Pmd 19 t vector cloning kit

M. jalapa RNA was extracted from plants grown with petroleum contamination for 45 days using the RNAprep Pure Plant Total RNA Extraction Kit (TianGen, Beijing, China) following the manufacturer’s instructions. After detection by 1% gel electrophoresis, the total RNA was first treated to remove the genomic DNA and then transferred into the first strand of cDNA using the PrimeScript RT reagent kit with gDNA Eraser (TaKaRa, Dalian, China) according to the manufacturer’s instructions. The first strand of synthesized cDNA was used as a template. The 2× PCR mix (TaKaRa, Dalian, China) was used to perform the PCR (Supplementary Table 1). The amplified product was recovered, directly ligated to the pMD-19T vector using the TA cloning method according to the operating instruction of pMD™ 19-T Vector Cloning Kit (TaKaRa, Dalian, China), and then transformed into TOP 10 competent cells. The positive clones, identified by bacterial liquid PCR using the sequencing primers M13 (M13F: TGTAAAACGACGGCCAGT, M13R: CAGGAAACAGCTATGACC) in the pMD-19T vector, were sent to Biobios (Shanghai, China) for sequencing.

The copy number of MbMNPV was calculated by absolute quantification. In short, MbMNPV genomic DNA was extracted from the liquefied larvae infected with MbMNPV and used as the template for the amplification of the conserved polyhedrin gene by PCR. The PCR products were sub-cloned into the pMD™19-T Vector Cloning Kit (TaKaRa). The recombinant plasmid polyhedrin-pMD-19T was used to calculate the plasmid copy number and generate a standard curve for RT-qPCR. CT value was obtained by RT-qPCR using the specific primer of polyhedrin for the fat body of mock- and MbMNPV-infected larvae for 72 h, and then the copy number of MbMNPV was determined by standard curve. The β-actin gene was used as an endogenous reference. The primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) (Table S1).

Ten housekeeping genes (α-TUB, AK, GAPDH, β-TUB1, RPL13, GST, β-TUB2, TBP, G6PDH and UBC) were selected as candidate RGs from the transcriptome database of E. onukii. These genes were cloned and sequenced, then confirmed by BLASTX. Primer premier 5 was used for primer design to clone these genes. Full-length cDNA was amplified by PCR using primers shown in Table 2. Each reaction included 50 ng cDNA, 1 μl of each primer (10 μM) and 2X PrimeSTAR Max Premix (TaKaRa, Tokyo, Japan), and the volume was adjusted with nuclease-free water to 20 μl. The PCR program contained a preliminary step at 95°C for 5min, 40 cycles of denaturation at 95°C for 10 s, an annealing temperature for 6s and 58°C for 1–3 min (depending on the length of mRNA). PCR products were examined by gel electrophoresis, purified using a TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver 4.0 (TaKaRa, Tokyo, Japan), connected to the pMD 19-T Vector Cloning Kit (TaKaRa, Tokyo, Japan), following the manufacturer’s protocol, and cloned in Escherichia coli; three of the bacteria solutions were then sent to Genscript (Nanjing, China) for sequencing. The obtained sequences were compared to the transcriptomic database to confirm the sequence prediction, and an online tool (https://www.genscript.com/tools/real-time-pcr-tagman-primer-design-tool) was used for qPCR primer design (Table 3).

Gel purified PCR products were ligated using the pMD19-T Vector Cloning Kit (TaKaRa, Dalian, China) and transformed into E. coli DH5α competent cells according to the manufacturer’s instructions. After transformation, E. coli DH5α cells were incubated overnight on LB-agar plates containing 100 μg/mL ampicillin, 40 μg/mL X-gal and 24 μg/mL IPTG at 37 °C. White colonies were selected and PCR-screened for the presence of inserts by using M13F and M13R vector primers. Thirty positive clones per library were randomly selected for sequencing.

Some of the sequences obtained from the metagenomic approach were shorter than expected. To obtain complete CDS sequences or RNA dependent RNA polymerase (RdRp) genes, consensus primers were designed by comparing the obtained sequences with known viral sequences (Supplementary Materials, Table S3). After nested RT-PCR or semi-nested RT-PCR, the products were analyzed on 1.0% agarose gels. Single fragments were recovered and purified using an Minibest Agarose Gel DNA Extraction Kit (TaKaRa Bio, Kusatsu City, Japan). The purified products were then ligated into pMD19-T Vector Cloning Kit (TaKaRa Bio, Japan) and transformed into E. coli DH5α cells. Clones were randomly picked and sent for Sanger DNA sequencing (Sangon Biotechnology Company, Shanghai, China).

The total RNA was extracted as described previously. The primers used for cloning are listed in Table S3. The coding sequence of CgVPE1 (Cs4g18790) was subcloned into the pMD™19-T vector using the pMD™19-T Vector Cloning Kit (TaKaRa, Beijing, China) and was sequenced by Sangon Biotech (Guangzhou, China). The 1458 bp cDNA sequence was obtained and translated into an amino acid sequence using DNAMAN 8.0 (Lynnon Biosoft, San Ramon, CA, USA). The phylogenetic analysis of CgVPE1 was conducted using MEGA-X v1.2.6 software and the neighbor-joining method.