Genes and vector constructions

The BG genes including bgl1 (GenBank accession: {"type":"entrez-nucleotide","attrs":{"text":"D64088","term_id":"984785"}}D64088) from A. aculeatus (abbreviated as Aabgl1) and bgl1 (GenBank accession: {"type":"entrez-nucleotide","attrs":{"text":"KJ739789","term_id":"665510696"}}KJ739789) from A. niger (abbreviated as Anbgl1) were codon-optimized according to the T. reesei’s codon preference [28]. The genes, bgl1 (GenBank accession: {"type":"entrez-nucleotide","attrs":{"text":"U09580","term_id":"493579"}}U09580) and cbh2 (GenBank accession: {"type":"entrez-nucleotide","attrs":{"text":"M16190","term_id":"170540"}}M16190) from T. reesei which were abbreviated as Trbgl1 and Trcbh2, do not need codon-optimizing because they were expressed homologously. All these gene sequences, whose signal peptide sequences and introns were deleted, were synthesized by Generay Biotech (Shanghai, China).

By subcloning, these sequences were linked to the promoter Pcbh1 with the signal peptide sequence of cbh1 and the terminator Tcbh1 from T. reesei to form the strong expression cassettes, which were then linked to the plasmid pCAMBIA1300 to form the final vectors [4, 7], as shown in Fig. 1.

a The recombinant plasmid pCAMBIA1300-Ps-Trbgl1-T with the strong expression cassette of T. reesei bgl1 gene; b the recombinant plasmid pCAMBIA1300-Ps-Aabgl1-T with the strong expression cassette of A. aculeatus bgl1 gene; c the recombinant plasmid pCAMBIA1300-Ps-Anbgl1-T with the strong expression cassette of A. niger bgl1 gene; d the recombinant plasmid pCAMBIA1300-Ps-Trbgl1-T-Ps-Trcbh2-T with the strong expression cassettes of T. reesei bgl1 gene and T. reesei cbh2 gene; e the recombinant plasmid pCAMBIA1300-Ps-Aabgl1-T-Ps-Trcbh2-T with the strong expression cassettes of A. aculeatus bgl1 gene and T. reesei cbh2 gene; f the recombinant plasmid pCAMBIA1300-Ps-Anbgl1-T-Ps-Trcbh2-T with the strong expression cassettes of A. niger bgl1 gene and T. reesei cbh2 gene. The hygromycin B resistance marker (PtrpC-hygB) and the kanamycin resistance marker (Kan) in the plasmid were used for selection of fungal and bacterial transformants, respectively