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1.
Zhongguo Zhong Yao Za Zhi ; 44(22): 4830-4836, 2019 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-31872589

RESUMO

GRAS transcription factors play important roles in the regulation of plant root growth and GA signaling. In this study,SmGRAS3 gene was cloned,which open reading frame was 2 247 bp,and encoding 748 amino acids. The physicochemical properties and structure of SmGRAS3 and its encoded protein were analyzed by bioinformatics software. This gene belongs to the SCL9 subfamily of the GRAS family,and its promoter sequence mainly contains the light response,stress response,and hormone response elements. It may interact with the GA signal pathway and anti-stress related proteins. The subcellular localization showed that SmGRAS3 protein was mainly located in the nucleus. The expression pattern analysis showed that the expression of Sm GRAS3 was the highest in the root and the lowest in the stem,and both light and low temperature could induce the high expression level of SmGRAS3. This study provides a foundation for further study on the roles of SmGRAS3 gene in the root growth and stress tolerance of Salvia miltiorrhiza.


Assuntos
Salvia miltiorrhiza/genética , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Filogenia , Proteínas de Plantas , Fatores de Transcrição
2.
Yi Chuan ; 41(12): 1129-1137, 2019 Dec 20.
Artigo em Chinês | MEDLINE | ID: mdl-31857284

RESUMO

Insulin-degrading enzyme (IDE) is a highly conserved metallopeptidase that functions in the catabolism of bioactive peptides. In our previous study, we identified a putative circular transcript in that chicken insulin-degrading enzyme (IDE) gene through analyzing a high throughput sequencing result. Here we set to confirm the circular transcript of IDE (circIDE) and explore its expression regularity in normal barred Plymouth chicken. The circIDE was confirmed by PCR amplification and sequencing. The circular structure of circIDE was determined by RNase R processing and reverse transcription experiments. Then we analyzed the spatiotemporal expression pattern of circIDE and IDE mRNA and compared the differential expression of circIDE and IDE mRNA in the normal barred Plymouth chicken and the dwarf ones. The results showed that the full length of chicken circIDE was 1332 nt, divided form exon 2-11 of the IDE gene. RNase R tolerance analysis showed that chicken circIDE had the general characteristics of circular molecule, and was highly resistant to RNase R. The random primers had higher transcription efficiency than the oligo-d(T)18 primers, confirming that circIDE is a circular structured molecule without poly(A). circIDE was highly expressed in the liver and heart tissues but less in the muscle tissues of leg and breast in normal chickens at the age of 1 and 12 weeks. The expression profile of circIDE in liver tissue showed that circIDE level was lower in1 to 6 weeks and then became higher after 8 weeks of age. The expression of circIDE in liver tissue was significantly higher in normal chicken than that in dwarf barred Plymouth chicken (P<0.05). This study confirmed a circIDE strucutre in chicken IDE gene and uncovered its expression regularity. We demonstrated that the expression level of circIDE in the liver tissue was higher in normal barred Plymouth chicken compared to dwarf species. This study paves the way for further understanding the biological function of chicken circIDE, including its roles in regulating chicken growth and development.


Assuntos
Galinhas , Clonagem Molecular , Insulisina , Animais , Expressão Gênica , Perfilação da Expressão Gênica , Insulisina/genética , Fígado/metabolismo , RNA Mensageiro/genética
3.
Pol J Microbiol ; 68(4): 559-563, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31880899

