Peasy t3 cloning vector

The pcDNA3.1 expression plasmid was bought from ThermoFisher (USA) and stored in our laboratory. Rabbit ARV p17 polyclonal antibody was obtained and stored in our lab. Restriction endonucleases Bam HI and Eco RI were purchased from NEB (USA). Trans1-T1 receptor cells, Taq high-fidelity DNA polymerase, pEASY-T3 cloning vector, DNA T4 ligase, and high-glucose DMEM were obtained from TransGen Biotech (Beijing, China). DL Marker and DNA gel recovery kits were purchased from TaKaRa (Dalian, China). The Animal Tissue RNA Extraction Kit and Plasmid Extraction Kit were purchased from Kang Wei Century (Beijing, China). The immunoprecipitation kit and AceQ Universal SYBR qPCR Master Mix were purchased from Novozymes Biotechnology Co. The mouse anti-Myc monoclonal antibody, rabbit anti-Flag monoclonal antibody and mouse anti-GAPDH monoclonal antibody were purchased from Beyotime Biotechnology (Beijing, China); Caspase-1 antibodies were purchased from Cell signaling technology; HRP-labeled sheep anti-mouse IgG and HRP-labeled sheep anti-rabbit IgG were purchased from Sigma; and goat anti-mouse IgG (H + L), FITC conjugate, and goat anti-rabbit IgG (H + L), PE conjugate, were purchased from TransGen (Beijing, China). FBS was purchased from LONSERA. 40,6-Diamino-2-phenylindole (DAPI) was purchased from Beyotime Company (Nanjing, China).

The total RNA of ten third instar nymphs was isolated using the SV Total Isolation System kit (Promega Corporation, Madison, WI, USA). First-strand cDNA was synthesized using the PrimeScript™ 1st Strand cDNA Synthesis Kit (TaKaRa Biotechnology, Dalian, Co., Ltd. Dalian, China) according to the manufacturer’s instructions. The first-strand cDNA obtained from the insect was used as the PCR template. The gene-specific primers for AlAMPK were designed based on conserved regions found in AMPK from other insect species: Cimex lectularius (GenBank accession No. XM_014407037), Halyomorpha halys (XM_024358872) and Myzus persicae (XM_022324589). The primers were AMPK-F and AMPK-R (Table S1). Following amplification, the products were separated on a 1.0% agarose gel and purified using the TIANgel Midi DNA Purification System (TianGen Biotech CO., LTD, Beijing, China). Purified DNA fragments were cloned into the pEASY-T3 cloning vector (TransGen Biotech, Beijing, China). Recombinant plasmids were isolated using the Plasmid Mini kit and sequenced. The full-length cDNA of AlAMPK was obtained by the rapid amplification of cDNA ends (RACE) using a SMART^TMRACE cDNA Amplification Kit (Clontech, Palo Alto, CA, USA). PCR products were purified, cloned, and sequenced as above. All the primers used in this study are listed in (Table S1).

MiR-23b and ~150 bp of flanking sequence was amplified from human genomic DNA and cloned into pEASY^®-T3 Cloning Vector (Transgen Biotech, Beijng, China). After sequencing confirmation, the fragment was digested with EcoRI and BamHI and cloned into lentiviral vector pCDH-CMV-MCS-EF1-copGFP (LV-miR-23b; System Bioscience, CA, USA). Lentiviral miR-23b sponge (LV-sponge) was constructed by Hanbio Co. LTD (Shanghai, China). Virus was created and infected targeted cells using a polybrene reagent (Merck Millipore, Darnstadt, Germany) according to manufacture's instruction.

Two groups of specific primers (Supplementary Table 1B) were designed with Primer Premier 5.0 software (Premier, Canada), and the complete coding sequences of the IgWRKY50 and IgWRKY32 genes were amplified twice. The PCR products were connected to the pEASYT3 cloning vector (TransGen Biotech, Beijing, China) and then sequenced by the company (Sangon Biotech, Shanghai, China). The open reading frames of IgWRKY50 and IgWRKY32 were found by NCBI ORF Finder.¹ The protein functional domains of IgWRKY50 and IgWRKY32 were predicted by SMART software (Letunic and Bork, 2018 (link); Letunic et al., 2021 (link)).² The phylogenetic tree of amino acid sequences of other species with homology to IgWRKY50 and IgWRKY32 genes was constructed by MEGA 6 software using the neighbor-joining (NJ) method with 1,000 bootstrap replicates (Tamura et al., 2013 (link)). The accession numbers of the WRKYs are listed in Supplementary Table 2. The tertiary structure prediction of IgWRKY50 and IgWRKY32 proteins was completed by SWISS-MODEL software (Biasini et al., 2014 (link)), and the three-dimensional structure model of proteins was constructed by the I-TASSER website³ and Chimera 1.11.2 software⁴ (Pettersen et al., 2004 (link)).

Genomic DNA (gDNA) was isolated from whole BPH body or forewings to determine the heterozygous or homozygous genotypes of the BPHs. gDNA was extracted from one individual adult, as reported previously [63 (link)]. Briefly, one adult was homogenized in a 0.2-ml Eppendorf tube, followed by the addition of 50 μl of extraction buffer (10 mM Tris-HCl pH 8.2, 1 mM EDTA, 25 mM NaCl, 0.2 mg/ml proteinase K). The tubes were then incubated for 30 min at 37°C, followed by 2 min at 95°C to inactivate the proteinase K, and the supernatant solution was used directly as a template for PCR.
gDNA was isolated from forewings as reported previously [64 (link)], with slight modifications. Briefly, forewings were digested in 0.5 ml extraction buffer (0.01 M Tris-HCl, 0.01 M EDTA, 0.1 M NaCl, 0.039 M dithiothreitol, 2% sodium dodecyl sulfate, 20 μg/ml, pH 8.0) for 12h at 37°C, followed by 2 min at 95°C to inactivate proteinase K. The supernatant solution was then used directly as a template for PCR. PCR products spanning NlInR2^E4 and NlInR2^E5 sgRNA target sites were amplified from the extracted gDNA using primer pairs for E4-iF/E4-iR and E5-iF/E5-iR (S20 Table), respectively. The PCR products were then used for Sanger sequencing or subcloned into pEasy-T3 cloning vector (TransGen Biotech), and then single clones were picked for Sanger sequencing.

Genomic DNA was extracted using the phenol-chloroform method from SA and IMA pre-adipocytes. Bisulfite treatment was performed according to the manufacturer’s instruction of EZ DNA Methylation-Gold Kit^TM (Zymo Research, USA). Primers (BSP- Promoter 1 C, BSP- Promoter 1 H) for the amplification of the GR promoter region were designed by Methyl Primer Express v1.0. The PCR products were cloned in the pEASY-T3 Cloning Vector (Transgen Biotech, Beijing, China), then positive clones were sequenced.