Molecular cloning, characterisation, and expression of a neutral trehalase from the insect pathogenic fungus Metarhizium anisopliae.

A neutral trehalase gene (NTH1) was isolated from a lambdaEMBL3 genomic library of the insect pathogenic fungus Metarhizium anisopliae. Sequencing of the gene revealed extensive homology with other fungal neutral trehalases. The NTH1 gene exists as a single copy in the genome. Two STREs exist in the 5'UTR of NTH1, which may mediate transcriptional activation of the NTH1 gene in response to various stresses. The NTH1 gene encodes a protein of 737 amino acids with a calculated M(r) of 83.1kDa. A cyclic adenosine 3',5'-monophosphate-dependent phosphorylation consensus site and a putative calcium binding site were found in the amino-terminal domain of NTH1, consistent with a regulatory enzyme. Expression of the trehalase cDNA was achieved in Saccharomyces cerevisiae. Southern blot analysis of RT-PCR products indicated that the neutral trehalase gene is transcribed in vitro in cell-free haemolymph of the tobacco hornworm Manduca sexta and in vivo in the early stage of infection.