Sybr premix ex tag tli rnaseh plus

Gene-specific primers for qRT-PCR were designed by the Primer Express software and were listed in S4 Table. qRT-PCR was conducted with SYBR Premix Ex Tag (Tli RnaseH Plus) (TaKaRa) and 2 μl cDNA (diluted to 20X) as a template. Thermal cycling conditions were as follows: an initial enzyme activation of 30 s at 95°C, followed by 40 cycles of denaturation for 10 s at 95°C, annealing and extension for 20 s at 60°C, with a final melting gradient starting from 60°C and heating to 95°C for 15 s, 60°C for 1 min, and 95°C 15 s. The qRT-PCR reactions were conducted using a 7300 Fast Real Time PCR System (ABI). Primer specificity was confirmed by dissociation curves of the PCR amplification products. The mean Ct values were normalized against the reference, V. myrtillus glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (NCBI accession number: AY123769.1). Since primer efficiencies were approximately equal, the expression was calculated by the 2^–ΔCt method. To ensure the reliability of expression results, qRT-PCR was performed for all genes on three separate cDNA preparations for each organ and fruit developmental stage. For additional replication, all templates and standards were run in triplicate.

Trichoderma reesei Δtku70 (ATCC MYA-256), a nonhomologous end joining pathway-deficient strain, was used as the parent strain in this study [52 (link)]. All the other strains constructed in this study are listed in Additional file 8: Table S1. The E. coli strain GB05-dir was used for constructing all the plasmids [53 (link)].
Wheat bran was kindly provided by Longlive Bio-Technology Co., Ltd. (Yucheng, Shandong, China). The p-nitrophenyl-β-d-cellobioside (pNPC) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Uridine, PEG6000, sorbitol, and lactose were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). KOD FX DNA polymerase (Toyobo Co., Ltd., Osaka, Japan) was used for all polymerase chain reaction amplifications. RNAiso™ reagent, PrimeScript^® RT reagent Kit With gDNA Eraser (Perfect Real Time) and SYBR^® Premix Ex Tag™ (Tli RNase H Plus) were purchased from Takara Bio Inc. (Shiga, Japan). The DIG High Prime DNA Labeling and Detection Starter Kit I (Roche Diagnostics, Mannheim, Germany) was used for Southern blotting analysis. The restriction enzymes Ase I and Xho I used for genome digestion were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). All other chemicals were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).