Development Of BRET Systems For Abscisic Acid Detection

Abscisic acid(ABA)is involved in the regulation of key physiological processes including plant development and responses to biotic and abiotic stress.It is reported that abscisic acid receptor PYL and type 2C protein phosphatase(PP2Cs)are involved in the signal transduction of ABA.In the presence of ABA,PYL binding to ABA can inhibit the activity of PP2 C,resulting in the release of protein kinase SnRK2 s.Then SnRK2 s phosphorylates the downstream transcripts,causing a series of responses to ABA.Based on the mechanism of abscisic acid's signal transduction,PYL02 and 2C-type protein phosphatase PP2C06 from Nipponbare were selected in this thesis.The luciferase(Nluc)with high-luminescence and monomeric yellow-green fluorescent protein(mNeonGreen)with high fluorescence yield were chosen to fuse the N-terminus or C-terminus of PYL2 or PP2C06,respectively.Eight fusion proteins have been obtained.Based on the interaction of PYL and PP2 C mediated by ABA,the distance between donor and acceptor reaches the critical distance of energy transfer,resulting in BRET signal.And a new method for abscisic acid detection by BRET has been established.1.Construction of the BRET signal module.The recombinant vectors of pET28a-mNeonGreen-PYL2,pET28a-PYL2-mNeonGreen,pET28a-Nluc-PP2 C,pET28a-PP2C-Nluc,pET28a-mNeonGreen-PP2 C,pET28a-PP2C-mNeonGreen,pET28a-Nluc-PYL2 and pET28a-PYL2-Nluc have been constructed.And the eight fusion proteins were expressed in Escherichia coli BL21.After purification by HIS column,the protein expression was analyzed by SDS-PAGE.The spectral characteristics of the eight proteins were identified by UV-vis,fluorescence and bioluminescence techniques.The combination of the donors and the receptor proteins were investigated to optimize the BRET signal module.And it was found that the combination of mNeonGreen-PYL2 and Nluc-PP2 C achieved the highest bioluminescence efficiency.2.Optimization of BRET detection system.The salt ion concentration,BSA concentration and concentration ratio of donor protein and receptor protein were analyzed.The results showed that increased salt ion concentration and BSA concentration inhibited BRET signal.When the concentration ratio of energy donor vs.energy receptor was 1:6,the system had the highest energy transfer efficiency.Using the optimized BRET systems to detect ABA,the linear range was 0.005-0.1 ?M with the linear regression equation y = 0.53344 x + 0.06597(R2 = 0.88632)and the detection limit(LOD)was 3.8 nM.The specificity of the BRET system for abscisic acid was also investigated.Finally,the system was applied to detect abscisic acid concentration in leaf samples of drought and normal rice and the results were compared with HPLC-MS.The results showed that the concentration of ABA was 4.9150 ng/g in dry rice leaf and 3.3168 ng /g in normal rice leaf by BRET method and 12.724 ng/g for dry rice leaf and 12.678 ng/g in normal rice leaf by HPLC-MS method.It shows that the BRET method has been successfully established and can be used to detect abscisic acid in practical samples.3.Based on the fluorescence characteristics and specific characteristics of PYL2-mNeonGreen,a fluorescence detection method for abscisic acid has been established.Two recombinant vectors,pET28a-PYL2-mNeonGreen and pET28a-mNeonGreen,have been constructed.The fusion protein was expressed in Escherichia coli BL21.After purification by HIS column,the results were identified by SDS-PAGE.After adding abscisic acid to the buffer containing PYL2-mNeonGreen,the fluorescence intensity of the fusion protein was gradually decreased.The net change in fluorescence intensity caused by the addition of ABA by substracting the background caused by mNeonGreen autofluorescence photobleaching was obtained with the linear relationship of y=0.02679x+0.04728(R2 = 0.9978)and the detection limit(LOD)0.04728 ?M.The method can be used to measure the concentration of ABA.