HE-TAIL PCR, a powerfull tool for identification of Trichoderma harzianum lip1 gene

Abstract

Trichoderma harzianum is a widespread soil fungus, known as a biocontrol agent against soilborne plant pathogens. Species of Trichoderma are commercially applied as biological control agents against plant fungal pathogens based on different mechanisms, such as antibiosis, competition for nutrients and mycoparasitism. One of the mechanisms involved is the production of several lytic enzymes. In T. harzianum has been identified several glucanases, celulases, chitinases and proteases, but nothing are already known about its lipolytic system. In this work we described the identification of lip1 from T. harzianum, a gene encoding the first extracellular triacylglicerol lipase known in this specie. A cDNA library of T. harzianum CECT 2413 cloned in pBluescript SK+ was screened, and the expressed sequence tags (ESTs) with more similarity against lipase gene sequences in international databases selected and sequenced. Elucidation of complete gene nucleotide sequence of lip1, including 52 bp of the open reading frame at the N-terminal region and 693 bp of the promoter region were achieved by high-efficiency thermal asymmetric interlaced (HE-TAIL) PCR (Michiels et al., 2003), which confirmed to be a powerful tool to identify flanking regions from short known sequences. Lip1 codifies a 558 amino acids protein, with 59322,6 KDa and a calculated global iso-electric point value of 4,56. The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AM18087.