Development BRET/FRET Biosensor For Cytokinin In Plant

As one of the classic plant hormones,cytokinins(CK)play a key role in plant growth and development,growth-defense balance,and long-distance transmission of nutrition signals.Therefore,to develop an in-situ and real time detection method for CK is important to study plant physiological and biochemical processes and the mechanisms of its signal transmission networks.To overcome the limitations of the classical detection methods for CK in vivo,we aimed to develop biosensors for the detection CK in vivo based on the CK receptor,Arabidopsis histidine Kinases 4(AHK4),which has a high affinity for CK(Kd?2?4 n M),with the combination of fluorescence resonance energy transfer(FRET)and bioluminescence resonance energy transfer(BRET)technology.In this thesis,AHK4 is used as the recognition unit to construct the CK BRET and FRET biosensors,and new methods for CK detection in vivo in plants have been established.It mainly includes the following three parts:1.BRET biosensors has been developed and applied to detect cytokinin in vitro.In this study,AHK4 was fused with luciferase Nluc and green fluorescent protein m Neon Green to construct four cytokinin BRET biosensors.The fusion proteins were induced to express in E.coli by IPTG.Four BRET biosensor modules were obtained,and the optical characteristics of the biosensor module was investigated by the fluorescent protein fused on each module.Based on the principles of BRET,the BRET biosensor modules with the highest resonance energy transfer efficiency was screened and optimized,and the optimal combination of LUC-AHK4/AHK4-m Neon Green was obtained.This combination was further used to determine 2-i P concentration in vitro.2.Cytokinin BRET biosensor was genetically transformed to Arabidopsis.Based on the constructed and optimized cytokinin BRET biosensor of the previous work in my research,a binary expression vector was constructed to transform Arabidopsis thaliana.The vectors p U1301-AHK4-m Neon Green(hygromycin resistance)/p U1301-LUC-AHK4(geneticin resistance),the positive control p U1301-LUC-m Neon Green(geneticin resistance),and the negative control p U1301-LUC(geneticin resistance)have been constructed based on p U1301 plasmids with different resistances,and the constructed vectors were successfully transformed into Arabidopsis thaliana.Positive transformants were further selected by the double antibiotics and PCR.3.The prokaryotic expression vectors of cytokinin FRET biosensor was constructed using AHK4 as recognition unit and m Turquoise2 and m Neon Green as the energy donor and receptor,respectively.Four unimolecular and four biomolecular FRET biosensors have been developed by changing the order of the fluorophores and sensing module components.The fusion proteins were induced to express in E.coli by IPTG.Three unimolecular FRET biosensor proteins were purified and the optical characteristics of the proteins were investigated.The FRET biosensor protein with the highest resonance energy transfer efficiency was screened and optimized based on the principles of FRET.The optimal selection of AHK4-m Turquoise2-AHK4-m Neon Green was obtained and it was further used to determine 2-i P concentration in vitro.