Cfx connect optics module

Gene transcript levels were analyzed using Q-PCR with a BIO-RAD CFX Connect^TM Optics Module (Bio-Rad, USA). The cDNA was synthesized from RNA using PrimeScript^® RT reagent Kit With gDNA Eraser (TaKaRa, Japan). Actin was used as an internal control in gardenia (forward primer: 5′-GCGAGGAAACAAGTGGAAGACTA-3′; reverse primer: 5′-TGCCAACCACCATTTATTAGGAG-3′)^{29 (link)}. All gene-specific primers in this study were shown in Supplemental Table S1, and synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). Q-PCR was performed using the SYBR^® Premix Ex Taq^TM (Perfect Real Time) (TaKaRa, Japan) and contained 12.5 µL 2 × SYBR Premix Ex Taq^TM, 2 µL cDNA solution, 2 µL mix solution of target gene primers and 8.5 µL ddH₂O in a final volume of 25 µL. The amplification was carried out under the following conditions: 50 °C for 2 min followed by an initial denaturation step at 95 °C for 30 s, 40 cycles at 95 °C for 5 s, 50 °C for 15 s, and 72 °C for 30 s. Gene relative expression levels of target genes were calculated by the 2^−△△Ct comparative threshold cycle (Ct) method^{68 (link)}. The Ct values of the triplicate reactions were gathered using the Bio-Rad CFX Manager V1.6.541.1028 software.

Gene transcript levels were analyzed using qRT-PCR with a BIO-RAD CFX ConnectTM Optics Module (Bio-Rad, USA). cDNA was synthesized from RNA using the PrimeScript® RT Reagent Kit With gDNA Eraser (TaKaRa, Japan). All gene-specific primers in this study are shown in Supplementary Table S5. qRT-PCR was performed using SYBR® Premix Ex TaqTM (Perfect Real Time) (TaKaRa, Japan). The amplification was carried out under the following conditions: 55 °C for 2 min, followed by an initial denaturation step at 95 °C for 30 s, and 40 cycles at 95 °C for 5 s, 55 °C for 15 s, and 72 °C for 30 s. Relative expression levels of target genes were calculated by the 2^-△△Ct comparative threshold cycle method^{50 (link)}.

Five-year-old P. lactiflora plants in potting soil (loam:peat:perlite, 1:1:1) were employed for the DS-stimulated PlMYB108 expression pattern analysis. The P. lactiflora plants were placed in a glass greenhouse on the Wenhui Road campus of Yangzhou University. They were separated into 2 groups, which each contained six plants: one group received water at 17:00 daily and served as the control group, and the other group received natural DS. The top leaves were collected at 0, 7, 14, and 21 days post exposure for an assessment of the PlMYB108 levels, using qRT-PCR via the BIO-RAD CFX Connect^TM Optics Module (Bio-Rad, Hercules, CA, USA). All values were computed via the 2^−∆∆Ct relative threshold cycle (Ct) method [31 (link)]. Primer sequences are summarized in Table S2.