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1.
Int J Mol Sci ; 20(19)2019 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-31569557

RESUMO

As an important nongrain crop, the growth and yield of potato (Solanum tuberosum L.) is often affected by an unfavorable external environment in the process of cultivation. The MYB family is one of the largest and most important gene families, participating in the regulation of plant growth and development and response to abiotic stresses. Several MYB genes in potato that regulate anthocyanin synthesis and participate in abiotic stress responses have been identified. To identify all Solanum tuberosum L. MYB (StMYB) genes involved in hormone or stress responses to potentially regulate potato growth and development, we identified the MYB gene family at the genome-wide level. In this work, 158 StMYB genes were found in the potato genome. According to the amino acid sequence of the MYB domain and gene structure, the StMYB genes were divided into R2R3-MYB and R1R2R3-MYB families, and the R2R3-MYB family was divided into 20 subgroups (SGs). The expression of 21 StMYB genes from different SGs in roots, stems, leaves, flowers, shoots, stolons, young tubers, and mature tubers was determined by quantitative real-time polymerase chain reaction (qRT-PCR). The expression patterns of StMYB genes in potatoes treated with abscisic acid (ABA), indole-3-acetic acid (IAA), gibberellin acid 3 (GA3), NaCl, mannitol, and heat were also measured. We have identified several potential candidate genes that regulate the synthesis of potato flavonoids or participate in hormone or stress responses. This work provides a comprehensive understanding of the MYB family in potato and will lay a foundation for the future investigation of the potential functions of StMYB genes in the growth and development of potato.

2.
BMC Plant Biol ; 19(1): 342, 2019 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-31387526

RESUMO

BACKGROUND: GRAS are plant-specific transcription factors that play important roles in plant growth and development. Although the GRAS gene family has been studied in many plants, there has been little research on the GRAS genes of Tartary buckwheat (Fagopyrum tataricum), which is an important crop rich in rutin. The recently published whole genome sequence of Tartary buckwheat allows us to study the characteristics and expression patterns of the GRAS gene family in Tartary buckwheat at the genome-wide level. RESULTS: In this study, 47 GRAS genes of Tartary buckwheat were identified and divided into 10 subfamilies: LISCL, HAM, DELLA, SCR, PAT1, SCL4/7, LAS, SHR, SCL3, and DLT. FtGRAS genes were unevenly distributed on 8 chromosomes, and members of the same subfamily contained similar gene structures and motif compositions. Some FtGRAS genes may have been produced by gene duplications; tandem duplication contributed more to the expansion of the GRAS gene family in Tartary buckwheat. Real-time PCR showed that the transcription levels of FtGRAS were significantly different in different tissues and fruit development stages, implying that FtGRAS might have different functions. Furthermore, an increase in fruit weight was induced by exogenous paclobutrazol, and the transcription level of the DELLA subfamily member FtGRAS22 was significantly upregulated during the whole fruit development stage. Therefore, FtGRAS22 may be a potential target for molecular breeding or genetic editing. CONCLUSIONS: Collectively, this systematic analysis lays a foundation for further study of the functional characteristics of GRAS genes and for the improvement of Tartary buckwheat crops.


Assuntos
Fagopyrum/genética , Proteínas de Plantas/fisiologia , Fatores de Transcrição/fisiologia , Fagopyrum/crescimento & desenvolvimento , Fagopyrum/metabolismo , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Genoma de Planta , Família Multigênica , Filogenia , Desenvolvimento Vegetal/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sintenia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Triazóis/farmacologia
3.
BMC Plant Biol ; 19(1): 344, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31390980

