[Cloning and characterization of the neutral trehalase gene in Metarhizium anisopliae CQMa102].

The partial fragment of the neutral trehalase (NTL) in Metarhizium anisopliae CQMa102 was amplified by the polymerase chain reaction (PCR) with primers designed according to the sequences of the NTL in GenBank. The amplified fragment was cloned and sequenced. Based on the known sequence of NTL gene, the 5' and 3' rapid amplification of cDNA ends (RACE) were used to amplify the 5' and 3'regions of the NTL cDNA, then the whole cDNA sequence of NTL gene in M. anisopliae CQMa102 was obtained by combining the above sequences and its whole genomic DNA was amplified by PCR. In order to obtain more regulatory information of the NTL, panhandle polymerase chain reaction strategy was used to amplify the 5' flanking sequences adjacent to the known sequence of the neutral trehalase gene. The sequence analysis shows that the DNA sequence is 3484bp in size, includes three introns, and the cDNA sequence is 2385 bp in size, encodes a protein of 737 amino acid residues with a calculated Mr of 83.1kD, in which there is a cyclic adenosine 3', 5'-monophosphate-dependent phosphorylation consensus site and a putative calcium binding site, which is consistent with a regulatory enzyme. Comparison of this sequence with the NTL of M. anisopliae ME1 in GenBank (GenBank Accession: AJ298019, AJ298020) shows that the nucleotide homology is as high as 93%, and the amino acid homology comes up to 99%. Southern analysis indicated that NTL gene was present as a single copy in this M. anisopliae strain. A single transcript of approximately 2.5 kb was detected by Northern blot analysis. The NTL mRNA was present throughout spore germination in rich medium, but accumulated to its highest level at the early stage of exponential growth. Thereafter, the mRNA level declined at the late stage of exponential growth, but began to show an increase during the stationary phase of growth. A 982bp upstream sequence of NTL was amplified using panhandle PCR method, which contains one stress response element (STRE). The NTL nucleotide sequence and its amino acid sequence have been accessed by GenBank (Accession: AY557613, AY557612).