gloeosporioides. Collectively, our data demonstrate that MdWRKY100 is a positive regulator of Colletotrichum gloeosporioides resistance in apple.


Assuntos
Colletotrichum/fisiologia , Resistência à Doença/genética , Malus/genética , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Malus/metabolismo , Malus/microbiologia , Filogenia , Doenças das Plantas/microbiologia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Alinhamento de Sequência , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
19.
Chem Commun (Camb) ; 55(61): 8935-8938, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31286126

RESUMO

The Eubacterium saburreum serine protease inhibitor from the human gut microbiota inhibits the eukaryotic pancreatic elastase associated with acute pancreatitis. Interestingly, the inhibition efficiency and stability are markedly increased by the para-sulphonato-calix[8]arene capped silver nanoparticles. Moreover, this enzyme is distinguishable by its high inhibitory effect at broad pH range between 2-10 and temperatures from 10 to 40 °C, in the presence of para-sulphonato-calix[8]arene capped silver nanoparticles the enzyme remains active even at 70 °C.


Assuntos
Calixarenos/química , Nanopartículas Metálicas/química , Elastase Pancreática/antagonistas & inibidores , Serpinas/química , Prata/química , Sequência de Aminoácidos , Animais , Ensaios Enzimáticos , Eubacterium/química , Concentração de Íons de Hidrogênio , Estabilidade Proteica , Alinhamento de Sequência , Serpinas/isolamento & purificação , Ácidos Sulfônicos/química , Suínos , Temperatura Ambiente
20.
J Agric Food Chem ; 67(33): 9307-9313, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31352784

RESUMO

Porphyra is one of the most consumed types of red algae. Porphyran is the major polysaccharide extracted from Porphyra, and it is composed of alternating 4-linked α-l-galactopyranose-6-sulfate (L6S) and 3-linked ß-d-galactopyranose (G) residues. ß-Porphyranases are promising tools for degrading porphyran; however, few enzymes have been reported, and the biochemical properties of porphyranases are still unclear. Here, a novel GH16 ß-porphyranase, designated as Por16A_Wf, was cloned from Wenyingzhuangia fucanilytica and expressed in Escherichia coli. Its biochemical properties and hydrolysis pattern were characterized. Por16A_Wf exhibited stable activity on a wide pH scale from 3.5 to 11.0. Glycomics analysis using LC-MS revealed that Por16A_Wf specifically hydrolyzed the glycosidic linkage of G-L6S, whereas it tolerated 3,6-anhydro-α-l-galactopyranose and methyl-d-galactose in -2 and +2 subsites, respectively. Por16A_Wf could be applied as a biotechnological tool for tailoring porphyran, which would serve in directional preparation of its disaccharide, producing products with various molecular weights and facilitating investigation of the structural heterogeneity of Porphyra polysaccharides.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Flavobacteriaceae/enzimologia , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Água do Mar/microbiologia , Sefarose/análogos & derivados , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Biocatálise , Biotecnologia , Clonagem Molecular , Estabilidade Enzimática , Flavobacteriaceae/classificação , Flavobacteriaceae/genética , Flavobacteriaceae/isolamento & purificação , Glicosídeo Hidrolases/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Peso Molecular , Filogenia , Porphyra/química , Porphyra/metabolismo , Sefarose/química , Sefarose/metabolismo , Alinhamento de Sequência
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