Isolation, Purification And Characterization Of Polygalacturonase Of Rhizoctonia Solani Causing Rice Sheath Blight And Cloning And Expression Of Rspg1 Gene Of The Pathogen

Polygalacturonase (PG) produced by Rhizoctonia solani, the pathogen of rice sheath blight, was one of important pathogenitic factors. The crude PG in the culture filtrate could be precipitated with acetone and successively purified by DEAE Sepharose Fast Flow ion exchange column, Phenyl-Sepharose 6 Fast Flow hydrophobic column, Sephadex G-75 gel column and DE52 ion exchange column. The molecular weight of the purified PG was 39.81 kD by SDS-PAGE and the pI value was 4.58 by IEF-PAGE. The PG was demonstrated as a glycoprotein that contained 1.48% saccharide andα-amino acid, but no lipid and ?. The PG activity was observed in the range of pH value from 4 to 12, and the strongest one was at pH 5. The PG was not stable at the higher temperature (≥40℃) and lost its activity at 100℃for 20min. The PG was also sensitive to ultraviolet radiation, chloroform, trypsin and proteinase K, and its activity was decreased by 60.0%, 48.3%, 65.0% and 68.3% respectively after their treatment.A pair of primers was designed to amplify the complete sequence according to R. solani PG gene nucleotides in GenBank (FJ544455, FJ544456, DQ848983) by polymerase chain reaction (PCR). After being cloned and sequenced, PG gene Rspg1 of R. solani cauing rice sheath blight consisted of 1395bp, with five introns of 57bp, 57bp, 59bp, 66bp, 61bp. The Rspg1 sequence showed a strong similarity (99%) to that of pg1 (FJ544456) in corn sheath blight pathogen when being blasted.The Rspg1 gene was constructed in an expression vector pET-28a (+) resulted in plasmid pETRspg1, and expressed in E. coli BL21 (DE3) by IPTG induction. The result showed that the PG activity of 277.78 U/mg was detected when BL21 containing pETRspg1 was cultured in LB medium and induced with 0.4mM IPTG.