Fastquant cdna

The saliva collection and infiltration treatments were as described in Section 2.3. The leaves were collected 6 h and 24 h after infiltration and stored at −80 °C until use. Total leaf RNA was extracted using TRIzol reagent (Tiangen, Beijing, China), and 1 μg RNA was used to synthesize the first cDNA strand with FastQuant cDNA (Tiangen, Beijing, China) according to the manufacturer’s instructions. Real-time quantitative-polymerase chain reaction (RT-qPCR) was performed in 20 μL reaction volumes containing 10 μL 2× SYBR Premix (Tiangen, Beijing, China), 8 μL water, 1 μL gene-specific primers, and 1 μL cDNA template. The reaction was performed with a CFX Connect^TM Real-Time PCR System (Bio-Rad, Hercules, CA, USA). The temperature protocol was: 95 °C for 30 s, followed by 40 cycles at 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 30 s. The expression of five genes involved in the SA pathway and five genes involved in the JA pathway were detected. CitGAPC1 was used as an internal control. Sequences and description of primers are listed in Table 1 [34 (link),35 (link),36 ,37 (link)]. There were four replicates per treatment (each leaf was considered a replicate), and each replicate contains three technique replicates.

Primers (F and R) were designed according to the sequences of the target genes found on the Phytozome database (https://phytozome.jgi.doe.gov/pz/portal.html, accessed on 16 July 2018). Primer design was conducted using Primer 5 software (Premier, Mississauga, Ontario, Canada). The gene-specific primers are listed in Table S1. Total RNA was extracted from the leaves of B21-a-2-1-2 and B21-a-2-2-2 using an RNAprep pure plant kit (Tiangen Biotech, Beijing, China) and synthesized into cDNA using the FastQuant cDNA (Tiangen Biotech, Beijing, China). The following PCR program was used: 98 °C (10 s), 56 °C (15 s), and 68 °C (3 min) for 35 cycles. The reaction was conducted in a 20 µL volume containing 5 µL of 5× PrimeSTAR GXL buffer, 2 µL of dNTP mixture, 0.5 µL of each primer, 2 µL of genomic DNA, 0.5 µL of PrimeSTAR GXL DNA polymerase, and 8.5 µL of doubly distilled water. DNAMAN software (Lynnon Biosoft, San Ramon, CA, USA) was used for sequence alignment. Bioinformatics software was used for sequence analysis.