RESUMO

BACKGROUND: In the study, the trihelix family, also referred to as GT factors, is one of the transcription factor families. Trihelix genes play roles in the light response, seed maturation, leaf development, abiotic and biological stress and other biological activities. However, the trihelix family in tartary buckwheat (Fagopyrum tataricum), an important usable medicinal crop, has not yet been thoroughly studied. The genome of tartary buckwheat has recently been reported and provides a theoretical basis for our research on the characteristics and expression of trihelix genes in tartary buckwheat based at the whole level. RESULTS: In the present study, a total of 31 FtTH genes were identified based on the buckwheat genome. They were named from FtTH1 to FtTH31 and grouped into 5 groups (GT-1, GT-2, SH4, GTγ and SIP1). FtTH genes are not evenly distributed on the chromosomes, and we found segmental duplication events of FtTH genes on tartary buckwheat chromosomes. According to the results of gene and motif composition, FtTH located in the same group contained analogous intron/exon organizations and motif organizations. qRT-PCR showed that FtTH family members have multiple expression patterns in stems, roots, leaves, fruits, and flowers and during fruit development. CONCLUSIONS: Through our study, we identified 31 FtTH genes in tartary buckwheat and synthetically further analyzed the evolution and expression pattern of FtTH proteins. The structure and motif organizations of most genes are conserved in each subfamily, suggesting that they may be functionally conserved. The FtTH characteristics of the gene expression patterns indicate functional diversity in the time and space in the tartary buckwheat life process. Based on the discussion and analysis of FtTH gene function, we screened some genes closely related to the growth and development of tartary buckwheat. This will help us to further study the function of FtTH genes through experimental exploration in tartary buckwheat growth and improve the fruit of tartary buckwheat.


Assuntos
Fagopyrum/genética , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Mapeamento Cromossômico , Evolução Molecular , Fagopyrum/metabolismo , Duplicação Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Genoma de Planta , Filogenia , Proteínas de Plantas/genética , Fatores de Transcrição/genética
4.
BMC Plant Biol ; 19(1): 299, 2019 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-31286919

RESUMO

BACKGROUND: SPL (SQUAMOSA promoter binding protein-like) is a class of plant-specific transcription factors that play important roles in many growth and developmental processes, including shoot and inflorescence branching, embryonic development, signal transduction, leaf initiation, phase transition, and flower and fruit development. The SPL gene family has been identified and characterized in many species but has not been well studied in tartary buckwheat, which is an important edible and medicinal crop. RESULTS: In this study, 24 Fagopyrum tataricum SPL (FtSPL) genes were identified and renamed according to the chromosomal distribution of the FtSPL genes. According to the amino acid sequence of the SBP domain and gene structure, the SPL genes were divided into eight groups (group I to group VII) by phylogenetic tree analysis. A total of 10 motifs were detected in the tartary buckwheat SPL genes. The expression patterns of 23 SPL genes in different tissues and fruits at different developmental stages (green fruit stage, discoloration stage and initial maturity stage) were determined by quantitative real-time polymerase chain reaction (qRT-PCR). CONCLUSIONS: The tartary buckwheat genome contained 24 SPL genes, and most of the genes were expressed in different tissues. qRT-PCR showed that FtSPLs played important roles in the growth and development of tartary buckwheat, and genes that might regulate flower and fruit development were preliminarily identified. This work provides a comprehensive understanding of the SBP-box gene family in tartary buckwheat and lays a significant foundation for further studies on the functional characteristics of FtSPL genes and improvement of tartary buckwheat crops.


Assuntos
Fagopyrum/genética , Estudo de Associação Genômica Ampla , Família Multigênica , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Fagopyrum/crescimento & desenvolvimento , Fagopyrum/metabolismo , Frutas/genética , Frutas/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Filogenia , Proteínas de Plantas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição/metabolismo
5.
BMC Genomics ; 20(1): 483, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31185893

