Preliminary Study On Establishment Of Regeneration System Of Finger Blue Mold (penicillium Digitatum) And The Cyp51 Gene Function

The effect of some factors, including the appropriate materials for isolating protoplasts, the concentrations of enzyme, period of digestion and temperatures, and osmotic pressure stabilizers on the isolation and regeneration of protoplasts in Penicillium digitatum were studied. The results demonstrated that the purified protoplasts could regenerate through double layers of Czapek medium containing 0.7mol/L NaCl. The regeneration rate could reach 24.9%. Additionally, the mode of protoplast releasing and regeneration were investigated. It indicated that the primary protoplasts of P. digitatum were released mainly from the hyphalapex, and occasionally from the subapical or original sites. The regeneration of protoplasts could be classfied into two forms: the yeast-like cell developed from the protoplast and the mycelium grew from the protoplast directly.The CYP51 genes cloned from both isolates of imazalil-resistant and imazalil-sensitive of P. digitatum were cloned into the pCB 1004 expression vectors, respectively. The direction and number of inserted copy were confirmed by restriction enzyme digestion. These vectors were tried to transform into the P. digitatum imazalil-sensitive isolate by the methods of PEG-mediated or electroporation transformation. After that, the transformed protoplasts were regenerated on Czapek medium containing hygromycin at 300#g/ml. Unfortunately, the ideal transformants could not be obtained.In order to analyze the differences of the content of ergosterol in the imazalil-sensitive and imazalil-resistant isolates of P. digitatum, the technology of HPLC was used. The results showed that the contents of ergosterol between imazalil-sensitive and imazalil-resistant isolates were not significantly defferent (P=0.05, DMRT) when they grew in the medium without imazalil. Interestingly, when imazalil (0.1#g/ml) was added to the medium, the content of the ergosterol in imazalil-resistant isolate (pd07) was significantly higher than that in imazalil-sensitive isolate (pd23) (P=0.05, DMRT). These results suggested that higher accumulation of ergosterol might be induced by imazalil in the isolate of pd07, which contained4-126bp tandemly repeated in CYP51 gene more than that in the pd23. It also suggested that these tandemly repeated sequence might be an element that could regulate the expression of CYP51 gene.