Study On Detection Of 14-3-3 Gene Transcription And Protein Expression Of Transgenic Tobacco Based On Carbon And Nanogold Electrochemical Sensors

As a natural dimer protein,14-3-3 protein recognizes and binds target protein white molecules in plant cells,regulates various life activities in plant cells,and mainly assists in the exchange and transportation of nutrients and regulates plant growth And development,promote the metabolism of plant somatic cells,respond to external stress of plant bodies,control signal transmission pathways in plant cells,etc.In plant cells,the 14-3-3 protein is encoded by multiple different genes,and different 14-3-3 genes have their functional specificity.Therefore,when we analyze the functions of different 14-3-3 genes in plants,we must not only detect the expression level of 14-3-3 gene,but also analyze the content of protein expressed by 14-3-3 gene.Currently,RT-PCR is often used to detect gene transcription levels,while WB is used to detect protein expression levels.However,these detection methods have their own shortcomings.In the process of use,RT-PCR operation time is longer,and the cost of the instrument is higher.The WB detection method also has a relatively complicated operation process and insufficient detection limits.In contrast,the electrochemical sensor detection method has the advantages of higher detection sensitivity,shorter operation time,reusable storage and use,and lower detection cost.This study verified the gold standard 14-3-3 by constructing an electrochemical DNA sensor and protein immunosensor to detect and analyze the 14-3-3g gene transcription level and the content of expressed protein in the transgenic tobacco planted in our laboratory.The feasibility and practical application value of immunosensor and electrochemical DNA sensor prepared by protein antibody are as follows:1.Construct a DNA sensor based on the principle of complementary DNA base pairing to detect the content of 14-3-3g gene nucleic acid DNA.The screen-printed carbon electrode is used as the working electrode of the DNA sensor.Based on the electrostatic adsorption of colloidal gold and chitosan,the DNA single-stranded molecule is fixed on the working electrode and serves as the DNA probe sequence of the DNA sensor.The target sequence is finally assembled and immobilized on the working electrode of the sensor according to the principle of specific complementary pairing and binding between bases of DNA double-stranded molecules.The complementary double-stranded DNA is assembled into a complete DNA sensor by adsorbing the indicator methylene blue.Connect the constructed DNA sensor to the electrochemical analysis workstation,and add hydrochloric acid buffer Tris-HCl dropwise to the working electrode as an electrochemical reaction solution.Methylene blue then catalyzes the oxidation of hydrochloric acid buffer Tris-HCl to form an electrochemical catalytic signal current It was detected by the electrochemical analysis workstation.Based on different concentrations of DNA target sequences,different electrochemical signal current values are formed.The research results show that when the target DNA sequence is in the range of 0.1-1.15?mol/L,the current peak of the DPV voltammetry curve is good with the target DNA sequence concentration.Linear relationship,the linear regression equation is y=-13.333x+22.29,R~2=0.9951.By extracting the total RNA of the14-3-3 transgenic tobacco leaves,combining with the DNA single-strand probe sequence on the working electrode of the DNA sensor,and performing the base complementary pairing hybridization reaction,the transcription level of the 14-3-3g gene is detected.The results showed that compared with the wild control tobacco WT,the transcription level of the 14-3-3g gene in RNAi interference expressing plants i2 and i1 decreased by 63.2%and 68.4%,respectively;while overexpression of 14-3-3g in plants p2 and pl The level of gene transcription increased by 47.1%and 37.3%,respectively.This result is consistent with the results of RT-PCR analysis.When using this method for detection,there is no need to reverse the mentioned RNA into cDNA,which saves time and resources compared to RT-PCR.2.Prepared 14-3-3 protein detection immunosensor based on enzyme-antibody co-conjugated gold(AuNs)nanoprobe for signal amplification.Using gold nanoparticles coupled with hydrogen peroxide catalytic enzyme(CAT)and 14-3-3 antibody(anti-14-3-3)to prepare a nano-signal probe(CAT-Au-anti-14-3-3 signal probe,referred to as CASP).Assemble layers of 14-3-3 antibody(anti-14-3-3),14-3-3 protein(14-3-3)and nanosignal probe(CASP)into multi-walled carbon nanotubes(MWCNTs)And the surface of the screen-printed electrode(SPCE)modified with gold nanoparticles(Au),SPCE?MWCNTs/Au-anti-14-3-3/14-3-3/CASP immunotype sensors were prepared,which can produce reduction catalysis for H2O2 Current.At a room temperature of 20?,when the sensor detects the concentration of 14-3-3 protein in the range of 0.02-5ng/?L,the steady-state signal current value has a good linear relationship with the concentration of 14-3-3protein:y=356.16x+192.57,the correlation coefficient R2=0.9989 reached a very significant level;the standard deviations of the measurements were all less than 0.5%,with good repeatability.14-3-3 protein test results of genetically modified plants?In addition,the detection limit range of this method is wider than that of traditional Western blot analysis,and the sensitivity is higher.Therefore,this electrochemical method has the advantages of high sensitivity,strong immunity,good selectivity and repeatability,etc.,and can be used for quantitative detection of 14-3-3protein.