RESUMO

BACKGROUND: In reported plants, the bZIP family is one of the largest transcription factor families. bZIP genes play roles in the light signal, seed maturation, flower development, cell elongation, seed accumulation protein, abiotic and biological stress and other biological processes. While, no detailed identification and genome-wide analysis of bZIP family genes in Fagopyum talaricum (tartary buckwheat) has previously been published. The recently reported genome sequence of tartary buckwheat provides theoretical basis for us to study and discuss the characteristics and expression of bZIP genes in tartary buckwheat based on the whole genome. RESULTS: In this study, 96 FtbZIP genes named from FtbZIP1 to FtbZIP96 were identified and divided into 11 subfamilies according to their genetic relationship with 70 bZIPs of A. thaliana. FtbZIP genes are not evenly distributed on the chromosomes, and we found tandem and segmental duplication events of FtbZIP genes on 8 tartary buckwheat chromosomes. According to the results of gene and motif composition, FtbZIP located in the same group contained analogous intron/exon organizations and motif composition. By qRT-PCR, we quantified the expression of FtbZIP members in stem, root, leaf, fruit, and flower and during fruit development. Exogenous ABA treatment increased the weight of tartary buckwheat fruit and changed the expressions of FtbZIP genes in group A. CONCLUSIONS: Through our study, we identified 96 FtbZIP genes in tartary buckwheat and synthetically further analyzed the structure composition, evolution analysis and expression pattern of FtbZIP proteins. The expression pattern indicates that FtbZIP is important in the course of plant growth and development of tartary buckwheat. Through comprehensively analyzing fruit weight and FtbZIP genes expression after ABA treatment and endogenous ABA content of tartary buckwheat fruit, ABA may regulate downstream gene expression by regulating the expression of FtPinG0003523300.01 and FtPinG0003196200.01, thus indirectly affecting the fruit development of tartary buckwheat. This will help us to further study the function of FtbZIP genes in the tartary buckwheat growth and improve the fruit of tartary buckwheat.

6.
BMC Plant Biol ; 19(1): 248, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31185913

RESUMO

BACKGROUND: ZF-HD is a family of genes that play an important role in plant growth, development, some studies have found that after overexpression AtZHD1 in Arabidopsis thaliana, florescence advance, the seeds get bigger and the life span of seeds is prolonged, moreover, ZF-HD genes are also participate in responding to adversity stress. The whole genome of the ZF-HD gene family has been studied in several model plants, such as Arabidopsis thaliana and rice. However, there has been little research on the ZF-HD genes in Tartary buckwheat (Fagopyrum tataricum), which is an important edible and medicinal crop. The recently published whole genome sequence of Tartary buckwheat allows us to study the tissue and expression profiles of the ZF-HD gene family in Tartary buckwheat on a genome-wide basis. RESULTS: In this study, the whole genome and expression profile of the ZF-HD gene family were analyzed for the first time in Tartary buckwheat. We identified 20 FtZF-HD genes and divided them into MIF and ZHD subfamilies according to phylogeny. The ZHD genes were divided into 5 subfamilies. Twenty FtZF-HD genes were distributed on 7 chromosomes, and almost all the genes had no introns. We detected seven pairs of chromosomes with fragment repeats, but no tandem repeats were detected. In different tissues and at different fruit development stages, the FtZF-HD genes obtained by a real-time quantitative PCR analysis showed obvious expression patterns. CONCLUSIONS: In this study, 20 FtZF-HD genes were identified in Tartary buckwheat, and the structures, evolution and expression patterns of the proteins were studied. Our findings provide a valuable basis for further analysis of the biological function of the ZF-HD gene family. Our study also laid a foundation for the improvement of Tartary buckwheat crops.


Assuntos
Fagopyrum/genética , Regulação da Expressão Gênica de Plantas/genética , Genes de Plantas/genética , Família Multigênica , Proteínas de Plantas/genética , Fagopyrum/metabolismo , Perfilação da Expressão Gênica , Filogenia , Proteínas de Plantas/metabolismo
7.
BMC Plant Biol ; 19(1): 84, 2019 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-30786863

RESUMO

BACKGROUND: AP2/ERF transcription factors perform indispensable functions in various biological processes, such as plant growth, development, biotic and abiotic stresses responses. The AP2/ERF transcription factor family has been identified in many plants, and several AP2/ERF transcription factors from Arabidopsis thaliana (A. thaliana) have been functionally characterized. However, little research has been conducted on the AP2/ERF genes of tartary buckwheat (Fagopyum tataricum), which is an important edible and medicinal crop. The recently published whole genome sequence of tartary buckwheat allowed us to study the tissue and expression profiles of AP2/ERF genes in tartary buckwheat on a genome-wide basis. RESULTS: In this study, 134 AP2/ERF genes of tartary buckwheat (FtAP2/ERF) were identified and renamed according to the chromosomal distribution of the FtAP2/ERF genes. According to the number conserved domains and gene structure, the AP2/ERF genes were divided into three subfamilies by phylogenetic tree analysis, namely, AP2 (15 members), ERF (116 members) and RAV (3 members). A total of 10 motifs were detected in tartary buckwheat AP2/ERF genes, and some of the unique motifs were found to be important for the function of AP2/ERF genes. CONCLUSION: A comprehensive analysis of AP2/ERF gene expression patterns in different tissues and fruit development stages by quantitative real-time PCR (qRT-PCR) showed that they played an important role in the growth and development of tartary buckwheat, and genes that might regulate flower and fruit development were preliminarily identified. This systematic analysis establishes a foundation for further studies of the functional characteristics of FtAP2/ERF genes and improvement of tartary buckwheat crops.


