Studies On The Expression Of The Rice Cytokinin Oxidase And Its Interaction With The Isopentenyl Adenine By Fluorescence Method

Cytokinin(CTK)is widely distributed in plants with a specific biological function.The accurate detection of CTKs is very difficult due to its low concentration.In this thesis,we have expressed rice cytokinin oxidase in E.coli successfully.Through studing the interaction between CKX and isopentenyl adenine and obtained the thermodynamics parameters telated their interaction.We have also developed a new fluorescence method to detect isopentenyl adenine.This thesis contains three parts as following:Firstly,we downloaded the amino acid sequence of rice CKX from NCBI and predicted that the CKX is a soluble protein with a molecular weight of 58 k Da and an isoelectric point of 6.19 by bioinformatics analysis.We obtained the tertiary structure of CKX by homology modeling and assessed the model to be reliable by PROCHECK evaluation.Secondly,we extracted RNA from Nipponbare and obtained the CKX gene by RT-PCR.We have constructed corresponding recombinant p ET28a-CKX and expressed CKX in E.coli BL21(DE3).The expression condition was optimized and the best level of expression was obtained after 8 h at 15? with addition of 1 m M IPTG and 5 ?g/L riboflavin in culture.The expressed enzyme was detected by SDS-PAGE and Western-blot.Thirdly,we have applied fluorescence method to study the interaction between CKX and isopentenyl adenine in vitro,and obtain some parameters of their interaction.We found that addition of isopentenyl adenine can quench the fluorescence of CKX and the quenching mechanism was to be a static quenching procedure.We have measured the number of binding sites and apparent binding constant,and have calculated the thermodynamics parameter ?H,?G and ?S by fluorescence quenching method.Based on thermodynamics parameter's results we concluded that their binding reaction was both entropy-driven and the enthalpy-driven,and the Van der Waals force and hydrogen bond force played major role in the interaction.Based on the synchronous fluorescence spectrometry results we demonstrated that the binding site between isopentenyl adenine and CKX is in the microenvironment of both tryptophan and tyrosine.The fluorescence signal of cofactor FAD decreases gradually with the addition of isopentenyl adenine.And this method can be used for isopentenyl adenine routine assay.Under optimized experimental parameters,the linear segment increases from 0.6 ?M to 100 ?M with a regression equation of ?F = 0.04 + 0.15cip(r = 0.999,cip ?M)with the detection limit of 0.15 ?MiP.