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. Author manuscript; available in PMC: 2017 May 19.
Published in final edited form as: Fungal Genet Biol. 2017 Jan 16;100:22–32. doi: 10.1016/j.fgb.2017.01.005

Figure 4. CATP gene silencing in P. brasiliensis.

Figure 4

(A) Transfer DNA (T-DNA) inserted into the genome of P. brasiliensis yeast cells via ATMT. The antisense oligonucleotide was targeted to exon four (dashed box), with a length of 80 bp. This AS oligonucleotide was placed under control of the calcium binding protein (CBP-1), the terminator CAT-B and harboring hygromycin B phosphotransferase (HPH) under control of Aspergillus nidulans glyceraldehyde 3-phosphate (PGPDA) and with the terminator TTRCP. (B) PbCATP gene expression levels obtained by RT-qPCR assay. The measurement was normalized with the housekeeping gene β-tubulin in the wild-type (PbWT60855), the empty vector control (PbEV60855) and the antisense RNA strain (PbCATP-aRNA) grown at exponential phase. Mitotic stability was confirmed by sub-culturing P. brasiliensis PbCATP-aRNA yeast cells, checking for low expression levels in this isolate after successive sub-cultures. (C) Light microscopy of P. brasiliensis yeast cells morphology. (D) Growth curve in PbWT60855, PbEV60855, PbCATP-aRNA. Yeast cells were grown in BHI liquid medium at 36°C, OD600 nm was determined at each time point. (E) Vitality in PbWT60855, PbEV60855 and PbCATP-aRNA. The pH change was monitored in yeast suspensions at three minutes intervals, for 30 minutes. Results represent the mean of three individual experiments. (F) Profile expression of CAT isoforms, alternative oxidase (AOX) and glutathione (GSH) in PbWT and PbCATP-aRNA yeast cells during batch culture growth. Gene expression levels were determined by RT-qPCR assay and normalized with the housekeeping gene β-tubulin. Results are the mean of three individual experiments.