Assuntos
Fagopyrum/genética , Genes de Plantas/genética , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Mapeamento Cromossômico , Regulação da Expressão Gênica de Plantas , Genes de Plantas/fisiologia , Genoma de Planta/genética , Filogenia , Proteínas de Plantas/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Alinhamento de Sequência , Fatores de Transcrição/fisiologia
8.
BMC Genomics ; 20(1): 113, 2019 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-30727951

RESUMO

BACKGROUND: The NAC (NAM, ATAF1/2, and CUC2) transcription factor family represents a group of large plant-specific transcriptional regulators, participating in plant development and response to external stress. However, there is no comprehensive study on the NAC genes of Tartary buckwheat (Fagopyrum tataricum), a large group of extensively cultivated medicinal and edible plants. The recently published Tartary buckwheat genome permits us to explore all the FtNAC genes on a genome-wide basis. RESULTS: In the present study, 80 NAC (FtNAC) genes of Tartary buckwheat were obtained and named uniformly according to their distribution on chromosomes. Phylogenetic analysis of NAC proteins in both Tartary buckwheat and Arabidopsis showed that the FtNAC proteins are widely distributed in 15 subgroups with one subgroup unclassified. Gene structure analysis found that multitudinous FtNAC genes contained three exons, indicating that the structural diversity in Tartary buckwheat NAC genes is relatively low. Some duplication genes of FtNAC have a conserved structure that was different from others, indicating that these genes may have a variety of functions. By observing gene expression, we found that FtNAC genes showed abundant differences in expression levels in various tissues and at different stages of fruit development. CONCLUSIONS: In this research, 80 NAC genes were identified in Tartary buckwheat, and their phylogenetic relationships, gene structures, duplication, global expression and potential roles in Tartary buckwheat development were studied. Comprehensive analysis will be useful for a follow-up study of functional characteristics of FtNAC genes and for the development of high-quality Tartary buckwheat varieties.


Assuntos
Evolução Molecular , Fagopyrum/metabolismo , Regulação da Expressão Gênica de Plantas , Família Multigênica/genética , Análise de Sequência de DNA , Fatores de Transcrição/genética , Fagopyrum/genética , Fagopyrum/fisiologia , Perfilação da Expressão Gênica , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/fisiologia , Análise de Sequência de RNA , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia
9.
Planta ; 249(5): 1301-1318, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30617544

RESUMO

MAIN CONCLUSION: Genome-wide identification, expression analysis and potential functional characterization of previously uncharacterized MADS family of tartary buckwheat, emphasized the importance of this gene family in plant growth and development. The MADS transcription factor is a key regulatory factor in the development of most plants. The MADS gene in plants controls all aspects of tissue and organ growth and reproduction and can be used to regulate plant seed cracking. However, there has been little research on the MADS genes of tartary buckwheat (Fagopyrum tataricum), which is an important edible and medicinal crop. The recently published whole genome sequence of tartary buckwheat allows us to study the tissue and expression profiles of the MADS gene in tartary buckwheat at a genome-wide level. In this study, 65 MADS genes of tartary buckwheat were identified and renamed according to the chromosomal distribution of the FtMADS genes. Here, we provide a complete overview of the gene structure, gene expression, genomic mapping, protein motif organization, and phylogenetic relationships of each member of the gene family. According to the phylogenetic relationship of MADS genes, the transcription factor family was divided into two subfamilies, the M subfamily (28 genes) and the MIKC subfamily (37 genes). The results showed that the FtMADS genes belonged to related sister pairs and the chromosomal map showed that the replication of FtMADSs was related to the replication of chromosome blocks. In different tissues and at different fruit development stages, the FtMADS genes obtained by real-time quantitative PCR (RT-qPCR) showed obvious expression patterns. A comprehensive analysis of the MADS genes in tartary buckwheat was conducted. Through systematic analysis, the potential genes that may regulate the growth and development of tartary buckwheat and the genes that may regulate the easy dehulling of tartary buckwheat fruit were screened, which laid a solid foundation for improving the quality of tartary buckwheat.


