Primer software version 5

Total RNA was extracted from the frozen samples of benzothiazole-treated and control larvae using the TransZol Up Kit (Transgen, Beijing, China). For each sample, approximately 1.0 μg of total RNA was used for first-strand cDNA synthesis using the TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR Kit (Transgen). Gene-specific primers were designed using Primer Software Version 5.0 (Premier Biosoft International, CA, USA). The sequences of the F and R primers used are shown in Table S1. qRT-PCR was performed using a Bio-Rad CFX Connect Real-Time System (Bio-Rad Laboratories Inc., Hercules, CA, USA) in a total volume of 20 μL volume with 1.0 μL of cDNA, 1.0 μL of each primer, 10 μL of Tip Green qPCR SuperMix (Transgen) and 7.0 μL of double distilled water. The cycling conditions were 30 s at 95 °C, followed by 40 cycles of amplification (95 °C for 5 s, 58 °C for 15 s, and 72 °C for 10 s). Three technical replicates and two biological replicates were conducted for all experiments. For the normalization of gene expression, ribosomal protein S3 (RPS3) gene was used as an internal standard, and the formula 2^−∆∆Ct was used to determine the relative expression. The data were statistically analyzed using the Student-Newman-Keuls test (SPSS v. 13.0 for Windows) (P < 0.05).

Based on the functional category and differential expression fold, nine genes were chosen by RT-PCR. Specific primers for differentially expressed protein-related genes were designed by Primer software version 5.0 (Premier Biosoft International, Palo Alto, CA, USA) (Table 3). Total RNA from two treatments was extracted using the kit (Takara Biomedical Technology Co., Beijing, China). AceQ qPCR SYBR GREEN Master Mix (Vazyme Biotechnology Co., Ltd., Nanjing, China) was used for RT-PCR analysis. The results were estimated using the 2-△△CT method. Every sample had three biological replicates. Three reactions were performed for all reactions of each sample, and the β-actin gene was used as an internal standard.

Based on the functional category and the fold change in expression in the LT and HT groups relative to the control group, 15 genes were chosen for qRT-PCR analysis. Primer software version 5.0 (Premier Biosoft International, CA, USA) was used to design gene-specific primers, and primer sequences are indicated in Table 1. qRT-PCR analysis was performed using AceQ qPCR SYBR GREEN Master Mix (Vazyme Biotechnology Co., Nanjing, China), and relative expression levels were calculated using the 2^−△△Ct method [70 (link)]. The BnaActin gene was used as the control. Each experiment was replicated 3 times.

About 300 mg of frozen leaves collected equally from six pots (one plant per pot, one leaf per plant) were mixed as a biological replicate. There were three biological replicates for each treatment. Total RNAs were independently extracted thrice from +Al and control frozen leaves using the Recalcitrant Plant Total RNA Extraction Kit (Centrifugal column type, Bioteke Corporation, Beijing, China) according to the manufacturer’s instructions. Gene-specific primers were designed using Primer Software Version 5.0 (PREMIER Biosoft International, Palo Alto, CA, USA) according to the corresponding sequences of selected proteins in the citrus genome (http://www.phytozome.net/cgi-bin/gbrowse/citrus/). The sequences of the F and R primers used are given in Table S3. qRT-PCR was performed according to Zhou et al. [88 (link)]. Each sample was run in two technical replicates. For the normalization of gene expression, citrus actin (GU911361.1) was used as an internal standard, and the leaves from control plants were used as the reference sample, which was set to 1.

Total RNA was extracted from leaves by using TRIzaol reagent (Thermo Fisher Scientific Inc.) [73 (link)], and cDNA was synthesized by HiScript II Q RT SuperMix for qPCR (Cat No. r223–01, Vazyme, Nanjing, China). Then analyzed by qPCR using SYBR Green Premix Pro Taq HS qPCR Kit II (Rox Plus) (Cat No. AG11719, Accurate Biotechnology (Hunan) Co.,Ltd., China) on StepOnePlus system (Applied Biosystems, USA). The relative expression of genes was analyzed by the 2^−ΔΔCT method [74 (link)] and were normalized to the internal control gene BrPP2A for pakchoi. Each reaction was performed in three technical replicates and three independent biological replicates (biological replicates: leaves of the same part of three independent plants; technical replicates: repeat detection and analysis of the same sample). Primers for qPCR analysis were designed by Primer Software Version 5.0 (Premier Biosoft International, CA, USA) and shown in Table S8.