Development FRET And Electrochemical Methods For The Detection Of Abscisic Acid In Plants

Abscisic acid（ABA）is a kind of isoprenoids（steroids）hormones,which regulates the stress response and physiological activities of plants.Therefore,the establishment of an in-situ and real-time detection method for abscisic acid in vivo in plants is of a great significance for studying the mechanism of signal transmission of ABA.Many ABA signaling elements have been identified so far,and their activity is regulated by many factors,including environmental,developmental,and self-expression regulation.Proteins from the PYR-PYL-RCAR family are considered to be ABA receptors.In response to ABA binding,a conformational rearrangement of PYLs generates a new surface epitope that contributes to subsequent dissociation of PYL dimers.Based on the principle of signal transduction pathway and molecular conformation change,this experiment combined electrochemical technology and fluorescence resonance energy transfer（FRET）technology to develop biosensors to detect ABA in vivo in plants.It mainly includes the following four parts:1.The ABA receptor PYL2 from Nipponbare has been expressed prokaryoticly.the PYL2 gene was cloned to construct the p ET26b（+）-PYL2 prokaryotic expression vector,which was transformed into E.coli BL21（DE3）.The protein PYL2 was expressed and purified by HIS column.2.The electrochemical sensor was used to detect abscisic acid.Protein PYL2 was immobilized to the electrode surface by self-assembly method.The addition of ABA causes the conformation of PYL2 molecule to change from dimer to monomer.The conformation of PYL2 changed with the addition of ABA,the electrode needs to continuously give electrons to reduce Cu²⁺（His）to Cu⁺（His）,so the reduction current increases.According to the change of reduction current of Cu²⁺（His）at-0.005 V,ABA can be detected by the electrochemical biosensor.The linear range was 5-25 n M with the linear regression equation was y=0.1297 x+1.487（R²=0.978）and the detection limit 4 n M.The specificity of the electrochemical biosensor for ABA was high.The gold modified carbon fiber electrode（CFE）electrode was selected after being sealed by electrophoretic paint and gold plated.After assembled with PYL2 protein,the abscisic acid releasing from rice protoplasts under abiotic stress was monitored by the gold modified CFE with Amperometric i-t technology.The results were validated by LC-MS.3.Combinating the ABA induce of the abscisic acid receptor PYL2 and phosphatase PP2C Vx GФL domain interaction characteristics with FRET technology,a FRET biosensor for abscisic acid in vitro and in vivo has been developed.Unimolecule FRET sensor was constructed using PYL2 and PP2C AA as recognition unit,cyan fluorescent protein m Turquoise2 and yellow fluorescent protein cp173Venus as reporting unit.The prokaryotic expression vectors p ET28a（+）-PYL2-m Turquoise2-PP2C AA-cp173Venus and p ET28a（+）-m Turquoise2-PYL2-PP2C AA-cp173Venus were constructed using fusion genes by overlapping PCR.The recombinant protein was expressed and purified by Ni column.The in vivo vector p U1301-m Turquoise2-PYL2-PP2CAA-cp173Venus was constructed and genetically transformed into Arabidopsis thaliana is expected to ABA detection and imaging in vivo.4.The FRET biosensor was used to detect abscisic acid in vitro.The optical characteristics of the two FRET biosensors was investigated.Because of the highest sensing efficiency the protein m Turquoise2-PYL2-PP2C AA-cp173Venus was selected.The results showed that the fluorescence emission ratio of the FRET sensor gradually increased with the increase of ABA concentration in the range of 0.1 n M-100μM.The linear regression equation was y=0.0597 x+0.999（R²=0.934）.The sensor has small response to other common plant hormones and it indicated the high selectivity of the FRET biosensor.