RESUMO

We demonstrate here for the first time the use of an IncP-1ß plasmid, R751, as a gene capture vehicle for recombineering/conjugation strategies to clone large segments of bacterial genomes (20 - 100 + Kb). We designed R751 derivatives containing alternative markers for greater flexibility when using the R751 vehicle across different bacteria. These markers are removable if desired as part of the cloning procedure (with no extra steps needed). We demonstrated utility via cloning of 38 and 22 kb genomic segments from Salmonella enterica serovar Typhimurium and Escherichia coli, respectively. The plasmids expand the options available for use in recombineering/conjugation-based cloning applications.We demonstrate here for the first time the use of an IncP-1ß plasmid, R751, as a gene capture vehicle for recombineering/conjugation strategies to clone large segments of bacterial genomes (20 ­ 100 + Kb). We designed R751 derivatives containing alternative markers for greater flexibility when using the R751 vehicle across different bacteria. These markers are removable if desired as part of the cloning procedure (with no extra steps needed). We demonstrated utility via cloning of 38 and 22 kb genomic segments from Salmonella enterica serovar Typhimurium and Escherichia coli, respectively. The plasmids expand the options available for use in recombineering/conjugation-based cloning applications.


Assuntos
Clonagem Molecular , Conjugação Genética , Escherichia coli/genética , Plasmídeos/genética , Salmonella typhimurium/genética , DNA Bacteriano/genética , Recombinação Genética
4.
Zhongguo Zhong Yao Za Zhi ; 44(14): 3015-3021, 2019 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-31602848

RESUMO

Three Chrysanthemum-chalcone-isomerase genes( CmCHI) were successfully cloned by PCR from the database of Chrysanthemum transcriptome and named CmCHI1,CmCHI2 and CmCHI3,respectively. Bioinformatics analysis showed that the base numbers of CmCHI1-3 open reading frame were 708,633 and 681 bp,encoding 235,210 and 226 amino acids,respectively. Three fusion proteins of about 30 kDa were successfully induced by prokaryotic expression technology,and the corresponding recombinant fusion proteins were isolated and purified by Ni-NTA resin column. Clustering analysis showed that the 3 CmCHI were homologous with Compositae plants,and CmCHI1 and CmCHI3 belonged to type Ⅰ CHI. CmCHI2 belongs to type Ⅳ CHI. Using ß-actin as an internal reference gene,RT-qPCR was used to detect and analyze the expression of CmCHI1-3 genes in Hangju. The results showed that the expression levels of CmCHI1 and CmCHI3 were higher,while the expression levels of CmCHI2 were lower. It was concluded that CmCHI1 and CmCHI3 were the main chalcone isomerase genes involved in the synthesis of flavonoids in Hangju,and CmCHI2 was a helper gene. Flooding treatment significantly promoted the expression of CmCHI1 and CmCHI3 genes,but had no regulatory effect on CmCHI2. The above results provided a basis for further study of the molecular regulation mechanism of CHI gene in the metabolism of flavonoids in Hangju,which laid a foundation for improving the content of flavonoids in Hangju and finally improving the medicinal quality of Hangju.


Assuntos
Chrysanthemum/genética , Liases Intramoleculares/genética , Proteínas de Plantas/genética , Chrysanthemum/enzimologia , Clonagem Molecular
5.
Zhongguo Zhong Yao Za Zhi ; 44(15): 3253-3260, 2019 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-31602880

RESUMO

Flavonoids are a group of secondary metabolites found in plants. They have many pharmacological functions and play an important role in Chinese sumac( Rhus chinensis),which is a well-known traditional Chinese medicinal plant. Chalcone isomerase( CHI,EC 5. 5. 1. 6) is one of the key enzymes in the flavonoids biosynthesis pathway. In this paper,the full-length c DNA sequence encoding the chalcone isomerase from R. chinensis( designated as Rc CHI) was cloned by RT-PCR and rapid-amplification of c DNA Ends( RACE). The Rc CHI c DNA sequence was 1 058 bp and the open reading frame( ORF) was 738 bp. The ORF predicted to encode a 245-amino acid polypeptide. Rc CHI gene contained an intron and two exons. The sequence alignments revealed Rc CHI shared47. 1%-71. 6% identity with the homologues in other plants. Real-time PCR analysis showed that the total flavonoid levels were positively correlated with tissue-specific expressions of Rc CHI mRNA in different tissues. The recombinant protein was successfully expressed in an Escherichia coli strain with the p GEX-6 P-1 vector. In this paper,the CHI gene was cloned and characterized in the family of Anacardiaceae and will help us to obtain better knowledge of the flavonoids biosynthesis of the flavonoid compounds in R. chinensis.


