RESUMO
Carotenoid cleavage oxygenases (CCOs) are a family of dioxygenases, which specifically catalyze the cleavage of conjugated double bonds in carotenoids and apocarotenoids in plants. In this study, genome-wide analysis of CCO genes in pepper plants was performed using bioinformatic methods. At least 11 members of the CCO gene family were identified in the pepper genome. Phylogenetic analysis showed that pepper and tomato CCO genes could be divided into two groups (CCDs and NCEDs). The CCD group included five sub-groups (CCD1, CCD4, CCD7, CCD8, and CCD-like). These results indicate that there is a close genetic relationship between the two species. Sequence analysis using the online tool, Multiple Expectation Maximization for Motif Elicitation (MEME), showed that the CCO proteins comprise multiple conserved motifs, with 20 to 41 amino acids. In addition, multiple cis-acting elements in the promoter of CCO genes were identified using the online tool PlantCARE, and were found to be involved in light responsiveness, plant hormone regulation, and biotic and abiotic stresses, suggesting potential roles of these proteins under different conditions. RNA-seq analysis revealed that the CCO genes exhibit distinct patterns of expression in the roots, stems, leaves, and fruit. These findings suggest that the CCO genes have important roles in the vegetative and reproductive development of pepper plants.
Assuntos
Capsicum/enzimologia , Capsicum/genética , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Família Multigênica , Oxigenases/genética , Filogenia , Motivos de Aminoácidos , Sequência Conservada/genética , Éxons/genética , Perfilação da Expressão Gênica , Genes de Plantas , Íntrons/genética , Solanum lycopersicum/enzimologia , Solanum lycopersicum/genética , Oxigenases/metabolismo , Regiões Promotoras Genéticas/genética , Alinhamento de Sequência , Análise de Sequência de RNARESUMO
We conducted a case-control study to investigate the role of 3 single-nucleotide polymorphisms of the gene encoding transforming growth factor-b1 (TGFB1) in the development of metastatic brain tumors in non-small cell lung cancer patients. The polymorphisms in TGFB1 rs4803455, rs1800469, and rs1800470 were evaluated by polymerase chain reaction-restriction fragment length polymorphism. Odds ratios and their corresponding 95% confidence intervals were used to assess the influence of TGFB1 rs4803455, rs1800469, and rs1800470 on metastatic brain tumors. We found that cases were more likely to have a later disease stage when compared with control subjects, without brain metastasis. Individuals carrying the TGFB1 rs1800469 TT and CT+TT genotypes had an increased risk of developing brain metastasis compared with the rs1800469 CC genotype. Moreover, a significant interaction was observed between the rs1800469 polymorphism and disease stage. However, no significant association between polymorphisms rs4803455 and rs1800470 and the risk of developing brain metastasis were observed. We found that the TGFB1 rs1800469 polymorphism may be predictive biomarker for the risk of developing brain metastasis in non-small cell lung cancer patients.
Assuntos
Neoplasias Encefálicas/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Predisposição Genética para Doença/genética , Neoplasias Pulmonares/genética , Polimorfismo de Nucleotídeo Único , Fator de Crescimento Transformador beta1/genética , Adulto , Idoso , Biomarcadores Tumorais/genética , Neoplasias Encefálicas/secundário , Carcinoma Pulmonar de Células não Pequenas/patologia , Estudos de Casos e Controles , Feminino , Frequência do Gene , Genótipo , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Razão de Chances , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Fatores de RiscoRESUMO
In this study, 2 approaches were adopted to obtain good single-strand conformation polymorphism (SSCP) data for autotetraploid alfalfa; primers were added to PCR products, and fluorescent-labeled primers were utilized. PCR-SSCP conditions for a 331-bp fragment in the coding region of polygalacturonase-inhibiting protein gene 2 in alfalfa (MsPGIP2) were optimized, and the results showed that the best SSCP gel pattern could be obtained when the loading mixture was made by mixing 1 µL PCR products, 0.2 to 0.8 µL unlabeled primers (50 µM) and 4 to 16 µL loading buffer. Furthermore, the use of the fluorescent-labeled primers resulted in 2 separated electrophoresis images from 2 complementary single DNA strands, thus making the determination of alleles and idiotypes a relatively easy task. In addition, the results of sequencing prove that the determination of alleles and idiotypes were accurate based on SSCP analysis. Finally, a total of 9 alleles with 18 SNP sites were identified for MsPGIP2 in the alfalfa variety 'Algonquin'. In conclusion, MsPGIP2 possessed great genetic variation, and the addition of primers to the PCR products in combination with the fluorescent labeling of primers could significantly improve the sensitivity and resolution of SSCP analysis. This technique could be used for genetic diversity detection and marker-assisted breeding of useful genes in autopolyploid species such as alfalfa.
Assuntos
Impressões Digitais de DNA/métodos , Medicago sativa/genética , Proteínas de Plantas/genética , Alelos , Primers do DNA/química , Fluorescência , Polimorfismo de Nucleotídeo Único , Polimorfismo Conformacional de Fita SimplesRESUMO
The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3) can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML) cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss) inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO) cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05) 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy.
Assuntos
Animais , Cricetinae , Feminino , Humanos , Citoplasma/metabolismo , Produtos do Gene tat/metabolismo , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/metabolismo , Cromatografia de Afinidade , Diferenciação Celular/genética , Citoplasma/genética , Eletroforese em Gel de Poliacrilamida , Vetores Genéticos , Produtos do Gene tat/genética , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/genética , TransfecçãoRESUMO
The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3) can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML) cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss) inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO) cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05) 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy.