Primestar high fidelity dna polymerase

The cDNA fragments corresponding to Sw-5b (AY007366.1), NbHSP90 (Niben101Scf15166g03015.1), NbRAR1 (LC314308.1), NbEDS1 (Niben101Scf06720g01024.1), NbNDR1 (AY438029.1), NbNPR1 (Niben101Scf14780g01001.1), NbNRC2a (KT936525.1), NbNRC2b (KT936526.1), NbNRC3 (MK692736.1), NbNRC4 (MK692737.1), NbNRG1 (DQ054580.1), and NbADR1 (Niben101Scf02422g02015.1) were amplified by polymerase chain reaction (PCR) using PrimeSTAR High-Fidelity DNA Polymerase (TaKaRa, Dalian, China) and cloned into pTRV2 [37 (link)]. For constructing TRV-NbNRC2/3/4 and TRV-NbNRG1/NbADR1, gene fragments were fused by overlap PCR. The pTRV2-NbPDS, pTRV2-NbSGT1, and pTRV2-GUS vectors have been described previously [52 (link)].

Total RNA was extracted using TRIzol Reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocols. RNA quality, purity and concentration were evaluated by measuring the absorbance at OD_260/280 using a NanoVue spectrophotometer (GE Healthcare Biosciences, Uppsala, Sweden). The cDNA for cloning and qPCR was synthesized using 1 μg of total RNA with the PrimeScript^® RT reagent Kit (Takara Biotechnology Dalian Co., Ltd., Dalian, China) following the manufacturer’s protocol.
Five heat shock protein genes were identified based on the L. bostrychophila transcriptome database [31 (link)]. The transcripts, which contain the full-length open reading frames (ORFs), were verified by PCRs with specific primers (Table 1). PCRs were conducted using PrimeSTAR high-fidelity DNA polymerase (Takara, Dalian, China), in accordance with the following protocol: 95 °C for 3 min, followed by 34 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 2 min and then with a final extension at 72 °C for 10 min. The amplified products were purified by electrophoresis on agarose gels (1.0–1.5%) and cloned into the pGEM-T Easy vector (Promega, Fitchburg, MA, USA). After confirmation by PCR using primers M13F and M13R, the positive clones containing target genes were isolated and sequenced (BGI, Beijing, China).

Gene-specific primers for amplification of gene full-length coding regions were designed (Supplementary Table S1) based on published B. dorsalis transcriptome data (Shen et al., 2013 (link)) using Primer Premier 5.0 (Premier Biosoft International, Palo Alto, CA, United States), with DNAMAN v.6.03 (Lynnon Biosoft, San Ramon, CA, United States) being used for sequence alignment. The open-reading frame (ORF) sequences of BdKr-h1 and BdMet were PCR-amplified using PrimeSTAR high-fidelity DNA polymerase (Takara, Dalian, China), in accordance with the following protocol: initial denaturation at 98°C for 2 min; followed by 35 cycles of 98°C for 15 s, 60°C for 15 s, and 72°C for 3 min; with final extension at 72°C for 10 min followed by holding at 12°C. The resulting PCR products were separated by electrophoresis on agarose gels (1.0–1.5%). PCR products of the expected size were excised from the gels, ligated into a pGEM-T Easy vector (Promega, Beijing, China), and transformed into Trans5α chemically competent cells (TransGen Biotech Co., Ltd., Beijing, China). Transformants were selected on Luria–Bertani agar plates containing 0.1% ampicillin and sequenced (BGI, Beijing, China).

To verify the ORF of Idgf6 in B. correcta, primers were designed based on the conserved regions of Idgf6 in B. oleae, Ceratitis capitata, and D. melanogaster (sequence from GenBank) and the sequence of Idgf6 from the B. correcta transcriptome (No. MK450457). DNAMAN v.6.03 (Lynnon Biosoft, San Ramon, CA, United States) was used for sequence alignment. The primers for cloning are listed in Table 1. The open-reading frame (ORF) sequence of Idgf6 was amplified using PrimeSTAR high-fidelity DNA polymerase (Takara, Dalian, China) following the manufacturer’s protocol. The PCR products were isolated, purified and ligated into a pGEM-T Easy vector (Promega, Beijing, China) and sequenced by a company (BGI, Beijing, China).
The ORF and conserved domain were identified with ORF Finder software¹ and NCBI BLAST results². To predict the conserved domains of B. correcta Idgf6, Idgf6 protein sequences from 23 species in Drosophilidae and Tephritidae were collected by BlastP in GenBank (Supplementary Table S1) and aligned with the sequence of B. correcta Idgf6 with ClustalX 2 software and GeneDoc 2.7.0 (Nicholas et al., 1997 ; Larkin et al., 2007 (link)).