Assuntos
Flavonoides/biossíntese , Liases Intramoleculares/genética , Rhus/enzimologia , Clonagem Molecular , DNA Complementar , Plantas Medicinais/enzimologia , Plantas Medicinais/genética , Rhus/genética
6.
Zhongguo Zhong Yao Za Zhi ; 44(16): 3588-3593, 2019 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-31602927

RESUMO

Tripterygium wilfordii is a medicinal plant commonly used in the treatment of rheumatoid arthritis,and with pharmacological activities in anti-tumor and obesity treatment. The known active ingredients in T. wilfordii are mainly terpenoids,but with very low content. Therefore,the analysis of the biosynthesis pathway of terpenoids in T. wilfordii has become a research hotspot to solve the problem of its resources. Terpenoid synthase( TPS) is a key enzyme that catalyzes the formation of a wide variety of terpenoid skeletons. In this study,a gene fragment with an ORF of 1 785 bp was cloned from T. wilfordii. Bioinformatics analysis was performed using NCBI's BLASTP,ProtParam and Interpro online tools and MEGA 6.0 software. The response of this gene to methyl jasmonate was also detected by real-time fluorescent quantitative PCR,and its catalytic function was verified by prokaryotic expression and in vitro enzymatic assay. Bioinformatics analysis indicated that the amino acid sequence encoded by this gene had both N-terminal domain and C-terminal domain of TPS,as well as the DDxx D conserved domain of the class I of TPS family. And Tw MTS gathered together with TPS-b subfamily in the Neighbor-Joining Tree constructed with known homologous TPSs. The results of RT-PCR showed that 50 µmol·L-1 MeJA 12 h could increase the expression of Tw MTS to 735 times in the control group at 12 h,and 1 644 times at 24 h. In addition,in vitro enzymatic reaction results showed that Tw MTS can catalyze the production of ß-citronellol with GPP as substrate,indicating that Tw MTS was a monoterpene synthase. The above results provided a new element for the synthetic biology database of T. wilfordii terpenoids,and laid the foundation for future biosynthesis research.


Assuntos
Liases Intramoleculares/genética , Proteínas de Plantas/genética , Tripterygium/genética , Clonagem Molecular , Tripterygium/enzimologia
7.
Nucleic Acids Res ; 47(16): 8874-8887, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31616952

RESUMO

Localized arrays of proteins cooperatively assemble onto chromosomes to control DNA activity in many contexts. Binding cooperativity is often mediated by specific protein-protein interactions, but cooperativity through DNA structure is becoming increasingly recognized as an additional mechanism. During the site-specific DNA recombination reaction that excises phage λ from the chromosome, the bacterial DNA architectural protein Fis recruits multiple λ-encoded Xis proteins to the attR recombination site. Here, we report X-ray crystal structures of DNA complexes containing Fis + Xis, which show little, if any, contacts between the two proteins. Comparisons with structures of DNA complexes containing only Fis or Xis, together with mutant protein and DNA binding studies, support a mechanism for cooperative protein binding solely by DNA allostery. Fis binding both molds the minor groove to potentiate insertion of the Xis ß-hairpin wing motif and bends the DNA to facilitate Xis-DNA contacts within the major groove. The Fis-structured minor groove shape that is optimized for Xis binding requires a precisely positioned pyrimidine-purine base-pair step, whose location has been shown to modulate minor groove widths in Fis-bound complexes to different DNA targets.


