Fast Determination Of Signaling Molecules In Rice Under Bacterial Blight Pathogen Stress Based On Fluorescence Spectroscopy

In recent years,the problem of rice disease has become increasingly prominent.Taking Zhejiang Province as an example,rice bacterial leaf blight disease is showing a trend of increasing popularity,and thus it is urgent to develop new methods for effectively solve the disease problem.The detection of key signaling molecules under the stress of rice bacterial leaf blight is particularly important for analysis of rice disease mechanism.Fluorescence analysis,as a powerful technique,has the advantages of high sensitivity,fast response and low cost.It has great potential and application prospects in plant molecular analysis.In this study,the signaling molecules related to rice bacterial blight stress were screened based on non-targeted metabonomics technology.Then the fluorescence analysis methods for signaling molecules under rice bacterial blight stress was constructed by using developed fluorescent probes combining fluorescent quantum dots,metal organic framework materials with specific recognition strategies,so as to provide technical support for rice health assessment and analysis of rice disease mechanism.The main research contents are summarized as follows:（1）Based on the non-targeted metabonomics technology,the effects of bacterial blight stress on rice metabolism were studied,and the screening of differential metabolites between infected and non infected groups of rice was realized,which provided data support and theoretical basis for the construction of subsequent fluorescence detection methods for signaling molecules.Non-targeted metabonomics technology was used to comprehensively detect the metabolites of rice samples from the infected group and the healthy group,and the difference between the metabolites of the two groups was compared by multivariate statistical analysis.Compared with the healthy group,rice had significant differences in metabolites after seven days of bacterial blight stress.756 differentially expressed metabolites were screened by OPLS-DA model,with 392 and 364 metabolite contents up and down.According to the KEGG database pathway annotation,328 metabolites were annotated into the 15 metabolic pathways of KEGG,including 3 differential metabolites related to signal transduction,namely abscisic acid,jasmonic acid and salicylic acid.In addition,hydrogen peroxide can participate in rice response to bacterial blight as a signal molecule and is an important substance in signal transduction pathway.Therefore,the above four molecules were regarded as key signaling molecules of rice under bacterial blight stress.（2）Based on the aptamer modification technology,a fluorescence probe with high affinity for abscisic acid was developed,which realized the selective detection of abscisic acid content in rice,solved the problem of the lack of specificity of the existing rapid detection methods for abscisic acid,and provided a new idea for the design of a high specific ratiometric fluorescent probe and the sensitive detection of abscisic acid.The carbon quantum dot@zinc based metal organic framework（CQDs@ZIF-8）with double emission fluorescence properties was prepared by hydrothermal method,and gold nanoparticles（Au NPs）were modified with aptamers that can specifically recognize abscisic acid to achieve specific capture of abscisic acid.ZIF-8 not only avoided the self-aggregation of CQDs,but also made the fluorescence peak of CQDs red shift through intramolecular charge transfer（ICT）,and changed the quenching property of gold nanoparticles（Au NPs）on the fluorescence of CQDs.When abscisic acid is added to the system,the modified aptamers on Au NPs can selectively recognize abscisic acid and cause the aggregation of Au NPs,resulting in the change of the fluorescence signal of CQDs@ZIF-8.According to this change,a fluorescence sensor based on fluorescence resonance energy transfer（FRET）mechanism was constructed for quantitative detection of abscisic acid.The detection limit（LOD）of the proposed method for abscisic acid is as low as 0.11 nmol/L.