| 14-3-3 protein is the core of the interaction between proteins in plant cells.14-3-3 protein regulates all kinds of life activities by binding to target proteins,and participates in the transport of substances,growth and development,nutritional metabolism and cell cycle in plants.Regulation of physiological processes such as stress response and light signal transduction pathway.Biological and abiotic stress stimulation not only changed the transcription and protein expression level of 14-3-3 protein gene in plants,but also changed the interaction between 14-3-3 protein and target protein.Many studies have shown that 14-3-3 protein can affect its activity by regulating the expression of antioxidant enzyme genes.In addition,14-3-3 protein regulates carbon and nitrogen metabolism through interaction with metabolic enzymes.The 14-3-3 proteins in plants are encoded by many genes,and different 14-3-3 genes are functionally specific.Therefore,in order to investigate the function of different 14-3-3 genes in plants,we should not only analyze the expression level of 14-3-3 gene,but also examine the changes of 14-3-3 protein regulation target protein expression level.At present,RT-PCR is used to detect gene transcription level and Western blot is used to detect protein expression in scientific research and diagnosis.However,these methods have their own shortcomings,and the operation cost of fluorescent quantitative PCR instrument is high.The cost of both instruments and consumables is not low.Western Blot analysis method often has some disadvantages such as complicated operation and low detection limit.The electrochemical analysis method has the advantages of good selectivity,no pretreatment,continuous on-line monitoring,and the cost of determination is much lower than that of large analytical instruments.A highly sensitive electrochemical DNA sensor and an electrochemical immunosensor were constructed to analyze the transcriptional and protein expression levels of 14-3-3c and 14-3-3g and their regulatory target protein genes in transgenic tobacco previously obtained in our laboratory.The effects of 14-3-3c and 14-3-3g on the response of tobacco to formaldehyde stress were investigated.The main results were as follows:1.Based on the principle of base complementary pairing,the DNA sensor was constructed by screen printing electrode.The carbon nanotubes and single-stranded DNA probes were immobilized on the working electrode surface by chitosan.Under suitable conditions,double strand DNA was formed by hybridization with complementary sequences in solution.Electrochemical methods were used to detect the changes of electrochemical signals before and after hybridization.The results show that there is a good linear relationship between the peak current and the concentration of the target sequence when the concentration of the target sequence is in the range of 0.5-1 μmol/L,and the linear regression equation is y =-8.225x+34.84(R2 = 0.836).Total RNAs were extracted from transgenic tobacco leaves and hybridized with single-stranded DNA probes on the electrodes to detect the transcriptional level of 14-3-3c and g genes.The results showed that the transcriptional levels of 14-3-3c(ic4)and 14-3-3g(ig2)were decreased by 62.5% and 67.4%,respectively,compared with WT;The transcriptional levels of 14-3-3c and g genes increased by 46.5% and 36.8%,respectively in 14-3-3c(kc1)and 14-3-3g(kg2).2.Based on the principle of highly specific binding between antigen and antibody,the electrochemical immunosensor of 14-3-3 protein was constructed by using screen printing electrode,and the quantitative detection of 14-3-3 protein was realized.There was a good linear relationship between the peak current of IT curve and 14-3-3 protein concentration in the range from 64 ng/m L to 2000ng/ml concentration.The linear regression equation was y = 1.0113 x + 582.34(R2 = 0.9986).The detection limit of Western Blot analysis could only reach μg/ml level.The expression level of 14-3-3 protein in transgenic tobacco was quantitatively detected by electrochemical immunosensor with 14-3-3 protein.The results showed that the expression level of 14-3-3 protein in transgenic tobacco was compared with WT.The expression of 14-3-3c and g protein decreased by 54.7% and 23.7% in ic4 and ig2 plants,and increased by 27.7% and 27.8% in kc1 and kg2 plants,respectively.The detection results are consistent with the traditional Western Blot method,but this method has high sensitivity,low cost and less time consuming.3.The transcription levels of antioxidant enzymes and formaldehyde metabolism-related enzymes in 14-3-3 transgenic tobacco leaves were quantitatively analyzed by electrochemical DNA sensor.In ic4 plants,the APX transcription level increased by 40.2% and the Cu/Zn SOD transcription level increased by 37.8%,and the Cu/Zn SOD transcription level increased by 29.6% and 50%,which significantly increased the activity of ic4 SOD and POD,and the decrease of POD transcription water by 15% in ig2 resulted in a significant decrease in POD activity.In kc4,the Cu/Zn SOD transcriptional water increased the activity of Cu/Zn SOD by 20%.The expressions of malate dehydrogenase and isocitrate dehydrogenase were up-regulated in ic4 plants,down-regulated in kc4,and up-regulated in isocitrate dehydrogenase.The results of 13C-NMR analysis showed that the up-regulated expression of 14-3-3c under formaldehyde stress resulted in more carbohydrate and nitrogen transport amino acids in the formaldehyde metabolism of transgenic tobacco leaves,while interfering with the expression of the gene inhibited the synthesis of carbohydrate and nitrogen transport amino acids.Interference with 14-3-3g expression inhibited the production of carbohydrates from formaldehyde metabolism,but promoted the synthesis of nitrogen transport amino acids. |
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