Assuntos
Bacteriófago lambda/genética , Cromossomos Bacterianos/química , DNA Nucleotidiltransferases/química , DNA Bacteriano/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Fator Proteico para Inversão de Estimulação/química , Proteínas Virais/química , Sítio Alostérico , Bacteriófago lambda/metabolismo , Sequência de Bases , Sítios de Ligação , Cromossomos Bacterianos/metabolismo , Clonagem Molecular , Cristalografia por Raios X , DNA Nucleotidiltransferases/genética , DNA Nucleotidiltransferases/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fator Proteico para Inversão de Estimulação/genética , Fator Proteico para Inversão de Estimulação/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reparo de DNA por Recombinação , Alinhamento de Sequência , Termodinâmica , Proteínas Virais/genética , Proteínas Virais/metabolismo
8.
Nat Biotechnol ; 37(9): 1041-1048, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31477922

RESUMO

Cytosine or adenine base editors (CBEs or ABEs) can introduce specific DNA C-to-T or A-to-G alterations1-4. However, we recently demonstrated that they can also induce transcriptome-wide guide-RNA-independent editing of RNA bases5, and created selective curbing of unwanted RNA editing (SECURE)-BE3 variants that have reduced unwanted RNA-editing activity5. Here we describe structure-guided engineering of SECURE-ABE variants with reduced off-target RNA-editing activity and comparable on-target DNA-editing activity that are also among the smallest Streptococcus pyogenes Cas9 base editors described to date. We also tested CBEs with cytidine deaminases other than APOBEC1 and found that the human APOBEC3A-based CBE induces substantial editing of RNA bases, whereas an enhanced APOBEC3A-based CBE6, human activation-induced cytidine deaminase-based CBE7, and the Petromyzon marinus cytidine deaminase-based CBE Target-AID4 induce less editing of RNA. Finally, we found that CBEs and ABEs that exhibit RNA off-target editing activity can also self-edit their own transcripts, thereby leading to heterogeneity in base-editor coding sequences.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Desaminases APOBEC/genética , Desaminases APOBEC/metabolismo , Animais , Clonagem Molecular , Citometria de Fluxo , Regulação Enzimológica da Expressão Gênica , Marcação de Genes , Células HEK293 , Humanos , Petromyzon , Conformação Proteica , RNA , RNA Guia/genética , Streptococcus pyogenes , Transcriptoma
9.
J Agric Food Chem ; 67(40): 11035-11043, 2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31517486

RESUMO

Ca2+-binding proteins (CaBPs) are widely distributed as Ca2+ sensor relay proteins that regulate various cellular processes, including Ca2+ homeostasis. Diamide insecticides such as cyantraniliprole kill insects by disrupting the Ca2+ homeostasis in muscle cells. However, less attention has been paid to the roles of CaBPs in response to insecticides. In this study, two CaBP genes (BtCaBP1 and BtCaBP2) were identified in the whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), and their functions in response to cyantraniliprole were investigated. After expression of BtCaBP1 and BtCaBP2 in vitro, the results of Ca2+ imaging and cytotoxicity assay revealed that the overexpression of each of the BtCaBPs stabilized Ca2+ concentration in the cytoplasm after exposure to cyantraniliprole and decreased the toxicity of cyantraniliprole against Sf9 cells. However, the knockdown of BtCaBP1 or BtCaBP2 in vivo significantly increased the toxicity of cyantraniliprole to B. tabaci. Taken together, these results provide evidence that BtCaBP1 and BtCaBP2 play a role in response to cyantraniliprole exposure through stabilization of Ca2+ concentration in whiteflies.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Clonagem Molecular , Hemípteros/efeitos dos fármacos , Hemípteros/genética , Proteínas de Insetos/metabolismo , Inseticidas/farmacologia , Pirazóis/farmacologia , ortoaminobenzoatos/farmacologia , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Hemípteros/classificação , Hemípteros/metabolismo , Proteínas de Insetos/genética , Filogenia
10.
Sheng Wu Gong Cheng Xue Bao ; 35(9): 1771-1786, 2019 Sep 25.
Artigo em Chinês | MEDLINE | ID: mdl-31559758

