4.5.2. Gene Expression and Differential Gene Enrichment

The expression of unigenes was calculated with the of RPKM (reads per kb per million reads) method [47] according to the following formula: RPKM(A) = (1000000 ∗ C)/(N ∗ L/1000)

The RPKM(A) value stands for the expression of gene A; C values stands for the number of reads that uniquely aligned to gene A; N values stands for the total number of reads that uniquely aligned to all genes, and L stands for the number of bases on gene A.

According to the gene expression represented by the RPKM values for each sample, the significant differentially expressed genes (DEGs) among the samples were screened with edge R. The screening criteria were an FDR < 0.05 (*p*-value after calibration by FDR) and |log2FC| > 1, and GO functional analysis and KEGG pathway analysis were performed based on the results for the DEGs.

#### 4.5.3. Diversity and Collinearity of *S. furcifera* P450 Genes

Twenty-two relatively complete P450 amino acid sequences obtained from the transcriptome were compared with thirty-two P450 amino acid sequences of the white-back planthopper downloaded from NCBI and analyzed for the conserved functional domains with the motif (https://www.genome. jp/tools/motif/) and meme (http://meme-suite.org/tools/meme) tools, and their phylogenetic tree was constructed by using MEGA 6.0 software with the default settings and the neighbour-joining method. The results were visualized with TBtools software.

#### *4.6. Quantitative PCR (qRT-PCR)*

Total RNA of the Sus-Lab and SF-Sel strains was extracted using TRIzol reagent (Invitrogen™, ThermoFisher Scientific, USA) according to the instructions of the manufacturer's kit, and the reverse transcription reaction was performed with a cDNA Synthesis for qPCR (One-Step gDNA Removal) kit according to the instruction manual. The cDNA was kept at −20 ◦C for qRT-PCR.

The cDNAs of four P450 genes (*CYP6FD1*, *CYP6FD2*, *CYP4FD1* and *CYP4FD2*), one transporter (*Unigene0036498*), four transcription factors (*NlE78sf*, *C2H2ZF1*, *C2H2ZF3* and *C2H2ZF2*) and one reference gene (*RPL9*) [48] from the Sus-Lab and SF-Sel strains were amplified by PCR with twelve pairs of corresponding primers (Table 3). The qRT-PCR system and procedure were as described by Wang et al. [49]. All experimental results were analyzed in three independent replicates, and the treatment means and variances were analyzed via one-way ANOVA with PROC GLM of the SAS program. All means were compared by least squared difference (LSD) tests at a Type I error = 0.05.


**Table 3.** The primers of upregulation expression genes used in this study.

The biological function of *CYP6FD1* and *CYP4FD2* was verified through RNA interference as described by Mao et al. [31] and Wang et al. [49], with some modifications. A 168 bp fragment of *CYP6FD1*, 450 bp of *CYP4FD2* cDNA and a 657 bp green fluorescent protein (gfp) fragment were amplified by PCR using corresponding primer pairs (with the T7 promoter appended). PCR was performed with the primers listed in Table 4. The PCR products were purified for use as templates for dsRNA synthesis using the T7 MEGAscript kit (ThermoFisher, USA) according to the manufacturer's instructions. The dsRNA concentration was measured using a spectrophotometer (Nanodrop) after 1:10 dilution of the dsRNA product in water and adjustment of the ultimate concentration to 4 ng/μL for injection.


