Gene Cloning, Expression And Function Analysis Of β-1,4-endo-cellulase From Rhizoctonia Solani AG-1-IA

Corn sheath blight is a soil-borne disease in the Corn Belt around the world,which has become an important disease in corn production and increased gradually.The main pathogen of corn sheath blight is Rhizoctonia solani Kühn, which belonged to Rhizoctonia, having a widespread host range, including rice, corn, soybean, barley, wheat, and so on, causing sheath blight. Rhizoctonia.solani can produce many cell-wall degradation enzymes, such as Polygalacturonase (PG), cellulase(Cx), and become the main pathogenicity factor.In this study, degenerate primers were designed on the conserved domain of other reported glycoside hydrolase family 45 protein. Through RT-PCR, 3'RACE PCR and 5'-TAIL PCR, twoβ-1,4-endo-celluase genes were cloned from Rhizoctonia solani AG-1-IA, named EG-1 and EG-2 respectively. The full-length of EG-1 DNA gene is 1061bp, containing seven introns, which accession number of cDNA and DNA in GenBank is GU372731 and GU372728 respectively; The full-length of EG-2 DNA gene is 1086bp, containing seven introns, which accession number of cDNA and DNA in GenBank is GU372730 and GU372729 respectively. The gene of EG -1 and EG-2 constructed recombination expression plasmid pPIC9K/EG-1 and pPIC9K/EG-2, linearized by restriction enzyme Sac I, then transformed to Pichia pastoris GS115. The recombinant P. pastoris PEG-45 was obtained from pPIC9K/EG-1, its expression level was up to 0.35mg/mL at the eight day. The molecular mass of a single band of the enzyme was estimated to be 28.05kDa, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum reaction pH value of the expressed beta-1,4-endo-cellulase was 3.0, and the optimum reaction temperature was 45℃. The recombinant P. pastoris of pPIC9K/EG-2 was not detected target protein after fermentation.we inoculated Rhizoctonia.solani to corn leaves, and found the hypha have a powerful infection ability. Through RT-PCR, we confirmed thatβ-1,4-endo-cellulase EG-1, EG-2 and EGIII can express in the process. By changing inductive substrates of Marcus medium, we found the optimum substrate forβ-1,4-endo-cellulase is CMC-Na.we inoculated the corn leaves by purifiedβ-1,4-endo-cellulase from PEG-45, taken inactivated endocellulase as contrast, and found that there were obvious disease spots in vaccination sites and expanded along the veins while there were no expansion disease spot in the contrast. Through RT-PCR, we proved some defense genes expressed in the process, such as PR, POD, PAL. This study make a primary study from physiological detection and gene expression aspects for corn sheath blight, the research results will lay the foundation for further study the disease mechanism, foster new species and develop effective chemicals for sheath blight.