Studies On Electrochemical Biosensors For Cytokinin Analysis

Cytokinins(CK),one of the classical plant hormones,plays a key role in plant development,regulation of apical dominance and promotion of spike branching.The content of CK in plants is related to metabolic regulation.Cytokinin oxidase(CKX)is involved in irreversible catalytic reaction of CK degradation.Arabidopsis histidine Kinases 4 is a direct receptor of CK and can rapidly respond to its signal transduction.In this thesis,CKX and AHK4 have been chosen to serve as selectivity unit to develop electrochemical biosensors,and new methods for cytokinin detection have been established.The thesis contains four parts as following:1.Construction of cytokinin oxidase from Nipponbare displayed on the surface of bacteria.Three recombinant vectors pET28a-INP-CKX,pET26b-INP-CKX and pMAL-p2x-INP-CKX were constructed using ice nucleation protein(INP)as carrier protein and transformed into E.coli for the surface display.The displayed protein samples were analysed by SDS-PAGE.The experiment results showed that cytokinin oxidase in pET28a-INP-CKX and pET26b-INP-CKX plasmids expressed as inclusion body,while in pMAL-p2x-INP-CKX plasmid cytokinin oxidase was successfully displayed on the cell surface.And this solved the problem of CKX expressed as the inclusion body by prokaryotic expression.The size of the displayed protein was 116 kDa by Western-blot analysis,which consistent with the theoretical size of CKX.The cytokinin oxidase surface displayed bacteria were used to develop biosensor for isoamyl alkenyl adenine.2.The biosensor for isoamyl alkenyl adenine detection was constructed based on cytokinin oxidase displayed on the suface of bacteria.The direct electrochemistry of CKX with its coenzyme FAD was realized on the pyrolytic graphite electrode.The redox peak potential of CKX was-0.42 and-0.48 V,respectively.After addition of isoamyl alkenyl adenine,the oxidative peak current of FAD was increased and the reductve peak current was decreased.The isoamyl alkenyl adenine has been detected by the decreased reduction peak current of FAD with the addition of isoamyl alkenyl adenine.The biosensor possesses specificity to isoamyl alkenyl adenine with a linear range of 1~9 ?M.The linear regression equation is y = 0.12 x + 0.8997(R2 = 0.9833).The detection limit(LOD)was 0.7 ?M(S/N=3).3.Prokaryotic expression of AHK4 from Arabidopsis thaliana.In order to increase the soluble amount of AHK4 in prokaryotic expression,the recombinant plasmid pET26b-SUMO-AHK4 was constructed by fusion of ubiquitin related modifier SUMO from Saccharomyces cerevisiae.The recombinant bacteria were induced and the expressed fusion protein was purified.The active protein was used to develop biosensor to detect isoamyl alkenyl adenine.4.The biosensor for CK analysis was constructed using protein AHK4 as recognition unit.The protein was immobilized on Au electode by bovine serum albumin and glutaraldehyde with their strong crosslinking ability.Because of no electrochemical signal of AHK4,ferrocene was used as the electrochemical mediator of the biosensor.The AHK4 based biosensor has strong specificity to isopentene adenine and can avoid the interference of other plant hormones.The linear range of the biosensor for isoamyl alkenyl adenine is 3~15 ?M.The linear regression equation is y = 0.275 x + 0.1134(R2 = 0.9848).The detection limit(LOD)was 1 ?M(S/N=3).