RESUMO

Rv2742 is a novel gene identified from Mycobacterium tuberculosis H37Rv by the proteogenomics strategy. The aim of this study was to establish a system of soluble expression and purification of the missing protein Rv2742 in M. tuberculosis H37Rv, to provide reference for further research on the biological function of Rv2742. The soluble protein was not successfully induced by prokaryotic expression vectors pGEX-4T-2-Rv2742, pET-32a-Rv2742, pET-28a-Rv2742 and pMAL-c2X-Rv2742. After the codon of novel gene Rv2742 was optimized according to E. coli codon usage frequency, only the recombinant strain containing plasmid pMAL-c2X-Rv2742 could produce soluble products of Rv2742 encoding gene. In addition, the expression effects of the desired fusion protein were also analyzed under different conditions including hosts, culture temperatures and IPTG concentrations. The optimum expression conditions were as follows: Rosetta (DE3) host, 16 °C culture temperature and 0.5 mmol/L IPTG. After being purified by affinity chromatography with amylose resin, the fusion protein sequence was confirmed by LC-MS/MS. These results indicated that the novel gene Rv2742 product could be successfully induced and expressed in a soluble form by the expression system pMAL-c2X with MBP tag. Our findings provide reference for studies on potential interaction and immunogenicity.


Assuntos
Mycobacterium tuberculosis , Cromatografia Líquida , Clonagem Molecular , Escherichia coli , Mycobacterium tuberculosis/genética , Proteínas Recombinantes de Fusão , Espectrometria de Massas em Tandem
11.
Cell Biochem Biophys ; 77(4): 357-366, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31562588

RESUMO

This study aimed to investigate for the first time, the profile of Physarum microplasmodial phosphatase (PPH) activity toward the phosphorylated light chain of Physarum myosin II (PLCM) at pH 7.6, the velocity of cytoplasmic streaming, and PPH expression in spherule formation during dark starvation (DS). In this study, we cloned the full-length cDNA of PPH using polymerase chain reaction, based on the N-terminal amino acid sequence of the purified enzyme. The cDNA contained an open reading frame (ORF) of 1245 bp, corresponding to 415 amino acids. We confirmed that a rapid increase in PPH activity toward PLCM and a rapid decrease in cytoplasmic streaming velocity precede spherule formation by Physarum microplasmodia. The profiles of increase in PPH activity toward PLCM, PPH expression, and PPH accumulation during DS were correlated with spherule formation in the Physarum microplasmodia. Moreover, application of the wheat germ cell-free expression system resulted in the successful production of recombinant PPH and in the expression of phosphatase activity toward PLCM. These results suggest that PPH is involved in the cessation of cytoplasmic streaming in Physarum microplasmodia during DS.


Assuntos
Corrente Citoplasmática/fisiologia , Monoéster Fosfórico Hidrolases/metabolismo , Physarum/enzimologia , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Miosina Tipo II/metabolismo , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Fosforilação , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
12.
Plant Mol Biol ; 101(4-5): 455-469, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31541388

RESUMO

Regulation of abscisic acid (ABA) signaling is crucial in balancing responses to abiotic stresses and retaining growth in planta. An ABA receptor (PYL/RCAR) and a protein phosphatase (PP2C), a co-receptor, form a complex upon binding to ABA. Previously we reported that the second and fourth positions in the VxGΦL motif of PP2Cs from Oryza sativa are critical in the interaction of PP2Cs with PYL/RCARs. Considering substantial effects of the VxGΦL motif on ABA signaling outputs, further comprehensive characterization of residues in the second and fourth positions are required. Here we surveyed the second and fourth positions of the VxGΦL motif by combination of biochemical, structural and physiological analyses. We found that the fourth position of the VxGΦL motif, highly conserved to small hydrophobic residues, was a key determinant of the OsPP2C50:OsPYL/RCAR interactions across subfamilies. Large hydrophobic or any hydrophilic residues in the fourth position abrogated ABA responsiveness. Analysis of crystal structures of OsPP2C50 mutants, S265L/I267V ("LV"), I267L ("SL") and I267W ("SW"), in complex with ABA and OsPYL/RCAR3, along with energy calculation of the complexes, uncovered that a bulky hydrophobic residue in the fourth position of the VxGΦL motif pushed away side chains of nearby residues, conferring side-chain rotameric energy stress. Hydrophilic residues in this position imposed solvation energy stress to the PP2C:PYL/RCAR complex. Germination and gene expression analyses corroborated that OsPP2C50 AS and AK mutants modulated ABA responsiveness in Arabidopsis. Our results suggest that ABA responsiveness could be fine-tuned by the fourth position of the VxGΦL motif on PP2Cs. KEY MESSAGE: We comprehensively surveyed the VxGΦL motif to find that the fourth position, highly conserved to small hydrophobic residues, was critical in regulating ABA responsiveness.


Assuntos
Motivos de Aminoácidos , Oryza/fisiologia , Fosfoproteínas Fosfatases/química , Proteínas de Plantas/química , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacologia , Arabidopsis/genética , Clonagem Molecular , Cristalografia por Raios X , Oryza/genética , Oryza/metabolismo , Reguladores de Crescimento de Planta/metabolismo , Reguladores de Crescimento de Planta/farmacologia , Plantas Geneticamente Modificadas/metabolismo , Transdução de Sinais
13.
Biotechnol Appl Biochem ; 66(5): 880-899, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31397000

RESUMO

The present study was aimed to explore the molecular and structural features of UDP-N-acetylglucosamine pyrophosphorylase of Bombyx mori (BmUAP), an essential enzyme for chitin synthesis in insects. The BmUAP cDNA sequence was cloned and expression profiles were monitored during the molting and feeding stages of silkworm larvae. The effect of 20-hydroxyecdysone (20E) on BmUAP expression, and on silkworm molting was studied, which revealed that 20E regulates its expression. Multiple sequence alignment of various pyrophosphorylases revealed that the residues N223, G290, N327, and K407 of human UAP (PDB ID: 1JV1) were found to be highly conserved in BmUAP and all other eukaryotic UAPs considered for the study. Phylogenetic analysis inferred that the UAPs possess discrete variations in primary structure among different insect Orders while sharing good identity between species of the Order. The structure of BmUAP was predicted and its interactions with uridine triphosphate, N-acetylglucosamine-1-phosphate, and UDP-N-acetylglucosamine were analyzed. Virtual screening with a library of natural compounds resulted in five potential hits with good binding affinities. On further analysis, these five hits were found to be mimicking substrate and product, in inducing conformational changes in the active site. This work provides crucial information on molecular interactions and structural dynamics of insect UAPs.


Assuntos
Bombyx/enzimologia , Bombyx/genética , Clonagem Molecular , Simulação por Computador , Regulação Enzimológica da Expressão Gênica/genética , Simulação de Acoplamento Molecular , Nucleotidiltransferases/química , Nucleotidiltransferases/genética , Animais , Humanos , Nucleotidiltransferases/metabolismo , Conformação Proteica
14.
J Agric Food Chem ; 67(37): 10373-10379, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31453692

RESUMO

Agarose can be hydrolyzed into agarooligosaccharides (AOSs) by α-agarase, which is an important enzyme for efficient saccharification of agarose or preparation of bioactive oligosaccharides from agarose. Although many ß-agarases have been reported and characterized, there are only a few studies on α-agarases. Here, we cloned a novel α-agarase named CaLJ96 with a molecular weight of approximately 200 kDa belonging to glycoside hydrolase family 96 from Catenovulum agarivorans. CaLJ96 has good pH stability and exhibits maximum activity at 37 °C and pH 7.0. The hydrolyzed products of agarose by CaLJ96 are analyzed as agarobiose (A2), agarotetraose (A4), and agarohexaose (A6), in which A4 is the dominant product. CaLJ96 can hydrolyze agaropentaose (A5) into A2 and agarotriose (A3) and A6 into A2 and A4 but cannot act on A2, A3, or A4. This is the first report to characterize the α-agarase action on AOSs in detail. Therefore, CaLJ96 has potential for the manufacture of bioactive AOSs.


