RESUMO
Corn DNA was introduced into dry seeds of rice (cv. 'YuJing-6') by ion beam irradiation. Proteinase activities in rice seedling roots were subsequently analyzed by renaturation electrophoresis at pH 4.5, 7.0, and 8.5. Proteinase activity was more pronounced on gels at higher pH. Irradiation of rice seedling roots caused the loss of some proteinase bands at all pH conditions although a novel 50-kDa band was found at both pH 7.0 and 8.5. No new proteinase activity was detected at pH 4.5. However, novel bands and bands showing stronger activity were observed at pH 7.0 and 8.5. The data indicate that the expression of proteinases in rice seedling roots was altered following low energy ion beam mediated transformation with corn DNA.
Assuntos
DNA de Plantas/genética , Oryza/genética , Peptídeo Hidrolases/metabolismo , Proteínas de Plantas/metabolismo , Sementes/genética , Transformação Genética , Zea mays/química , Eletroforese em Gel de Poliacrilamida , Ensaios Enzimáticos , Expressão Gênica , Concentração de Íons de Hidrogênio , Íons , Cinética , Nitrogênio/química , Oryza/enzimologia , Peptídeo Hidrolases/genética , Proteínas de Plantas/genética , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Radiação não Ionizante , Plântula/enzimologia , Plântula/genética , Sementes/enzimologiaRESUMO
The effect of sinomenine (SIN) on the toll-like receptor (TLR) signal transduction pathway as well as the expression of myeloid differentiation factor 88 (MyD88) and tumor necrosis factor (TNF) receptor-associated factor-6 (TRAF6) was investigated. SIN inhibition of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) proliferation and RA cartilage and subchondral bone destruction was also investigated. RA-FLS were cultured in vitro and the intracellular alkaline phosphatase (ALP) activity was determined in order to obtain the optimal drug concentration. The rate of cell proliferation was determined. Fluorescence quantitative polymerase chain reaction (PCR) was applied to determine the MyD88 and TRAF-6 gene expression and western blot was used to detect the MyD88 and TRAF-6 protein expression. The ALP activity in the SIN groups was lower than that in the control group, among which the 0.5 mM SIN group had the lowest ALP activity (P < 0.01). The rate of RA-FLS proliferation detected by CCK-8 assay in the 0.5-mM SIN group was lower than that in the control group (P < 0.01) and was the highest 4 days after SIN induction. Gene and protein expression of MyD88 and TRAF-6 were downregulated significantly in the 0.5-mM SIN group compared to that in the control group (P < 0.01). SIN effectively inhibited MyD88 and TRAF-6 expression in RA-FLS, which may be one of the important molecular mechanisms involved in RA treatment and prevention of cartilage and subchondral bone destruction.
Assuntos
Analgésicos/farmacologia , Anti-Inflamatórios/farmacologia , Fibroblastos/efeitos dos fármacos , Morfinanos/farmacologia , Fator 88 de Diferenciação Mieloide/antagonistas & inibidores , Fator 6 Associado a Receptor de TNF/antagonistas & inibidores , Fosfatase Alcalina/antagonistas & inibidores , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Artrite Reumatoide/genética , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Artrite Reumatoide/cirurgia , Artroplastia , Proliferação de Células/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica , Humanos , Cápsula Articular/metabolismo , Cápsula Articular/patologia , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Cultura Primária de Células , Transdução de Sinais , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismoRESUMO
Rice variation induced by the introduction of exogenous DNA has become an important method of improving rice varieties and creating new germplasms. In this study, we transferred maize genomic DNA fragments to the receptor of Nipponbare rice using a modified "pollen-tube pathway" method. Material from mutant rice B1 and B2 were acquired and 14 specific bands were obtained from the material using amplified fragment length polymorphism analysis. From the 14 specific sequences obtained, there were 3791 bp, including 144 base mutations with a base mutation rate of 3.80%. Specific bands resulted from base mutation of selective bases or restriction endonuclease recognition sequences, or insertion or deletion of DNA fragments. The frequency of single-base mutations was significantly higher than that of double-base mutations, three-sequential base mutations, and multiple-sequential base mutations. The site frequency of base substitution (87.04%) was significantly higher than that of base insertion (3.70%) or deletion (9.26%). In all cases of base substitution, the frequency of transition (76.47%) was significantly higher than transversion (23.53%). The above results indicate that transferring foreign-species DNA into rice cells can induce base mutations in the receptor, with base substitutions occurring at the highest frequency, and the dominant type of base substitutions being transition. Preliminary analysis reveals that the molecular mechanism of transferring exogenous DNA into rice causes mutations, which provides theoretical data on biological mutagenesis for further research.