Peasy t1 cloning vector

PmGrx2 was screened for partial fragments and identified through the NCBI database BLAST in the cDNA library of P. monodon in the laboratory, upon which the primers were established through Premier 6.0. The PCR program, which facilitated the expansion of PmGrx2 open reading frame (ORF), underwent three procedures: 95 °C (3 min), 35 cycles of 95 °C (15 s), 55 °C (15 s), 72 °C (15 s), and 72 °C (5 min). In addition, the PCR amplification was completed in 25 μL of reaction mixture made up of forward primer (1 μL), reverse primer (1 μL), cDNA template (1 μL), double-distilled water (12.5 μL), and 2 × Taq Plus Master Mix Ⅱ (12.5 μL) (Vazyme, Nanjing, China). Nested PCR primers featuring 3’ and 5’ ends end were generated by Premier 6.0 based on PmGrx2 ORF. The procedures of the PCR program referred to the literature [11 (link)].
All PCR products were cloned into pEASY^®-T1 Cloning Vector (TransGen, Beijing, China), and a positive monoclonal colony was sequenced. RACE technology was taken to calculate the full-length cDNA of PmGrx2, while Ruibiotech (Guangzhou, China) was used for the synthesis of primers and the sequencing of PCR products. Supplementary Tables S1 and S2 list the primers above.

For the isolation of potential viral pathogens infecting tobacco plants, the symptomatic leaf samples were collected from Luzhou, Sichuan province, China in 2018. Total RNA was extracted using RNAiso Plus reagent (TaKaRa, Dalian, China) from tobacco leaves following the manufacturer’s guidelines. Viruses were detected by RT-PCR with four pairs of degenerate primers [27 ], including CMVCPf/CMVCPr (for cucumber mosaic virus), Tob-Uni1/Tob-Uni2 (for tobamoviruses), PotyF/PotyR (for potyviruses) and TSWVf/TSWVr (for tomato spotted wilt virus) (Additional file 1: Table S1). The amplified PCR products were purified with EasyPure Quick Gel Extraction kit (Transgen Biotech, Beijing, China), cloned into pEASY-T1 Cloning Vector (Transgen Biotech) and directly sequenced (Sangon Biotech, Shanghai, China), respectively.

The full-length cDNA was obtained by Rapid Amplification of cDNA Ends (RACE). 3’-RACE and 5’-RACE cDNA were synthesized using RACE kits (3’-Full RACE Core Set with PrimeScript RTase, 5’-Full RACE Kit, Takara, Japan). The RACE primers(Additional file 1: Table S1) were designed basing on EST sequence of DHN, which acquired by searching “Dehydrin” annotation in L. chinense’s transcriptome database [27 (link)]. All the PCR reactions were carried out in a 50 μL reaction mix according to the PCR protocol (Additional file 1: Table S1). The reaction mix consisted of 5 μL 10 × LA PCR Buffer II (Mg²⁺ Free), 5 μL MgCL₂ (25 mmol·L^− 1), 8 μL dNTP Mixture (2.5 mmol·L^− 1 each), 2 μL of each primer (2.5 mmol·L^− 1 each), 0.5 μL TaKaRa LA Taq (5 U·L^− 1), 2 μL each cDNA, 25.5 μL ddH₂O. PCR products were separated on a 1.5% agarose gel and purified using the DNA gel extraction kit (Transgen Biotech, China). The purified PCR products were cloned into pEASY®-T1 Cloning Vector and transferred into Trans5α Chemically Competent Cells for white-blue plaque selection (Transgen Biotech, China). Positive monoclonal was screened for sequencing (Genscript, China). Finally, the gene full-length was assembled according to the 3′ and 5’sequencing results.

The open reading frames (ORFs) of guppy it, avt, and itrs were obtained from the genome database (PRJNA238429), and the sequences were confirmed by PCR followed by Sanger sequencing. PCR was performed according to the protocol described in a previous report using cDNA samples from the brain or ovary (38 (link)). Briefly, initial denaturation was performed at 94°C for 3 min followed by 35 cycles at 94°C for 30 s, 55°C to 60°C for 30 s, and 72°C for 1 min. The reaction was terminated with an extension for 5 min at 72°C. The products were purified using a TIANgel Midi purification kit (TIANGEN, Beijing, China), subcloned into the pEASY-T1 cloning vector (TransGen Biotech, Beijing, China) and transformed into DH5α cells. Positive clones containing the inserts of the expected size were selected for sequencing to confirm the results. The confirmed sequences were submitted to the NCBI. All primers used in the present study are listed in Table 1.
The signal peptide and precursor cleavage sites of IT and AVT were predicted by SignalP 5.0 (39 (link)) and NeuroPred software (40 (link)), respectively. Seven putative transmembrane domains were predicted by the TMHMM server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0/). Multiple sequences were aligned and analyzed using Clustal X software (41 (link)), and phylogenetic trees were constructed using MEGA 6 software (42 (link)).

The full-length open reading frame of CpMYC2 and CpbHLH13 was amplified by PCR from the cDNA of wintersweet flowers by using a specific pair of primers containing Kpn1 and Xba1 restriction sites (Table 5). The PCR conditions were 5 min, 95 °C; 30 s, 95 °C; 30 s, 60 °C, 30 cycles; 30 s, 72 °C; 5 min, 72 °C. The PCR product was purified using a Tiangen Midi Purification Kit (Tiangen, Wuhan, China) and cloned into a pEASY-T1 cloning vector (Transgene Wuhan, China), according to the manufacturer’s instructions, and three positive clones were selected and sequenced.

The sequence encoding human 14-3-3ε gene (GenBank accession number NR_024058) was generated by PCR. In order to facilitate the protein expression in E. coli, we modified the least preferred codons into the most preferred ones without changing amino acid sequence, according to the codon usage in E. coli[43] (link). A two-step strategy combining assembly PCR and overlap extension PCR process was used. A long codon-optimized DNA sequence was divided into three fragments with size from 200 to 350 bp, which was assembled via PCR reactions using 45 and 60 mer oligonucleotides (Table S1). There was a 15–20 bp overlap for each of the oligonucleotides used. These three fragments were assembled into a full-length DNA sequence by overlap extension PCR. The 768 bp fragments was ligated into the pEASY-T1 cloning vector (TransGen Biotech, Beijing, China) by TA cloning and confirmed by DNA sequencing.

A FoccCSP cDNA fragment was found in the F. occidentalis transcriptome sequenced at the Beijing Genomics Institute (BGI). We designed forward and reverse primers (Table 1) for PCR amplification of the fragment for sequencing. transcriptome transcriptomeThe amplification conditions were 3 min at 95°C for initial denaturation followed by 30 cycles of 30 s at 94°C, 30 s at 55°C, 1 min at 72°C, and a 10 min final extension at 72°C. Amplified DNA was purified using an AxyPrep DNA gel extraction kit (Axygen), inserted into the pEASY-T1 Cloning Vector (TransGen, Beijing, China) and transformed into E. coli Trans1-T1 competent cells (TransGen, Beijing, China). Positive clones were analyzed by PCR using vector-specific primers and Taq DNA polymerase (Promega). Clones of interest were amplified, and the plasmids were purified and sequenced at Biomad. Three independent clones were sequenced to detect potential PCR mutations.