Primers and copper responsive promoter design and data of real-time RT-PCR assay in filamentous fungus Trichoderma reesei

This data article contains data related to the research article entitled “Copper-mediated on-off control of gene expression in filamentous fungus Trichoderma reesei” (Wang et al., 2017) [1]. Four kinds of copper responsive promoters were designed. Quantitative PCR (qPCR) was performed to determine relative mRNA levels of red fluorescent protein gene (rfp) extracted from cells grown under different concentrations of CuSO4. Three deletion vectors were constructed by using a copper-mediated self-excision cassette instead of a xylose-mediated self-excision cassette (Zhang et al., 2016) [2] to knock out xyn1, one of the two major specific endo-β-1,4-xylanases (Rauscher et al., 2006) [3], xyr1, the key transcriptional activator of cellulolytic and xylanolytic genes (Lichius et al., 2015) [4], and ace3, a factor essential for cellulase production (Häkkinen et al., 2014) [5]. This data article reports the primers, vector construction, and qPCR assay.


a b s t r a c t
This data article contains data related to the research article entitled "Copper-mediated on-off control of gene expression in filamentous fungus Trichoderma reesei" (Wang et al., 2017) [1]. Four kinds of copper responsive promoters were designed. Quantitative PCR (qPCR) was performed to determine relative mRNA levels of red fluorescent protein gene (rfp) extracted from cells grown under different concentrations of CuSO 4 . Three deletion vectors were constructed by using a copper-mediated self-excision cassette instead of a xylose-mediated self-excision cassette (Zhang et al., 2016) [2] to knock out xyn1, one of the two major specific endo-β-1,4-xylanases (Rauscher et al., 2006) [3], xyr1, the key transcriptional activator of cellulolytic and xylanolytic genes (Lichius et al., 2015) [4], and ace3, a factor essential for cellulase production (Häkkinen et al., 2014) [5]. This data article reports the primers, vector construction, and qPCR assay.
& Sequencing data were acquired through NCBI. In silico analysis of gene using online Real-time PCR (TaqMan) Primer Design (GenScript, China) and primer design software version 6.0 (Premier Biosoft, USA).

Data format
Raw, analyzed Experimental factors Gene sequences were retrieved from GenBank database; Plasmid were constructed; rfp expression were analyzed by qRT-PCR Experimental features Four kinds of copper responsive promoters were designed. qRT-PCR was performed to determine relative red mRNA levels of rfp extracted from cells grown under different concentrations of CuSO 4 . Three deletion cassettes were constructed to knockout xyn1, xyr1, and ace3, respectively.

Shanghai, China
Data accessibility Data is provided with this article

Value of the data
The modified copper responsive promoter Ptcu1c from T. reesei was used for the copper-dependent on-off control of DNA transcription and protein expression.
The relative levels of rfp transcripts increased~500-fold in the absence or presence of copper. The copper-mediated self-excision cassette was more widely used than a xylose-mediated selfexcision cassette in some T. reesei disruptants for the screening of candidate regulators for cellulase and hemicellulase production.

Data
Four copper responsive promoters were designed. Quantitative real-time PCR (qRT-PCR) was performed to determine relative mRNA levels of rfp extracted from cells grown under different concentrations of CuSO 4 . By using the copper-mediated self-excision cassette, three deletion plasmids were constructed to knockout xyn1, xyr1, and ace3.

Expression levels of rfp in T. reesei transformants
About 100 mg of T. reesei mycelium was harvested, and grown under different concentrations of CuSO 4 for 36 h. Total RNA was extracted using a FastRNA Pro Red Kit (MPbio, Irvine, CA, USA), according to the manufacturer's instructions. Reverse transcription was performed with 1000 ng of total RNA, using the TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (Trans-Gen, Beijing, China), according to the manufacturer's instructions. For RT-qPCR, the TransStart TipTop Green qPCR SuperMix (TransGen) was used with 200 nM of forward and reverse primers ( Table 2) and 1 μL of 10-fold diluted cDNA in a final volume of 20 μL. For gene transcription analysis, SYBR green assays, using primers with the reference gene sar1, were performed as described in the previous publication [6]. The primers of rfp were designed using GenScript Real-time PCR (TaqMan) Primer Design (https://www.genscript.com/tools/real-time-pcr-tagman-primer-design-tool). Thermocycling was performed in an ABI StepOne Plus thermocycler (Applied Biosystems, Foster City, CA, USA).
Quantitative real-time PCR (qRT-PCR) was performed using Ptcu1c-rfp [1] to determine relative rfp mRNA levels extracted from cells grown under different concentrations of CuSO 4 (Fig. 1). The relative levels of rfp transcripts increased~500-fold in the absence or presence of high levels of copper, indicating that the on-off control functions by affecting target RNA levels.

Name
Sequences Table 1 Detailed information on copper responsive promoter primers.
Name Table 3 Primers used in deletion plasmids construction. Name