Dianthus spiculifolius Schur,as an emerging ornamental plant,has extensive applications and economic values.In this study,the DsCBL4 gene was successfully cloned,and its tissue-specific expression,expression patterns ...Dianthus spiculifolius Schur,as an emerging ornamental plant,has extensive applications and economic values.In this study,the DsCBL4 gene was successfully cloned,and its tissue-specific expression,expression patterns under various abiotic stresses,subcellular localization,and bioinformatics analysis of the encoded amino acid sequence were conducted.The results showed that the coding region of the DsCBL4 gene was 675 bp long,encoding 224 amino acids.It had high homology with the amino acids encoded by Amaranthus tricolor,Chenopodium quinoa and Spinacia oleracea.The predicted relative molecular mass of DsCBL4 was 25.61 ku,with an isoelectric point of 4.58,and it had phosphorylation sites,belonging to an unstable hydrophilic protein.Its secondary structure includedα-helices,irregular coils and extended chains.The tertiary structure prediction revealed that DsCBL4 had four EFhand calcium-binding domains necessary for Ca2+binding in plant calmodulin-like proteins and the FPSF motif for calcineurin B-like protein(CBL)-interacting protein kinase(CIPK)activation.The expression level of the DsCBL4 gene showed tissue specificity,with the highest expression in roots.It was induced by drought,low temperature,combined drought and low temperature,salt stress,nitrogen stress,phosphorus stress,calcium ion stress,high temperature stress,and abscisic acid(ABA)stress.Both transient infection in Nicotiana tabacum L.and stable expression in transgenic Arabidopsis thaliana showed that the DsCBL4 protein was localized to the cell membrane.These results suggested that DsCBL4 might be involved in the abiotic stress response of Dianthus spiculifolius through the calcium signaling pathway,providing a theoretical basis for understanding its molecular mechanism.This study provided an important reference for further exploring the role of the DsCBLs gene family in plant stress resistance.展开更多
Objective： To explore the molecular mechanism of puerarin （Pue） in improving insulin resistance through observing its effect on the insulin resistance of 3T3-Li lipocyte induced by free fatty acid （FFA）. Methods...Objective： To explore the molecular mechanism of puerarin （Pue） in improving insulin resistance through observing its effect on the insulin resistance of 3T3-Li lipocyte induced by free fatty acid （FFA）. Methods： 3T3-L1 preadipocyte was induced by a culture solution containing insulin, isobutyo-menthyl-xanthine, and dexamethasone to mature lipocyte, and it was divided into six groups： the control group （normal cells）, the model group （untreated model cells）, and the four drug treatment group exposed to dimethyl biguanide （Met group）, high- dose pueradn （PueH group）, low-dose puerarin （PueL group）, and propylene glycol （PG group）, respectively. Mature lipocytes in various groups, except those in the normal group, were established into insulin resistance model by FFA induction and treated respectively with corresponding drugs. Peroxisome proliferator-activated receptor- γ （PPAR- γ） mRNA expressions at the fourth, sixth, and eighth day were observed using reverse transcription polymerase chain reaction （RT-PCR）; glucose transportation in various groups were observed by 2-deoxy-[3H]-D-glucose intake method; mRNA expression of Cbl binding protein （CAP） was determined by RT-PCR; and glucose transporter-4 （Glut-4） transposition was detected by immune-fluorescence method. Results： PPAR- γmRNA expression increased gradually, and it showed lower levels at the fourth, sixth, and eighth day in all treatment groups than that in the model group. Glucose transportation determination showed that the transportation in the model group was 2.23 ± 0.63, significantly lower than that in the normal group 5.05 ± 0.66 （P〈0.01）; as compared with the model group, they were significantly higher in the PueH and the PueL groups. In addition, the CAP mRNA expression and membranous distribution of Glut-4 were higher in the two Pue treated groups than those in the model group, respectively. Conclusion： Pue could markedly improve the insulin resistance of 3T3-L1 lipocyte, which is realized possibly by way of inactivating CAP path, promoting Glut-4 transposition to cell membrane to increase the transportation of glucose.展开更多
