| It is beneficial to study the lignin degradation by filamentous fungi in gene level. Agrobacterium tumefaciens-mediated transformation (ATMT) is an efficient tool for filamentous fungi genomic study because of its high frequencies of transformation, stable DNA integration, random insertion and wide application comparing to traditional methods.Penicillium simplicissimum H5 was isolated from soil, with the lignocellulose-degrading capability, while the degradation mechanism and characters of produced enzyme were not clear. In this study, ATMT of Penicillium simplicissimum H5 was successfully constructed, which will facilitate the functional genome study and gene engineering modification for this organism.A plasmid named pBHtl containing the hygromycin resistance gene was tranfered to Agrobacterium tumefaciens AGL-1 by freeze-thaw method and then co-cultured with spores of H5 for two days. The transformants were screened by hygromycin-containg medium. The integration of the hph gene into H5 genome was determined by PCR and Southern blot analysis. The results showed that T-DNA was inserted into the transformant randomly and mainly with single copy.The fungal transformants were then charcterized via Congo red decolorization assay. Cmparing to the wild type, one strain showed better growth status in nutrient-limitated media and obvious high cellulose degrading capability was found. Transformants were cultured in liquid medium for 7 days, then the culture liquid was concentrated and proteins were separated and purified through ammonium sulfate precipitation and dialysis. SDS-PAGE analysis showed that the transformants produced more extracellular protein than the wild-type strain, especially for the proteins at about 30kD. Further study to explain above phenomenon is necessary.
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