Abstract: One-dimensional electrophoresis (1-DE) of proteins, two-dimensional electrophoresis (2-DE) of proteins and cloning of cDNA sequence were used to study the virulence differentiation of Curvularia lunata (Wakker) Boed. isolated from maize (Zea maydis L.) in China. From 1-DE gel profiles of proteins, 110 reproducible bands were separated from six isolates of C. lunata CX-3, SD-6, C-152, C107-1, DD-60 and W-18. Sixty-eight bands (61.82%) were polymorphic, suggesting huge biodiversities among the isolates. All isolates for the experiment were clustered into three groups consisting of different virulent types by coefficient value of 0.605. Group 1, consisting of CX-3, SD-6 and C-152 with high virulence displayed more protein bands than Groups 2 and 3, consisting of C107-1 and DD-60 with low virulence. Proteomics approaches based on 2-DE techniques were applied to identify specific proteins associated with the virulence differentiation in CX-3 and DD-60. A total of 423 protein spots were separated. Out of them 75 specific protein spots were displayed in 2-DE gels. Among them 28 protein spots were unique in CX-3 and eight in DD-60, and 39 protein spots were shown on both 2-DE gels but expressed differently in intensity. Twenty protein spots including three unique protein spots and 17 differentially expressed protein spots (more than two-fold DD-60) in CX-3 were further identified with MALDI-TOF MS/MS. Results indicated that most of the identified proteins were found to be associated with virulence differentiation, metabolisms, stress response and signal transduction. One of them was identified as Brn1 protein, which had been reported to be related to melanin biosynthesis and the virulence differentiation in fungi. Combined with our previous findings, we assumed that Brn1 protein and its regulating products might be involved in the virulence differentiation of C. lunata. Consequently, we cloned a Brn1 cDNA fragment and aligned it with the fragments in other fungi. Results indicated that the 633-bp sequence of Brn1 cloned in C. lunata was highly homological with the compared fungi. Further work for the exact gene roles of Brn1 in our case is underway.

Key words: Brn1 cDNA fragment, Curvularia lunata, mass spectrometry, proteomics, two-dimensional electrophoresis, virulence differentiation, Zea maydis.