Assuntos
Fagopyrum/metabolismo , Frutas/metabolismo , Proteínas de Plantas/metabolismo , Fagopyrum/genética , Frutas/genética , Regulação da Expressão Gênica de Plantas , Filogenia , Proteínas de Plantas/classificação , Proteínas de Plantas/genética
10.
Int J Mol Sci ; 19(11)2018 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-30423920

RESUMO

Auxin signaling plays an important role in plant growth and development. It responds to various developmental and environmental events, such as embryogenesis, organogenesis, shoot elongation, tropical growth, lateral root formation, flower and fruit development, tissue and organ architecture, and vascular differentiation. However, there has been little research on the Auxin Response Factor (ARF) genes of tartary buckwheat (Fagopyrum tataricum), an important edible and medicinal crop. The recent publication of the whole-genome sequence of tartary buckwheat enables us to study the tissue and expression profile of the FtARF gene on a genome-wide basis. In this study, 20 ARF (FtARF) genes were identified and renamed according to the chromosomal distribution of the FtARF genes. The results showed that the FtARF genes belonged to the related sister pair, and the chromosomal map showed that the duplication of FtARFs was related to the duplication of the chromosome blocks. The duplication of some FtARF genes shows conserved intron/exon structure, which is different from other genes, suggesting that the function of these genes may be diverse. Real-time quantitative PCR analysis exhibited distinct expression patterns of FtARF genes in various tissues and in response to exogenous auxin during fruit development. In this study, 20 FtARF genes were identified, and the structure, evolution, and expression patterns of the proteins were studied. This systematic analysis laid a foundation for the further study of the functional characteristics of the ARF genes and for the improvement of tartary buckwheat crops.

11.
BMC Genomics ; 19(1): 648, 2018 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-30170551

RESUMO

BACKGROUND: Tartary buckwheat (Fagopyrum tataricum Gaertn.) is a widely cultivated medicinal and edible crop with excellent economic and nutritional value. The development of tartary buckwheat seeds is a very complex process involving many expression-dependent physiological changes and regulation of a large number of genes and phytohormones. In recent years, the gene regulatory network governing the physiological changes occurring during seed development have received little attention. RESULTS: Here, we characterized the seed development of tartary buckwheat using light and electron microscopy and measured phytohormone and nutrient accumulation by using high performance liquid chromatography (HPLC) and by profiling the expression of key genes using RNA sequencing with the support of the tartary buckwheat genome. We first divided the development of tartary buckwheat seed into five stages that include complex changes in development, morphology, physiology and phytohormone levels. At the same time, the contents of phytohormones (gibberellin, indole-3-acetic acid, abscisic acid, and zeatin) and nutrients (rutin, starch, total proteins and soluble sugars) at five stages were determined, and their accumulation patterns in the development of tartary buckwheat seeds were analyzed. Second, gene expression patterns of tartary buckwheat samples were compared during three seed developmental stages (13, 19, and 25 days postanthesis, DPA), and 9 765 differentially expressed genes (DEGs) were identified. We analyzed the overlapping DEGs in different sample combinations and measured 665 DEGs in the three samples. Furthermore, expression patterns of DEGs related to phytohormones, flavonoids, starch, and storage proteins were analyzed. Third, we noted the correlation between the trait (physiological changes, nutrient changes) and metabolites during seed development, and discussed the key genes that might be involved in the synthesis and degradation of each of them. CONCLUSION: We provided abundant genomic resources for tartary buckwheat and Polygonaceae communities and revealed novel molecular insights into the correlations between the physiological changes and seed development of tartary buckwheat.

