Labelling The Phosphate-Solubilizing Strain P8 Of Penicillium Oxalicum With GFP And Hygromycin Resistance Genes And Its Colonization In Rhizosphere Of Crops

Penicillium oxalicum strain P8 has a high Phosphate-solubilizing ability in soil which was screened out by professor Fan Bingquan from Soil and Fertilizer institude of CAAS(Chinese Academy of Agriculture Science). In greenhouse experiment Penicillium oxalicum strain of P8 can promote growth of maize, peanut and canola. The main purpose of the study is to mark the strain P8 with GFP and hygromycin resistance genes and study the rhizosphere colonization wih fluorescent microscope and confocal laser scanning microscop (CLSM).Fungus expressing vector pHYGEGFP was obtained with gene recombination technology. The backbone of pHYGEGFP derives from the plasmid pNOM102 .The fusion gene hph-egfp (hygromycin resistance gene and enhance green fluorescence protein gene) was fused into the downsteam of the GPD (glyceraldehydes-3-phosphate dehydrogenase) promoter and the upsteam of TtrpC terminator on the backbone.Conditions for isolation and regeneration of protoplasts from P. oxalicum strain P8 were studied. The optimum protoplast isolation was the 18-h-cultured hypha digested with 25mgmL"1 snailase and cellulase in 0.6 mol/L NaCl (pH 6.0) for 2.5-3 hours at 34℃, the yield of the protoplasts reached to 10⁶ 10⁷cfu mL^-1. In this study, the maximum regeneration rate of more than 60% was obtained in the CM medium containing osmolutes of 0.6molL^-1'NaCl.Plasmid pHYGEGFP was transformed into the protoplasts of P. oxalicum strain P8 by PEG mediated transformation. Five transformants which could all express GFP and hygromycin B resistance genes effectively and steadily were obtained. The morphology and P-solubilizing ability of five transformants did not show significant difference with the wildtype strain of P8. The result of southern blot conformed that the exogenous DNA had been integrated into the genomes of the transformants heterogeneously.Since the GFP- marked strain of P8-b could be visualized directly in siol with CLMS, it was used to study the colonization in rhizosphere of maize. Results indicated that...