In this study, Metarhizium collagen -like protein (MCL1) promoter from Metarhizium anisopliae was analysed and truncated into different sizes through series of targeted and random deletions based on the presence of various transcription factor-binding sites. Synthetic Green Fluorescent Protein (sGFP) was being utilized as a reporter gene to study the relative expression driving capability of unmodified and truncated promoters. Conserved promoter sequence analysis revealed similarity between the paralogous promoters from M. brunneum and M. acridum. sGFP expression in the haemolymph was directed with the help of mcl1 signal peptide sequence. Deleting the promoter region from - 2764 to - 1583 bp increases the promoter mcl1 (Pmcl1) activity by twofolds, while deletions of the regions upstream of - 1150 bp and - 840 bp caused a decrease of sGFP expression level (80% and 70%, respectively). Transcriptional binding sites predicted for the deleted region revealed the loss of upstream repressing sequences such as Matalpha2 along with ROX1 and Rap1 repressor-binding sites located - 2234 bp, - 1754 bp and - 1724 bp from the TSS. Compared with Pmcl1-wild type (2.7 kbp), Pmcl1-1583 bp had a shorter sequence and showed statistically significant expression in M. anisopliae. This study introduces a highly efficient strong inducible promoter for over-expression of target genes in M. anisopliae.
Keywords: Entomopathogenic fungi; Gene expression; Mcl1 promoter; Metarhizium collagen-like protein; Promoter truncation.