Cdna synthesis kit

The chemicals used in the experiments were reagent or High-Performance Liquid Chromatography (HPLC) grade. Restriction enzymes were purchased from New England BioLabs; RNA extraction kit was purchased from TransGen Biotech; cDNA synthesis kit and one-step cloning kit were purchased from Novo Protein Scientific (Shanghai); PCR kit was purchased from Toyobo Biotech; Premix TaqTM DNA polymerase was purchased from TaKaRa Bio; Taq master mix was purchased from Shanghai Wonton Biotech. I. indigotica was planted at Shanghai University of Traditional Chinese Medicine (SHUTCM), and two-month-old plants were used for target gene cloning.

Total RNA was isolated from the cells and tissues by a TRIZOL kit (Invitrogen) based on the manufacturer's protocol. RNA was quantified and then reverse‐transcribed into cDNA by a cDNA synthesis kit (Novoprotein). qRT‐PCR was conducted by SYBR Green (Novoprotein) in an ABI 7000 thermal cycler. RNA expression was analyzed using the 2^−ΔΔCT method. GAPDH or U6 was used as the endogenous control for normalization. The primer sequences were synthesized by Tsingke Biotechnology. The sequence of the primers is outlined in Table S2.

RNA extraction was conducted as described above, and RNA reverse transcription was conducted using a cDNA Synthesis Kit (All-in-one 1st Strand cDNA Synthesis SuperMix, Novoprotein, Suzhou, China). Sixteen genes were randomly selected, and their Ct values were measured using the qTOWER3 detecting system (Analytik, Jena, Germany) with SYBR qPCR SuperMix Plus (E096, Novoprotein, Suzhou, China). As shown in Table S1, the primers were designed using Primer Premier 5 (Primier Biosoft International, Palo Alto, CA, USA). Moreover, the internal control gene was IPP2 as reported in [8 (link)]. The relative expressions were calculated using the 2^−ΔΔCt method [61 (link)]. The Log 2-fold change was calculated by comparing the salt treatment group with the control group (0 h) using the method reported in [62 (link)].