Assuntos
Alteromonadaceae/enzimologia , Proteínas de Bactérias/química , Glicosídeo Hidrolases/química , Alteromonadaceae/química , Alteromonadaceae/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Estabilidade Enzimática , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Peso Molecular , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Sefarose/química , Sefarose/metabolismo , Especificidade por Substrato
15.
Plant Physiol Biochem ; 142: 490-499, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31442880

RESUMO

ICE1 (inducer of CBF expression 1) encodes a typical MYC-like basic helix-loop- helix (bHLH) transcription factor that acts as a pivotal component in the cold signalling pathway. In this study, DlICE1, a novel ICE1-like gene, was isolated from the southern subtropical fruit tree longan (Dimocarpus longan Lour.). DlICE1 encodes a nuclear protein with a highly conserved bHLH domain. DlICE1 expression was slightly upregulated under cold stress. Overexpression of DlICE1 in Arabidopsis conferred enhanced cold tolerance via increased proline content, decreased ion leakage, and reduced malondialdehyde (MDA) and reactive oxygen species (ROS) accumulation. Expression of the ICE1-CBF cold signalling pathway genes, including AtCBF1/2/3 and cold-responsive genes (AtRD29A, AtCOR15A, AtCOR47 and AtKIN1), was also significantly higher in DlICE1-overexpressing lines than in wild-type (WT) plants under cold stress. In conclusion, these findings indicate that DlICE1 is a member of the bHLH gene family and positively regulates cold tolerance in D. longan.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Genes de Plantas/genética , Proteínas de Plantas/genética , Sapindaceae/genética , Arabidopsis/genética , Arabidopsis/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Clonagem Molecular , Resposta ao Choque Frio , Regulação da Expressão Gênica de Plantas , Genes de Plantas/fisiologia , Malondialdeído/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/fisiologia , Plantas Geneticamente Modificadas , Prolina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sapindaceae/fisiologia , Análise de Sequência de DNA
16.
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
17.
World J Microbiol Biotechnol ; 35(9): 135, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31432264

RESUMO

The feather-degrading strain Thermoactinomyces sp. YT06 secretes an extracellular keratinolytic protease (KERTYT); however, the gene encoding this protease remains unknown. The kerT1 gene (1170 bp) encoding keratinase was cloned and expressed in Escherichia coli BL21(DE3). Purified recombinant keratinase (rKERTYT) was achieved at a yield of 39.16% and 65.27-fold purification with a specific activity of 1325 U/mg. It was shown that rKERTYT has many similarities to the native enzyme (KERTYT) by characterization of rKERTYT. The molecular weight of rKERTYT secreted by recombinant E. coli was approximately 28 kDa. The optimal temperature and the pH values of rKERTYT were 65 °C and 8.5, respectively, and the protein remained stable from 50 to 60 °C and pH 6-11. The keratinase was strongly inhibited by phenyl methane sulfonyl fluoride (PMSF), suggesting that it belongs to the serine protease family. It was significantly activated by Mn2+ and ß-mercaptoethanol (ß-Me). rKERTYT showed stability and retained over 80% activity with the existence of organic solvents such as acetone, methylbenzene and dimethyl sulfoxide. These findings indicated that rKERTYT will be a promising candidate for the enzymatic processing of keratinous wastes.


Assuntos
Clonagem Molecular , Escherichia coli/metabolismo , Expressão Gênica , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Thermoactinomyces/enzimologia , Ativadores de Enzimas/análise , Inibidores Enzimáticos/análise , Estabilidade Enzimática , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Temperatura Ambiente , Thermoactinomyces/genética
18.
Gene ; 717: 144046, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31434006

