Hiscript 2 q rt supermix gdna wiper

In order to determine DON content of strains, the conidia solution of PH-1 and FgMet3, FgMet14 deletion mutants (2×10³ conidia/mL) were transferred into the toxin production medium GYEP and cultured at 28°C, 175rpm in the dark condition for 7 days (Bian et al., 2021 (link)). Then the DON content of strains was determined by DON ELISA Plate Kit (Weisai) (Duan et al., 2020 (link)). The DON content (μg/g) of the strain was expressed by the DON content per unit weight of hyphae.
To determine the expression of Tri5, Tri6, which related to DON synthesis, the conidia of PH-1 and FgMet3, FgMet14 deletion mutants were placed in GYEP medium at 28°C, 175rpm for 36 hours in the dark. The hyphae were collected, RNA was extracted by RNA extraction kit, and inversely converted to cDNA with HiScript II Q RT SuperMix (+ gDNA wiper) (Vazyme Biotech Co. Ltd)., then the expression of Tri5, Tri6 in the strain was determined by qPCR (Bian et al., 2021 (link)). FgGadph was used as the reference gene. The experiment was repeated three times.

The RNA-seq results were validated using qRT-PCR and the expression of 12 randomly selected genes. The cDNA was obtained by reverse transcription using the extracted total RNA as a template. The reverse transcription reagent for qRT-PCR was the Hiscript II Q RT Super Mix (+gDNA Wiper; Vazyme, Nanjing, China; # R223-01). Then, the designed qRT-PCR primers (Table S2) were used to carry out the polymerase chain reaction with the fluorescent reagent AceQ qRT-PCR SYBR Green Master Mix (Vazyme; # Q111-02/AA). The reaction conditions for the amplification were: 95 °C for 5 min; 95 °C for 10 s, and 58 °C for 30 s, for 40 cycles; 95 °C for 15 s, 60 °C for 1 min, 95 °C for 15 s; and 40 °C for 5 min. The reaction volume consisted of 5 μL of AceQ qPCR SYBR Green Master Mix, 0.2 μL of primer 1, 0.2 μL of primer 2, 1 μL of 10 × diluted cDNA, and 4.6 μL of distilled water. Data were analyzed using three replicates, and the relative expression was calculated using the 2^−ΔΔCT method.

Quantitative RT-PCR was used to explore the expression patterns of related genes in self-pollination and cross-pollination of Camellia oleifera. Total RNA was extracted using E.Z.N.A Plant RNA Kit (Omega Bio-Tek, Norcross, Georgia), 1.2% agarose gel electrophoresis to judge the integrity and overall quality of total RNA. Reverse transcription of each sample was carried out with HiScript^® II Q RT SuperMix (+gDNA wiper) (Vazyme, Nanjing, China). PCRs were performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) on a CFX96 Real-time PCR system (BIO-RAD). Primers were designed for use in quantitative RT-PCR (qRT-PCR).
(CoCOI1: F5′-ATCAAAGAGAAAGATGGAGAATG-3′, R5′-CAGCACTAAAGAAACCAATAAGA-3′; CoJAZ1: F 5′-CCTCAGGCTCGCTAACTCA-3′, R5′-CGATGCCCTTCTTGCTATT-3′; CoMYC2: F 5′-CCCATCAGAGACCGAAAGACT-3′, R5′-ACCAAGGAGCCAGAACTAAAAC-3′). GAPDH was used as an internal control gene for normalization Quantitative variations were calculated by the relative quantification method (2^−ΔΔCT) and three independent.

Total RNA was extracted from different samples using the RNA-Solv® Reagent (Omega Biotek, USA), according to the manufacturer’s protocol. RNA was quantified using a NanoDrop 2000 UV Spectrophotometer (Thermo Fisher Scientific, USA). Total RNA (~ 1 μg RNA) was used for synthesizing cDNA using the HiScript® II Q RT SuperMix (+ gDNA wiper) (Vazyme, China). This was followed by storage at − 80 °C until use in further experiments.

The WT and mutants were cultured in LB medium containing Amp overnight, and then diluted 1:100 into fresh medium and grown to the exponential phase (OD₆₀₀ = 0.6) and stationary phase (OD₆₀₀ = 1.2). RNA was extracted with a Bacteria Total RNA Extraction Kit (Promega, Madison, WI, United States), and then HiScript II Q RT SuperMix (+gDNA wiper) (Vazyme, Nanjing, China) was used for reverse transcription after determining the RNA quality by Nanophotometer NP80 (IMPLEN, München, Germany). The final cDNA samples were analyzed by Quantitative Reverse Transcription PCR (qRT-PCR) using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). Primers used in the qRT-PCR analysis are listed in Table 1. Expression of the genes encoding helix-turn-helix transcriptional regulator VpsT, polysaccharides biosynthesis protein VpsM and VpsH, chemotaxis protein CheR, flagellar hook protein FlgE, flagellar basal body L-ring protein FliD, and alternative sigma transcription factor RpoN was determined in triplicate. The 16S rRNA gene was used as an endogenous control for normalization of expression values. The 2^–ΔΔCt method was used to quantify and compare each gene expression (Livak and Schmittgen, 2001 (link)).

Floral buds at stage 4 of development were incubated at 4 °C or 22 °C. Samples were frozen at –80 °C and ground in liquid nitrogen. Total RNA was extracted from the samples using an RNA extraction kit (TaKaRa, Ohtsu, Japan). Reverse transcription was performed using HiScript^® II Q RT SuperMix +gDNA wiper (Vazyme, Nanjing, China) using 2000 ng cDNA in a 40 μl reaction volume. After reverse transcription, 160 μl H₂O was added to dilute the sample to 200 μl. Gene expression was detected in 1 μl cDNA using an M5 HiPer Real-time PCR mix (Mei5bio, Beijing, China) and the ABI StepOne system (Thermo Fisher Scientific). RhUBI2 and RhACTIN5 were used as reference genes (Chen et al., 2023 (link)). All of the above experiments consisted of at least seven biological and three technical replicates. The related primers are shown in Supplementary Table S1.