Design And Application Of Salicylic Acid Protein Binding Engineering Enzyme

Tobacco is an annual or limited perennial herb of Solanaceae,which is widely used as a model plant in the field of biotechnology.Salicylic acid-binding protein 2（SABP2）was first discovered in tobacco.It belongs to theα/βfolding hydrolase family,which can bind specifically to salicylic acid and play an important role in salicylic acid signaling pathway.SABP2 is a methyl salicylate esterase that converts methyl salicylate into SA,inducing systemic acquired resistance in plants.SA is an important signal that activates plant defense against pathogen infection.SABP2 was identified from tobacco as protein that shows a high affinity for SA and plays a key role in activating systemically acquired resistance to plant pathogens.In this experiment,a recombinant plasmid named pET-21a（+）-SABP2 was constructed using the genomic DNA of SABP2 in tobacco as a template and transformed into DH5αreceptor cells.The wild-type SABP2 gene sequence was designed with site-directed mutagenesis by introducing mutations into primers.From the four designed mutants,the most active one was leucine at position 82 mutated to serine（L82S）.The recombinant bacteria were induced by 10μM IPTG to express the protein in E.coli BL21（DE3）.Protein was purified by agarose affinity column and dialyzed by phosphoric acid buffer.PAGE analysis showed that the recombinant protein was 29 k Da,and the molecular weight of SABP2 protein was almost the same.The enzyme activity of mutant L82S was 10.88 times higher than that of wild-type SABP2by high performance liquid chromatography with Me SA as substrate.Ellman reagent was used to detect enzyme activity with ASM as substrate,and the enzyme activity of L82S was increased by 555.26 times,indicating that the slow reaction,low activity and the need to accumulate a large amount of SA after pathogen infection to activate SAR signaling pathway could be solved by site-directed mutation.In addition,a plant expression vector containing PBI121-SABP2-L82s was constructed,and the genetic transformation of tobacco was carried out by agrobacterium-mediated method.The L82S mutant was overexpressed in tobacco under the control of the promoter,which laid a foundation for the further study of transgenic plants.Bioinformatics analysis predicted that SABP2 protein has a molecular weight of29 k Da and p I of 5.39.Analysis of the amino acid sequence of SABP2 showed that its polypeptide chain is hydrophilic and is a hydrophilic protein.there is no signal peptide sequence at the N-terminal end of the amino acid sequence of SABP2 and there is no digestion site for the N-terminal signal peptide.The prediction of the secondary structure showed that the composition of SABP2 was 41.15%forα-helix,15.77%for extended chain,37.69%for random coiling,and 5.38%forβ-folding.The whole peptide chain is located on the surface of the cell membrane without transmembrane structure.The three-dimensional structural model of SABP2 shows that it has typical structural characteristics of theα/βfolding hydrolase family.ASM is a functional analog of salicylic acid,it can activate defense genes by catalyzing the formation of Acibenzolar and methanethiol（CH₃SH）by SABP2.In recent years,it has been widely used in modern agriculture to protect plants from pests and diseases.ASM can react with methanol transesterification to form methanethiol（-SH）,thereby breaking the disulfide bond of DTNB to form NTB^2-,which appears yellow in the water and is used for pesticide residue detection.The methanol ratio DTNB,EDTA reaction,temperature and reaction time were determined by single factor experiment.The optimized method was applied for the determination of ASM transesterification with methanol and the kinetic data determination of SABP2-catalyzed ASM hydrolysis.The calibration curve of ASM showed a good linear relationship in the range of 25.2μg g^-1-315μg g^-1.The intra-day and intra-day accuracy and precision data were in line with FDA acceptance criteria.Using ASM as substrate,with the K_cat value of 31.1 min^-1,the V_max value of 0.622μM min^-1 using the Michaelis–Menten equation.In tobacco plants treated with 100μM,it was decreased as time elapsed in treated tobacco,reaching negligible values 72 h after treatment.The above studies are expected to lay a theoretical foundation for the application of SABP2.