Cloning And Functional Analysis Of Polygalacturonases From Fusarium Oxysporum F. Sp.Nelumbicola

Rhizome rot of lotus is an important soil-borne disease on lotus that caused by Fusarium oxyporum f. sp. nelumbicola in China. In this research, a series of cell wall degrading enzymes(CWDEs) were found in Rhizome rot of lotus pathogens, including polygalacturonase(PG), polymethygalacturonase(PMG), cellulase(Cx), polygalacturonic acid trans-eliminase(PGTE) and pectin methyl trans-eliminase(PMTE). The PGs of pathogens were considered as important virulence factors in infecting host plant. In order to know the function of PG isozyme genes of Fusarium oxyporum f. sp. nelumbicola, the main research contents were arranged as follows:1. The determination of CWDEs activities. In order to know the major CWDEs of rhizome rot of lotus in pathogenic processes, activites of five main CWDEs were measured. The results showed that the highest activities of polygalacturonase(PG) and cellulase(Cx) were 4.89 and 6.38U/mg, and the maximum activities of polymethylgalacturonase(PMG), polygalacturonic acid transeliminase(PGTE) and pectin methyltranseliminase(PMTE) were 2.60, 0.531 and 0.093U/mg. The activities of PG, and Cx were significantly higher than PMG, PGTE and PMTE, which indicated that PG and Cx were the major CWDEs in pathogenic processes.2. The full-length c DNA named pg1 and pg5 of PG gene were cloned by RT-PCR and RACE from Fusarium oxyporum f. sp. nelumbicola respectively. The cloned pg1 consisted of a single open reading frame(ORF) encoding 371 amino acids, while the cloned pg5 consisted of a single ORF encoding 360 amino acids. The two genes both contained two highly conserved regions of PG. A BLAST search of the deduced amino acids sequences of pg1 and pg5 demonstrated similarities to PG of other species. Both pg1 and pg5 were the closest to pg gene of Fusarium oxyporum by UPGMA analysis. In addition, 1319 bp DNA code region of pg1 was also obtained, which is composed of 5 exons and 4 inrtons. 1131 bp DNA code region of pg5 was also obtained, which is composed of 2 exons and 1 inrton. All of the exon/intron junction sequences conform to the GT/AG rule.3. The expression of PG genes. The full length c DNA of pg1 and pg5 were cloned into prokaryote expression vector PET30 a. Then the recombinant vectors PET30a/pg1 and PET30a/pg5 were transformed to the host strain BL21. The recombinant vectors were screened by PCR method and the expression recombinants were induced by IPTG. A protein about 38 KDa was found by SDS-PAGE analysis of the recombinant pg1, while the protein produced by the recombinant pg5 about 37 KDa.