Peasy t3 vector

The full length cDNAs were obtained using a Taq DNA Polymerase amplification kit (TransGen, Beijing, China) according to the manufacturer’s instructions. The TRPA1, Painless, and Pyrexia homologous genes of B. mori (BAO53207.1, BAO53208.1, and NP_001296536.1) and D. melanogaster (NP_001261602.1, NP_001261176.1, and NP_612015.1) were used to query the transcriptome dataset of T. absoluta by blastp and tblastn. According to the corresponding sequences of T. absoluta, primers were designed by primer 5.0 (Table 1). The amplified fragments were purified using an AxyPrep TM DNA Gel Extraction Kit (Axygen, West Orange, NJ, USA). Finally, the distinct single-band amplification products were cloned into the pEASY-T3 vector (Transgen) and sequenced.

The recombinant plasmid used in this work was pPIC9-pg63, which contained the gene pg63 (HQ446162) from the strain Penicillium sp. CGMCC 166923 . Site-directed mutagenesis was carried out using the specific primers (Additional file 6) and two-step polymerase chain reactions (PCRs). All mutants were verified by double-stranded plasmid sequencing. Escherichia coli Trans I-T1 (TransGen, Beijing, China) and P. pastoris GS115 (Invitrogen, Carlsbad, CA) were used for plasmid amplification and heterologous expression, respectively. The pEASY-T3 vector (TransGen) and pPIC9 (Invitrogen) were used for plasmid construction, respectively. Culture media for His⁺ transformants selection and P. pastoris growth and induction were prepared according to the manual of the Pichia Expression kit (Invitrogen). Mono-, di-, and trigalacturonic acid (GalpA, GalpA2 and GalpA3) and the substrate PGA were purchased from Sigma-Aldrich (St. Louis, MO). All other standard chemicals were of analytical grade.

Primers were designed based on the transcriptome information of B. tabaci, and were used to amplify partial segments of the BtDnmt1 gene. Next, rapid amplification of cDNA ends (5′- and 3′-RACE) was performed to obtain the full-length cDNAs using a SMART RACE cDNA amplification kit (Clontech, Mountain View, CA, USA) according to the manufacturer’s instructions. The gene-specific primer sets (Table 1) were designed based on the B. tabaci transcriptome information from the NCBI website (http://www.ncbi.nlm.nih.Gov). The amplified fragments were purified using an AxyPrep^TM DNA Gel Extraction Kit (Axygen, West Orange, NJ, USA). Finally, the distinct single-band amplification products were cloned into the pEASY-T3 vector (Transgen, Beijing, China) and sequenced.

The complete cDNA sequences of S. argus Dmrt2a and Dmrt2b were obtained from our mixed tissues [35 (link)] and gonad [36 (link)] transcriptome data. Nile tilapia Dmrt2a (NCBI accession number: NP_001266696) and Dmrt2b (NCBI accession number: AAX08123) mRNA sequences were used as query sequences to blast our transcriptome sequence data.
Primers flanking the open reading frame (ORF) were designed with reference to the transcript obtained, and the full lengths of S. argus Dmrt2a and Dmrt2b were cloned as described in [3 (link)]. The PCR protocol used was as follows: initial denaturation at 94 °C for 3 min, 37 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s and extension at 72 °C for 2 min, followed by a final extension of 10 min at 72 °C. The PCR products were then examined on 2% agarose gel stained with ethidium bromide. Amplification bands were extracted from the gel, purified, and cloned into the p-Easy-T3 vector (TransGen Biotech, Beijing, China) as described previously [34 (link)]. All primers used and the GenBank accession numbers of Dmrt genes are listed in Tables S1 and S2, respectively.

Genomic sequence was amplified by nested PCR and assembled using DNAMAN. Total RNA was extracted from alfalfa using Trizol reagent. RNA concentration was determined with a NanoDrop 2000 spectrophotometer (Thermo Scientific, Santa Cruz, CA, USA). One μg of total RNA was used for the first-strand cDNA synthesis using the PrimeScript^TM 1st strand cDNA Synthesis Kit (Takara Biomedical Technology Corporation, Beijing, China). Degenerate primers designed according to the sequence of SQS genes in M. truncatula and Arabidopsis were used for amplification (Table S2). The PCR amplicons were purified after agarose gel (1%) separation and cloned into pEASY-T3 vector (TransGen Biotech Corporation, Beijing, China). Sequencing confirmed MsSQS was used for subcloning. The qRT-PCR analysis was performed using the SYBR Premix Ex Taq (TaKaRa, Dalian, China) was used on BIO-RAD CFX96TM Real-Time System (BioRad, Hercules, CA, USA). β-actin was used as to normalize the loading. Three biological replicates were conducted.

PtDXS was amplified by PCR using specific primers (Table 1); the PCR system included 2 µL forward and reverse primers, 2.0 µL cDNA as template, 5.0 µL 10× PCR buffer (Mg²⁺ plus), 1 µL 10 mM dNTPs, 0.5 µL rTaq DNA polymerase (Takara, Japan) and the ddH₂O was to a constant volume up to 50 µL. Also, the PCR reaction was performed as follows: 95 °C for 10 min, 35 cycles of 95 °C for 1.5 min, 58 °C for 1.5 min, and 72 °C for 2 min and, finally, 72 °C for 10 min. In addition, the PCR product was purified according to the manufacturer’s instructions (AXYGEN, Suzhou, China), and the purified product was cloned into the PEASY-T3 vector (TransGen Biotech, Beijing, China) based on the complementary cohesive end. The vector was transformed into Escherichia coli TransTI. Positive clones were selected by blue and white spot induced by X-Gal and IPTG and the reconstructed plasmids were sequenced by GenScript Company (Nanjing, China).
Rapid amplification of cDNA ends (RACE) was used to amplify the 3′ and 5′ untranslated regions of PtDXS. Next, the 5′ and 3′ fragments were sequenced using specific primers (Table 1). The 5′- and 3′-RACE amplified fragments were inserted into the PEASY-T3 vector and sequenced. The full-length sequence of PtDXS was obtained by aligning the obtained sequences.

The partial cDNA sequences of HcToll6 and HcToll7 were retrieved from our previous hepatopancreas transcriptome database (unpublished). In this experiment, 3′ and 5′ RACE were performed by using a Clontech Advantage^® 2 PCR kit from Takara (Japan) with two pairs of specific primers (HcToll6-F:5′-TGGATAAACGATGTGCTAAGCGACCCC-3′, HcToll6-R: 5′-CTGTGAAGACCACGCAAATAGAGACGGAAC-3′; HcToll7-F: 5′-GAGAGAACTTGGACAGGAAAGGGGGCT-3′, HcToll7-R: 5′-CGTATCGGCAGGTCGCCAAGGGTAAC-3′) to obtain the full-length cDNAs of HcToll6 and HcToll7. The amplification products were purified using a DNA gel extraction kit (Shanghai Generay Biotech Co., Ltd., Shanghai, China), inserted into the pEasy-T3 vector, and transformed into Escherichia coli Trans1-T1 cells (TransGen Biotech, Beijing, China). The putative clones were identified by PCR with M13F and M13R primers. The selected clones were sequenced by a commercial company (Springen, China).