Construction Of Abscisic Acid Sensor And Its Application In Detection Of Huanglongbing Infected Citrus

Citrus Huanglongbing?HLB?is one of the most serious diseases in citrus cultivation.The pathogen is a Gram-negative bacteria and is widely spread mainly through citrus psyllids.The occurrence and spread of the disease caused great economic losses to the citrus industry.Abscisic Acid?ABA?is a ubiquitous plant hormone in plants.It can control the shedding of plant organs,inhibit plant growth,and can produce defense and resistance under adverse environmental stress.In view of the important physiological role of abscisic acid in the growth and development of citrus and the stress resistance produced by Huanglongbing bacteria infection,strengthening the prevention and control of Huanglongbing and detection of abscisic acid is of greatpractical significance for the development of the citrus industry.For this reason,there is an urgent need for developing rapid and accurate quantitative detection methods for it.In this work,the electrode was modified by reduced graphene?r GO?and sodium alginate?SA?.The detection of ABA is realized by modifying the ABA monoclonal antibody on the electrode surface,and an electrochemical sensor of ABA is established.The sensor has good selectivity and sensitivity.Taking citrus infected by Huanglongbing bacteria as the research object,the prepared ABA electrochemical immunosensor was used to detect the changes in ABA content in navel oranges infected by Huanglongbing bacteria,and the changes in gene expression of key enzymes in the ABA synthesis pathway was also studied by fluorescence quantitative PCR method.The results is consistent with the result of our sensor,which proves the practicality of the sensor.The specific research results are as follows:?1?Preparation and identification of navel orange samples infected with Huanglong diseaseIn this study,firstly,in the biological laboratory of the Seed Industry Equipment Department of the Beijing Academy of Agriculture and Forestry Sciences,the navel orange was grafted and infected with Huanglongbing bacteria,and then the suspected citrus samples were detected by RT-PCR.The statistical data obtained showed that among the 3 suspected samples amplified,2 of the materials showed the target band consistent with the positive control?703 bp?,indicating that the tested sample was infected with Citrus Huanglong disease.We select the positive materials as samples for subsequent experiments.In another sample,there was no specific amplified target band.It was consistent with the negative control result,indicating that the plant was not infected with Huanglongbing or the pathogen concentration was low,which was not been detected yet.?2?Preparation of ABA immunosensor with high sensitivity and high stabilityThe detection performance of the sensor can be increased by modifying r GO and SA on the electrode surface.And optimize the concentration of r GO,SA,and ABA-Antibody,the optimal conditions for the three materials were 1.5mg/ml,1.25mg/ml,0.5mg/ml.The immunosensor is constructed based on the electrode impedance changes??Z₁-Z₀?due to the binding reaction of ABA antibody and antigen.It is found this sensor showed linear relationship with ABA in the response range of10p M?1?M.The linear equation is y=103.46x+480.73,R²=0.99927,and the detection limit is about 10p M.?3?Detection of ABA by the developed sensor in plants after Huanglongbing infectionThe electrochemical immunosensor prepared by us was used to determine the ABA content in citrus samples after Huanglongbing infection.The test results showed that the impedance changes??Z₁-Z₀?of healthy leaves and diseased leaves were 72 and 823,respectively,which indicated that the level of ABA in the infected plants increased significantly,which is consistent with the results of previous studies.Our sensor provides a powerful tool for the detection of plant hormone levels under disease stress.?4?Study on the expression level of ABA related genes in citrusCit ZEP is the gene of the key enzyme in the pathway of ABA synthesis.During the experiment,we used fluorescence quantitative PCR to detect the expression level of Cit ZEP gene.The results showed that the expression of Cit ZEP increased in plants infected with Huanglongbing disease.This indicates that the increase in Cit ZEP expression caused an increase in ABA synthase,which in turn caused an increase in ABA.This result is consistent with the detection result of our electrochemical sensor,which proves the practicality of the sensor.