Gene Cloning, Expression And Pathogenicity Of Endopolygalacturonase From Rhizoctonia Solani Kühn AG1-IA

Corn sheath blight is a soil-borne disease in the Corn Belt around the world, which impact on worldwide corn yield, and cause enormous economic loss. The main pathogen is Rhizoctonia solani Kühn, which belonged to Rhizoctonia. The host range of the pathogen is wide, including the rice, maize, soybean, barley, wheat, and so on. The pathogen can cause sheath blight.Pectin is the main components of middle layer structure of plant cell wall. It can form a tough barrier, together with cellulose and hemicellulose, to block the outside world. Plant pathogenic fungi and bacteria can produce many enzymes which can degrade components of plant cell wall, the enzymes is the main pathogenic factor that plant pathogenic fungi and bacteria penetrate plant cell wall and spread between cells.Polygalacturonase is one of the main enzymes that degrade the pectin of plant cell wall, playing an important role in the pathogenicity of plant pathogenic fungi.The object of this study was Rhizoctonia solani Kühn AG1-IA. The study obtained a endo-polygalacturonase coding gene, imported the coding gene into Pichia pastoris to construct a high-level expression of the gene engineering strain, and then inoculated maize leaves with the purified protein to explore the pathogenic factors of Corn sheath blight from the level of gene and protein.Degenerate primers designed on the conserved domain of other reported endo-polygalacturonases, and a cDNA fragment encoding the endopolygalac -turonase gene from Rhizoctonia solani Kühn AG1-IA was obtained through RT-PCR. The RACE and TAIL-PCR were used to generate full-length cDNA clones. The full length of endo-PG1 cDNA gene is 1095bp, which contained an ORF of 364 amino acids. Then complete DNA encoding the endopolygalac -turonase was cloned and it contained five introns. The cDNA and DNA sequence of gene endo-PG1 has been registered in GenBank with accession number FJ544455 and FJ544456 respectively.The endo-PG1 gene and expression vector pPIC9K were digested with EcoRⅠand NotⅠ, and ligation was carried out in vitro. The recombinant expression plasmid pPIC9K/pg1 was constructed and sequenced to confirm the correct reading frame. The constructed plasmid pPIC9K/pg1 was linearized with a restriction enzyme SalI(insertion at HIS4), and transformed into Pichia pastoris GS115 competent cell by electroporation methods, and Screened for His+Mut+ transformants on MD and MM plates. The parent vector was linearized with the same restriction enzyme and transformed GS115 as a control. PCR analysis of P. pastoris integrants and G418 screening determined the multicopy integrants to induce by methanol. These integrants were used to analyze expression levels of interest protein every 24 hours. The engineering strain with highest expression level was called GS-endoPG-49. The genetic and endo-polygalacturonase expression stability of recombinant P. pastoris GS-endoPG-49 was tested and characterized. Leaves of four-six leaves maize were inocubated with the purified protein, Leaves of maize were inocubated with pH10.0 glycine-sodium hydroxide buffer as a control. The result showed that leaves inoculated by endo-PG1 were diseased after 24h, however, the leaves inoculated by glycine-sodium hydroxide buffer was no symptom. This indicates that the endo-PG1 may be one pathogenic gene of the gene family encoding polygalacturonase from Rhizoctonia solani AG1-IA.