Fig. 4

Analyses of HAX1 versions in T. reesei QM6a, QM9414 and Rut-C30. a Schematic illustration of the 782 bp hax1 structural gene (black arrow). Start codon (ATG, red arrow), introns (grey boxes), site targeted by integration of the amdS cassette (yellow triangle) and 5′-region rich in Xyr1-binding sites (green box) are indicated. Previously detected transcripts (Fig. 2c; PCR 1, PCR 2 and PCR 4) are illustrated by orange lines; lower or higher transcript levels are indicated by one or two plus symbols, respectively. Approximate positions of the 3′-end (white arrow) and 5′-ends of HAX1_QM6a (blue arrow), HAX1_QM9414 (yellow arrow) and HAX1_Rut-C30 (purple arrow) defined by RACE-PCR are marked. Positions of the primers used for amplification of the respective hax1 versions displayed in b are given as thin, black arrows. Rev-3′, hax1 rev_3′QM6a; Q6a, hax1 for_QM6a_BcuI; QM, hax1 for_QM9414_BcuI; Rut, hax1 for_Rut-C30_BcuI. Numbers on top indicate position from ATG in base pairs. b Gel electrophoreses of PCRs of the HAX1 versions in different T. reesei strains using cDNAs of QM6a, QM9414 and Rut-C30 as templates. Strains were pre-grown and transferred to medium with 1% (w/v) d-glucose (G) or 1.5 mM sophorose (S). Used primer pairs are indicated on the left (i.e, the PCR for detecting hax1_QM6a is analysed in gel on top; for detecting hax1_QM9414 in the middle gel; and for detecting hax1_Rut-C30 in the gel at the bottom). The strength of each band was quantified using Image Lab version 5.2 and related to the sample of Rut-C30 on sophorose (numbers are given below the respective band). P, Positive control; N, negative control; L, 50-bp DNA ladder. c In silico structure prediction of HAX1_QM6a, HAX1_QM9414 and HAX1_Rut-C30 based on RACE-defined 3′- and 5′-ends. Structures of minimized free energy are displayed. The stability is increasing from violet, blue, green, and yellow to red