Cloning and characterisation of two subtilisin-like protease genes from Neotyphodium lolii : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Molecular Genetics at Massey University

Abstract

PCR amplification of Neotyphodium lolii genomic DNA with degenerate primers detected two different sequences with homology to subtilisin-like proteases. These two PCR products were used to screen a N. lolii Lp19 genomic library. The prt1 gene was isolated by screening the genomic library with the GH30 PCR product. This gene encodes a putative peptide of 434 amino acids that is most similar to subtilisin-like proteases from Aspergillus sp. The prt1 gene contained a single intron, which was in a position conserved with other fungal genes. 3'RACE was used to determine the polyadenylation site for the prt1 gene. Repetitive DNA was a feature of both the 3' untranslated region (UTR) and sequences downstream of the prtl gene. Within the 3' UTR, a complex microsatellite was found extending over 50 base pairs. Downstream of the gene, a minisatellite locus of 360 base pairs in size was found, consisting of 40 copies of a 9 base pair AT-rich repeat. Expression of prt1 was examined in cultures with various types of carbon and nitrogen sources. Although no conclusive results could be drawn, the type of carbon and nitrogen available did have some effect on prt1 expression. Repression of prt1 expression was only observed in media supplemented with sucrose and glutamate. A 500 bp fragment from the prt1 promoter was introduced into the vector pFunGus to create a translational fusion with gusA. This vector, pMM9, was transformed into Penicillium paxilli. Although transformation frequencies were low, the transformants obtained appeared to be stable for hygromycin resistance. Expression of GUS was observed in seven out of twelve of the stable transformants. This showed that the promoter fragment in pMM9 was sufficient for expression of GUS in a heterologous system. The prt2 gene was isolated by screening a genomic library with the GH3 PCR product. Partial sequence has been obtained for the prt2 gene. The prt2 gene contains at least three introns, the first of which is conserved with prt1. From the sequence obtained, prt2 encodes a peptide with strong similarity to subtilisin-like proteases from Metarhizium anisopliae, a fungal pathogen of insects.