Construction and application of yeast two hybrid cDNA libraries of Phanerochaete chrysosporium

There has been no report on protein - protein interaction in Phanerochaete chrysosporium, a model microorganism for studying lignin degradation. In this study, two yeast two-hybrid libraries containing 2.5 × 10⁵ and 3.0 × 10⁵ recombinants, were constructed using 2 d and 3 d mRNAs derived from P. chrysosporium cultures growing in low-nitrogen medium. 14-3-3 gene was used to screen the 3-day library by yeast two-hybrid technique and more than 600 clones were obtained on the SD/-Ade/-His/-Leu/-Trp plates. Thirty positive recombinants could activate three reporter genes. Plasmids isolated from yeast transformants were successfully transformed into Escherichia coli. The thirty clones could be classified into 4 types on the basis of the digestion patterns by Hae III and Sau3A I, as well as their sequence analyses. Results showed that one type of clones encoded 14-3-3 protein, implying that homodimer could be formed between two molecules of 14-3-3 protein. The other three types of clones encoded unknown proteins. However, the Y2H333 contained an OB-fold nucleic acid binding domain on the basis of analysis in SBASE. These results demonstrated that the yeast two-hybrid cDNA libraries were qualified for studying protein - protein interactions and 14-3-3 protein may play important roles in P. chrysosporium.