基金Supported by the National Key R&D Program(2022YFF1300500)the Spring Goose Support Program(CYQN24018)the National Natural Science Foundation of China(32572123)。
文摘Dianthus spiculifolius Schur,as an emerging ornamental plant,has extensive applications and economic values.In this study,the DsCBL4 gene was successfully cloned,and its tissue-specific expression,expression patterns under various abiotic stresses,subcellular localization,and bioinformatics analysis of the encoded amino acid sequence were conducted.The results showed that the coding region of the DsCBL4 gene was 675 bp long,encoding 224 amino acids.It had high homology with the amino acids encoded by Amaranthus tricolor,Chenopodium quinoa and Spinacia oleracea.The predicted relative molecular mass of DsCBL4 was 25.61 ku,with an isoelectric point of 4.58,and it had phosphorylation sites,belonging to an unstable hydrophilic protein.Its secondary structure includedα-helices,irregular coils and extended chains.The tertiary structure prediction revealed that DsCBL4 had four EFhand calcium-binding domains necessary for Ca2+binding in plant calmodulin-like proteins and the FPSF motif for calcineurin B-like protein(CBL)-interacting protein kinase(CIPK)activation.The expression level of the DsCBL4 gene showed tissue specificity,with the highest expression in roots.It was induced by drought,low temperature,combined drought and low temperature,salt stress,nitrogen stress,phosphorus stress,calcium ion stress,high temperature stress,and abscisic acid(ABA)stress.Both transient infection in Nicotiana tabacum L.and stable expression in transgenic Arabidopsis thaliana showed that the DsCBL4 protein was localized to the cell membrane.These results suggested that DsCBL4 might be involved in the abiotic stress response of Dianthus spiculifolius through the calcium signaling pathway,providing a theoretical basis for understanding its molecular mechanism.This study provided an important reference for further exploring the role of the DsCBLs gene family in plant stress resistance.
文摘Objective： To explore the molecular mechanism of puerarin （Pue） in improving insulin resistance through observing its effect on the insulin resistance of 3T3-Li lipocyte induced by free fatty acid （FFA）. Methods： 3T3-L1 preadipocyte was induced by a culture solution containing insulin, isobutyo-menthyl-xanthine, and dexamethasone to mature lipocyte, and it was divided into six groups： the control group （normal cells）, the model group （untreated model cells）, and the four drug treatment group exposed to dimethyl biguanide （Met group）, high- dose pueradn （PueH group）, low-dose puerarin （PueL group）, and propylene glycol （PG group）, respectively. Mature lipocytes in various groups, except those in the normal group, were established into insulin resistance model by FFA induction and treated respectively with corresponding drugs. Peroxisome proliferator-activated receptor- γ （PPAR- γ） mRNA expressions at the fourth, sixth, and eighth day were observed using reverse transcription polymerase chain reaction （RT-PCR）; glucose transportation in various groups were observed by 2-deoxy-[3H]-D-glucose intake method; mRNA expression of Cbl binding protein （CAP） was determined by RT-PCR; and glucose transporter-4 （Glut-4） transposition was detected by immune-fluorescence method. Results： PPAR- γmRNA expression increased gradually, and it showed lower levels at the fourth, sixth, and eighth day in all treatment groups than that in the model group. Glucose transportation determination showed that the transportation in the model group was 2.23 ± 0.63, significantly lower than that in the normal group 5.05 ± 0.66 （P〈0.01）; as compared with the model group, they were significantly higher in the PueH and the PueL groups. In addition, the CAP mRNA expression and membranous distribution of Glut-4 were higher in the two Pue treated groups than those in the model group, respectively. Conclusion： Pue could markedly improve the insulin resistance of 3T3-L1 lipocyte, which is realized possibly by way of inactivating CAP path, promoting Glut-4 transposition to cell membrane to increase the transportation of glucose.
