Research On Artificial Pepaptamers For Inhibition Of Fusarium Oxysporum F.sp.Cubense FOC4

Banana wilt is a problem that has plagued the tropical banana industries for many years.Fusarium oxysporum f.sp.cubense(FOC)is a pathogenic organism of Banana wilt,which can infect banana roots and cause vascular lesions,leading to lignification of vascular bundles.Because Fusarium oxysporum is widely distributed through the soil,it is recalcitrant to control and cause devastating damage to the banana industry.Researches on banana fusarium wilt have been the focus of research on tropical crop diseases.At present,more and more high-tech is applied to the control of banana fusarium wilt.Artificial peptide aptamers(or pepaptamers)are short peptides that can be selected from artificial synthesized random amino acid libraries and can bind target proteins with high specificity and high affinity,which can be used for the development of biological antifungal agents with potential application value.The application of artificial peptide aptamers in the control of banana fusarium wilt is a new breakthrough in the prevention and control of diseases in the banana industry.In this study,high-capacity peptide aptamer libraries were applied to conduct antibacterial experiments on Fusarium oxysporum spores.Specific inhibitors of Fusarium oxysporum were screened out,which were harmless and environment-friendly to banana plants,and preliminary study of its mechanism of action.The results of the study were summarized as follows:(1)Peptide aptamer SNP-D4 was screened with inhibitory effect on Fusarium oxysporum.The sequencing results showed that the total length of the peptide aptamer SNP-D4 was 576 bp and encoded 191 amino acids.The molecular weight of the whole protein was 21 kDa.(2)The prokaryotic expression vector of SNP-D4 and scaffold protein SN were constructed.Through the exploration of inducing conditions,SNP-D4 and SN protein were induced successfully,and purified in soluble forms.(3)Bacterial whole-protein extract expressing artificial peptide aptamer SNP-D4 was used for antibacterial test,and it reduced the germination rate of Fusarium spores to 19.60%,and it reached the extreme significant difference compared with the control group PBS.The scaffold protein SN of the artificial peptide aptamer did not significantly affect the spore germination rate(the germination rate of 72.10%),indicating that the antibacterial function of the artificial peptide aptamer was contributed by 16 aa of variable peptide segment in the artificial peptide aptamer SNP-D4.(4)The purified SNP-D4 increased the inhibition on the spore germination rate of Fusarium oxysporum spores accompanying with gradual protein concentrations.Using 4 μM or more of SNP-D4 reduced the germination rate to extremely significant levels.(5)To investigate whether the SNP-D4 can permanently kill F.oxysporum,the number of colonies was plated and grown on PDA medium after 8μM SNP-D4 treatment,resulting in the significantly lower growth compared with the control groups PBS and SN,indicating that SNP-D4 had a fungicide effect on FOC4.(6)To ensure whether SNP-D4 damaged the outer membrane of pathogenic spores,PI staining was performed.After treated by SNP-D4,the pathogenic spores emitted red fluorescence under excitation light,while the negative control had no such phenomenon,indicating SNP-D4 destroyed the outer membrane of spores.(7)The pull-down assay combined with mass spectrometry analysis was performed to study the interaction between SNP and the total protein of spores.Eight candidate proteins were identified from spores by mass spectrometry.The proteins included W9HRQ49,N1RI26,F9G3P4,W9PJB7,A0A0D2YBZ1,A0A0D2Y610,F9F542 and X0ADE9.(8)The convolutional neural network was used to predict the interaction proteins between SNP-D4 and pathogenic spores.Compared with the results of mass spectrometry,it was found that 3 candidate interaction proteins were repeatedly identified,including W9PJB7,the aldehyde dehydrogenase protein.As a key enzyme in the biometabolic pathway,many antibiotics were achieved to inhibit aldehyde dehydrogenase previously.(9)The molecular docking model of SNP-D4 and W9PJB7 was established.It was speculated that SNP-D4 might interacted with Tyr437(near the catalytic center of W9PJB7)via Thr66 on the peptide loop,indicating that SNP-D4 functioned as suppression of acetaldehyde dehydrogenase activity and consequently impaired metabolism of ethanol to achieve antifungal effects.