Using degenerate PCR and TAIL-PCR,a protein kinase gene OPK1(Genebank accession No.:EU417815) was cloned from mycoparasite fungi Olpitrichum tenellum.OPK1 has an open reading frame of 2 031 bp interrupted by two introns(108 bp,84 bp) and putatively encodes a protein of 676 aa.Phylogenetic analysis indicated that OPK1 was most similar to other serine-threonine protein kinase in fungi.Southern blot analysis indicated that OPK1 is present as a single copy in the genome.RT-PCR showed it could be transcribed both in the phase of spore germination and hyphal growth.
For studying on pathogenicity mechanism of Fusarium moniliforme,REMI was used to transform protoplasts of FT1 strain with the vector pUCATPH,which contained hygromycin B-resistant gene.More than 300 transformants had been obtained,most of them were quite stable after five rounds of successive culture.25 mutants of morphology and 2 weak pathogenicity mutants were gained by REMI.PCR amplification showed that the hygromycin B-resistant gene had integrated into genomes of the two pathogenicity mutants.The optimum conditions of preparing protoplasts were: the mycelia growing in PDB medium for 14 h,lywallzyme was used to digest the mycelia at 100 r/min,30℃ for 4 h,and 0.7 mol/L NaCl was used as the osmotic stabilizer.