RESUMO

Flavonoids are major polyphenol compounds in plant secondary metabolism. The hydroxylation pattern of the B-ring of flavonoids is determined by the flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H). In this paper, one CsF3'H and two CsF3'5'Hs (CsF3'5'Ha and CsF3'5'Hb) were isolated. The phylogenetic tree results showed that F3'H and F3'5'Hs belong to the CYP75B and CYP75A, respectively. The Expression pattern analysis showed that the expression of CsF3'5'Ha and CsF3'5'Hb in the bud and 1st leaf were higher than other tissues. However, the CsF3'H had the highest expression in the 4th and mature leaf. The correlation analysis showed that the expression of CsF3'5'Hs is positively associated with the concentration of B-trihydroxylated catechins, and the expression of CsF3'H is positively associated with the Q contentration. Heterologous expression of these genes in yeast showed that CsF3'H and CsF3'5'Ha can catalyze flavanones, flavonols and flavanonols to the corresponding 3', 4' or 3', 4', 5'-hydroxylated compounds, for which the optimum substrate is naringenin. The enzyme of CsF3'5'Hb can only catalyze flavonols (including K and Q) and flavanonols (DHK and DHQ), of which the highest activities in catalyzing are DHK. Interestingly, The experiment of site-directed mutagenesis suggested that two novel sites near the C-terminal were discovered impacting on the activity of the CsF3'5'H. These results provide a significantly molecular basis on the accumulation B-ring hydroxylation of flavonoids in tea plant.


Assuntos
Camellia sinensis/genética , Sistema Enzimático do Citocromo P-450/genética , Flavonoides/metabolismo , Camellia sinensis/metabolismo , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/metabolismo , Flavonoides/química , Regulação da Expressão Gênica de Plantas , Hidroxilação , Mutagênese Sítio-Dirigida , Filogenia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Saccharomyces cerevisiae/genética
19.
BMC Plant Biol ; 19(1): 371, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31438856

RESUMO

BACKGROUND: Propamocarb (PM) is one of the main pesticides used for controlling cucumber downy mildew. However, due to its volatility and internal absorption, PM can easily form pesticide residues on cucumber fruits that seriously endanger human health and pollute the environment. The breeding of new cucumber varieties with a low abundance of PM residues via genetic methods constitutes an effective strategy for reducing pesticide residues and improving cucumber safety and quality. To help elucidate the molecular mechanism resulting in a low PM residue abundance in cucumber, we used the cucumber cultivar 'D0351' (which has the lowest PM residue content) as the test material and identified genes related to low PM residue abundance through high-throughput tag-sequencing (Tag-Seq). RESULTS: CsMAPEG was constitutively expressed and showed both varietal and organizational differences. This gene was strongly expressed in 'D0351'. The expression levels of CsMAPEG in different cucumber tissues under PM stress were as follows: fruit>leaf>stem>root. CsMAPEG can respond to salicylic acid (SA), gibberellin (GA) and Corynespora cassiicola Wei (Cor) stress and thus plays an important regulatory role in plant responses to abiotic and biological stresses. The PM residue abundance in the fruits of CsMAPEG-overexpressing plants was lower than those found in antisense CsMAPEG plants and wild-type plants at all tested time points. The results revealed that CsMAPEG played a positive role in reducing the PM residue abundance. A CsMAPEG sense construct increased the contents of SOD, POD and GST in cucumber fruits, enhanced the degradation and metabolism of PM in cucumber, and thus effectively reduced the pesticide residue abundance in cucumber fruits. CONCLUSIONS: The expression patterns of CsMAPEG in cucumber cultivars with high and low pesticide residue abundances and a transgenic verification analysis showed that CsMAPEG can actively respond to PM stress and effectively reduce the PM residue abundance in cucumber fruits. The results of this study will help researchers further elucidate the mechanism responsible for a low PM residue abundance in cucumber and lay a foundation for the breeding of new agricultural cucumber varieties with low pesticide residue abundances.


Assuntos
Carbamatos/farmacologia , Cucumis sativus/genética , Fungicidas Industriais/farmacologia , Genes de Plantas , Resíduos de Praguicidas , Clonagem Molecular , Cucumis sativus/efeitos dos fármacos , Cucumis sativus/enzimologia , Cucumis sativus/fisiologia , Perfilação da Expressão Gênica , Vetores Genéticos , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transformação Genética
20.
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
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