Analysing Of Heterogenous PGIP Protein Against Rice Sheath Blight And Screening Of Toxin-degrading Bacteria

Rhizoctonia solani Kühn belongs to the Rhizoctonia genus,which is divided into14 anastomosis groups(AGs),that is,AG-1 to AG-13 and AG-BI,most of which are phytopathogenic fungi.Studies have shown that the main pathogenic factors are cell wall degrading enzyme and toxin.Rhizoctonia solani has a wide range of hosts and is of great harm.Because of Rhizoctonia solani infection rice yield is sharply reduced every year.At present,neither resistance material has been found in rice germplasm resources that is fully immune to sheath blight,nor have stable main effect quantitative trait loci were detected,and there is no good new resistance gene resource.Therefore,this research mainly focuses on two aspects: cloning and analysis of polygalacturonase inhibiting protein(PGIP)genes with potency of antifungal diseases,and sheath blight resistant rice were obtained through transgenic technology.Purifing,analyzing activity and identifing active components of toxins which is the main pathogenic factor of Rhizoctonia solani,and screening of toxin-degrading bacteria and candidate toxin-degrading enzymes.The main results are as follows:1.Eight different PGIP genes were cloned from soybean,tomato,melon and other plants: CucmPGIP1,TriPGIP,GlyPGIP1,GlyPGIP2,CurPGIP,CapPGIP,BraPGIP and SolPGIP.Through bioinformatics analysis,CapPGIP does not form a hydrophobic signal peptide at the N-termination,8 PGIPs meet the criteria of the PGIP protein family: they contain major functional structure LRR repeats,conserved cysteine residues mianly located at the N-or C-termination and potential NGlycosylation sites;a hydrophobic signal peptide is formed at the N-termination,and the signal peptide cleavage sites are all behind serine.2.Eight PGIP gene vectors were constructed and transformed into DH5? to obtain positive DH5? strains containing recombinant plasmids.Transgenic rice with SolPGIP gene was obtained by gene gun-mediated method.Transgenic rice showed strong resistance to sheath blight in greenhouse.3.HPLC,LC-MS/MS,and biological activity determination were used topurify the Rhizoctonia solani toxins,analyze their activities,and identify the active ingredients.The results showed that the toxin active ingredients contained 70 substances,including sugars,sugar alcohols,carboxylic acids and carboxylic acid esters(salts).the biological activity of crude toxin revealed that tobacco leaves were necrotic at a concentration of 20 mg/mL;when the concentration reached 100 mg/mL,the rice radicle could hardly grow;the toxin would change the permeability of plant cell membranes and cause electrolyte extravasation;25 mg/mL toxin significantly increased defense enzyme activity in rice,but with the treatment time prolonged,each enzyme activity decreased.3.Selected strains A-6,D-5 and E-8 belonging to Pantoea,Enterobacter and Arthrobacter,each of which can use toxins as the sole carbon source.Analyzing the effect of each strain to Rhizoctonia solani,the ends of Rhizoctonia solani close to the the A-6 and D-5 strains are obviously transected,and the sclerotia formation is later and the number less.Strain D-5 has the best ability to degrade toxins..A full-spectrum analysis of the protein showed that the composition of the strain A-6 protein solution was complicated,so UniProt search was performed on the D-5 and E-8 protein full--spectrum analysis results,and it was found that the D-5 protein solution contained 4important hydrolases: Carb1,Pept,Carb2,and Poly.It was found that the E-8 protein solution contained 3 glycoside hydrolases.