12.
Int J Mol Sci ; 19(9)2018 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-30217096

RESUMO

Tartary buckwheat is a type of cultivated medicinal and edible crop with good economic and nutritional value. Knowledge of the final fruit size of buckwheat is critical to its yield increase. In this study, the fruit development of two species of Tartary buckwheat in the Polygonaceae was analyzed. During fruit development, the size/weight, the contents of auxin (AUX)/abscisic acid (ABA), the number of cells, and the changes of embryo were measured and observed; and the two fruit materials were compared to determine the related mechanisms that affected fruit size and the potential factors that regulated the final fruit size. The early events during embryogenesis greatly influenced the final fruit size, and the difference in fruit growth was primarily due to the difference in the number of cells, implicating the effect of cell division rate. Based on our observations and recent reports, the balance of AUX and ABA might be the key factor that regulated the cell division rate. They induced the response of auxin response factor 2 (FtARF2) and downstream small auxin upstream RNA (FtSAURs) through hormone signaling pathway to regulate the fruit size of Tartary buckwheat. Further, through the induction of fruit expansion by exogenous auxin, FtARF2b was significantly downregulated. The FtARF2b is a potential target for molecular breeding or gene editing.

13.
Int J Biol Macromol ; 115: 1079-1087, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29709539

RESUMO

Cellulose is the most abundant and renewable biological resource on earth. As nonrenewable resources are becoming scarce, cellulose is expected to become a major raw material for food, energy, fuel and other products. 1,4-ß-glucosidase (Bgl), as a kind of cellulose, can be degraded cellulose into industrial available glucose. In this study, we constructed mutants of Bgl with enhanced activity based on homology modeling, molecular docking, and the site-directed mutagenesis of target residues to modify spatial positions, steric hindrances, or hydrophilicity/hydrophobicity. On the basis of the high-activity mutations were got (N347S and G235 M) by using site-directed mutagenesis and screening methods and introduced in the Pichia pastoris expression system, the enzymatic properties of mutant enzymes were analysed. Assays of the activity of the purified Bgl revealed that the two mutants exhibited increased activity. The pPICZαA-G235 M and pPICZαA-N347S mutants exhibited a >33.4% and 44.8% increase in specific activity respectively, with similar pH, temperature and metal ion requirements, compared to wild-type Bgl. These findings would be good foundation for improving production properties of Bgl in the future.


Assuntos
Celulase/genética , Celulase/metabolismo , Simulação de Acoplamento Molecular , Pichia/genética , Homologia de Sequência de Aminoácidos , Domínio Catalítico , Celulase/química , Escherichia coli/enzimologia , Escherichia coli/genética , Expressão Gênica , Plasmídeos/genética , Ligação Proteica , Especificidade por Substrato
14.
Mol Biol Rep ; 41(11): 7567-73, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25123895

RESUMO

A ß-glucosidase gene (bgl) from Aspergillus oryzae GIF-10 was cloned, sequenced and expressed. Its full-length DNA sequence was 2,903 bp and included three introns. The full-length cDNA sequence contained an open reading frame of 2,586 nucleotides, encoding 862 amino acids with a potential secretion signal. The A. oryzae GIF-10 bgl was functionally expressed in Pichia pastoris. After 7-day induction, protein yield reached 321 mg/mL. Using salicin as the substrate, the specific activity of the purified enzyme reached 215 U/mg. The purified recombinant ß-glucosidase was a 110-kDa glycoprotein with optimum catalytic activity at pH 5.0 and 50 °C. The enzyme was stable between 20 and 60 °C, and retained 65% of its activity after being held at 60 °C for 30 min. The recombinant ß-glucosidase was relatively stable in a broad range of pHs, from 4.0 to 6.5. It showed broad specific activity, hydrolyzing a range of (1-4)-ß-diglycosides and (1-4)-α-diglycosides, and Mn(2+) stimulated its activity significantly.


Assuntos
Aspergillus oryzae/enzimologia , beta-Glucosidase/genética , beta-Glucosidase/metabolismo , Sequência de Aminoácidos , Aspergillus oryzae/genética , Sequência de Bases , Álcoois Benzílicos/metabolismo , Clonagem Molecular , Biologia Computacional , Primers do DNA/genética , DNA Complementar/genética , Eletroforese em Gel de Poliacrilamida , Glucosídeos/metabolismo , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Pichia , Plasmídeos/genética , Análise de Sequência de DNA , Especificidade por Substrato , Temperatura Ambiente
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