Research On The Diagnosis Method Of Wheat Stripe Rust Based On Single-base Resolution Recombinase Polymerase Amplificatio

Fungal diseases cause disastrous crop losses in epidemic years and pose a major threat to sustainable food production.Among them,stripe rust(also known as yellow rust)is a severe fungal disease caused by Puccinia striiformis(Pst)(P.striiformis),resulting in significant wheat yield reductions in the pandemic years.With the extensive use of synthetic chemical fungicides,fungicide-resistant crop fungal isolates have been frequently reported in major crop-producing areas.Currently available techniques to detect crop fungal pathogens are mainly the enzyme-linked immunosorbent assay(ELISA),polymerase chain reaction(PCR),and isothermal nucleic acid amplification.These molecular detection techniques have greatly improved the diagnostic efficiency of pathogenic fungal infections,but it is still difficult to detect the drug resistance of pathogenic fungi.In this paper,the isothermal nucleic acid amplification method,CRISPR-Cas system,and targeted editing protein probe are used to develop a highly sensitive detection method of single base mutation and a preliminary exploration of single base resolved RNA modification analysis method,which can be applied to the early diagnosis of wheat stripe rust and pathogen resistance analysis,providing a new tool for the prevention and control of wheat disease.This paper mainly includes the following two parts:1.We enabled RPA to resolve single-nucleotide mutation by designing an amplification refractory mutation system(ARMS).ARMS has been integrated with PCR to detect mutations.We introduced it to isothermal nucleic acid amplification and constructed an isothermal ARMS(termed i ARMS)assay to identify mutation-carried fungicide-resistant crop fungal pathogens.By introducing and optimizing mismatch bases in primers,the ability of primers to distinguish single base mutations was significantly improved.In addition,CRISPR-Cas12a was introduced to specifically detect RPA amplicons further reducing interference in non-specific amplification.We optimized the reaction conditions of the experiment.The optimal primer pairs were P2 and R02G,the optimal time of trans cutting of Cas12a was 30 min,and the optimal concentration of g RNA and Reporter was 60 n M and 500 n M,respectively.Based on the cascade identification and amplification process of ARMS-RPA and Cas12a/g RNA,the LOD of i ARMS is as low as 25 a M and the detection ability of low-abundance mutations is 0.1%.Compared with traditional sequencing methods,the sensitivity of the i ARMS method is significantly improved,which is conducive to the detection of low-abundance drug-resistant fungal pathogens.i ARMS could be proceeded at a constant temperature and thus is promising to serve in in-field crop diagnostics.Based on the i ARMS method,the phenotypes of 83 strains of Puccinia striiformis were investigated.It was found that the proportion of triadimefon-resistant Puccinia striiformis was high in Qinghai Province,Sichuan Province and Xinjiang Autonomous region.2.Based on i ARMS detection,targeted editing proteins were further constructed to develop crop RNA methylation detection methods and to explore host RNA methylation as a novel marker of disease infection.When crops are infected with fungal diseases,the cell RNA methylation level is correspondingly changed.The detection of RNA N6-adenylate methylation(m~6A)methylation is expected to be used in the diagnosis of disease infection.A targeted editing protein probe was constructed by fusing the writer YTH domain of RNA m~6A with cytosine deaminase(APOBEC-1).The YTH domain specifically binds to the RNA m~6A methylation site,and APOBEC-1 converts cytosine to uracil near the binding site.Furthermore,the information on RNA modification is converted into information on single base mutation.The recombinant plasmid containing the p ET-28 vector,APOBEC-1 gene,and YTH gene was constructed by seamless cloning technology,and the fusion protein was expressed in Escherichia coli.Then the targeted editing protein was obtained by nickel column purification and ultrafiltration tube concentration.Through transcription,a sequence with methylation of RNA m~6A was synthesized as the target sequence,and the RPA primer was optimized to realize the specific differentiation of editing sites.Using the optimized RPA primer,the editing efficiency of fusion protein was tested by the i ARMS method using the RNA template before and after fusion protein editing.However,there was no significant difference in RNA sequence signals between the experimental and control groups.It was found by sequencing that currently constructed targeted editing proteins failed to edit the bases near the m~6A site,so further optimization of fusion proteins including linker and other structures was needed.This work proposed the use of host RNA modification as a marker of pathogenic fungal infection and provided a new way for the diagnosis of crop diseases.