（3）A novel ratiometric fluorescent probe（NCQDs@Co-MOFs@MIPs）based on molecular imprinting technique was prepared,which realized the low-cost and selective detection of jasmonic acid in rice,made up for the lack of an effective jasmonic acid aptamer and the high cost of the aptamer,and provided a new way for the specificity and accurate quantification of jasmonic acid content in rice.Nitrogen doped carbon quantum dots（NCQDs）with unique fluorescence properties were prepared by hydrothermal method.The NCQDs have two fluorescence peaks with different properties at different excitation wavelengths.Through the characterization of the chemical and physical properties of the probe,it was found that the introduction of cobalt based metal organic frameworks（Co-MOFs）changed the surface charge distribution of NCQDs,enabling NCQDs to interact with jasmonic acid.In order to improve the selectivity of the probe,molecular imprinting technology was used to modify the probe surface with molecular imprinted polymers（MIPs）for jasmonic acid.The MIPs provide imprinted recognition sites with a"key"structure,which can achieve specific capture of jasmonic acid molecules.Taking advantage of the fluorescence properties of NCQDs and the unique role of Co-MOFs and MIPs,a ratiometric fluorescent probe was constructed based on photo-induced electron transfer（PET）mechanism for the quantitative detection of jasmonic acid.The LOD of the probe for jasmonic acid was 1.66 nmol/L.（4）The preparation method of ratiometric fluorescent probe based on the concept of green chemistry was proposed,which realized the accurate and sensitive detection of salicylic acid in rice,solved the problems of high harmfulness and great pollution of traditional fluorescent probe,and provided a new method for the construction of green fluorescent probe and the sensitive detection of salicylic acid in rice samples.Natural silk and curcumin biomass were used as raw materials to obtain fluorescent products,and prepare ratiometric fluorescent probe with dual emission property by doping fluorescent products in iron based metal organic frameworks（Fe-MOFs）.The hollow Fe-MOFs can not only avoid the self-aggregation of silk carbon quantum dots（SCQDs）,but also act as a bridge between curcumin（Cur）and salicylic acid.The Fe²⁺ion in Fe-MOFs interacts with curcumin and salicylic acid to form a ternary complex of Cur-Fe²⁺-SA,which avoids the interference of other metal ions on the detection results.In addition,the formation of the complex changes the absorption characteristics of curcumin,causing the restoration of the inhibited fluorescence signal of SCQDs.According to the above results,a fluorescent sensing platform was constructed based on inner filter effect（IFE）mechanism for the quantitative detection of salicylic acid.The LOD of this probe for salicylic acid was 0.14μmol/L.（5）A fluorescent probe（MoOx QDs@Co/Zn-MOFs）with synergistic peroxidase like catalytic activity was constructed,which realized the ultra-sensitive detection of hydrogen peroxide,solved the problem of low sensitivity of existing hydrogen peroxide detection probes,and provided a new strategy for the preparation of fluorescent probes with high enzyme like catalytic activity and the sensitive detection of hydrogen peroxide in rice.Both molybdenum oxide quantum dots（MoOx QDs）and cobalt/zinc doped metal organic frameworks（Co/Zn-MOFs）have peroxidase-like activity,and the preparation of the proposed probe triggered the synergistic catalytic effect between them.In addition,the prepared probe has a flower-like structure of hydrangea,and the large specific surface area of the structure can provide sufficient sites for the interaction between the probe and the analyte.In o-phenylenediamine（OPD）-hydrogen peroxide（H₂O₂）system,MoOx QDs@Co/Zn-MOFs can efficiently catalyze H₂O₂ to produce hydroxyl radicals（·OH）,and oxidize OPD to obtain fluorescent oxidation products（Ox OPD）.Ox OPD can inhibit the fluorescence intensity of MoOx QDs through FRET mechanism.Based on the above phenomena,a fluorescent sensor with high catalytic activity was constructed for the quantitative detection of hydrogen peroxide.The LOD of the prepared probe for hydrogen peroxide was 32.60 pmol/L.Combined with non-targeted metabolomics and fluorescence spectroscopy,this study explored the influence of bacterial blight infection on rice signal molecules,and realized the rapid and sensitive detection of four key signaling molecules in rice under bacterial blight fungus stress.Ratiometric fluorescence probes with environmental stability was constructed utilizing the unique properties of quantum dots and metal-organic frameworks.The rapid and effective detection of abscisic acid,jasmonic acid,salicylic acid and hydrogen peroxide were achieved by using the aptamer technology,molecular imprinting technology,green synthesis and synergetic catalysis strategy.The successful preparation and application of the above fluorescent probes solved the problems of traditional fluorescent probes in sensitivity,selectivity and stability,and provided effective technical support for the molecular information acquisition of rice bacterial blight disease and the research of rice disease stress mechanism.