Pmd19 t simple vector

DNA was extracted from the pooled material for each sample with Ultra-Clean soil DNA kits (MoBio, USA). Bacteria-specific primers, 27F and 1387R primers [24] (link), were used for 16S rRNA gene amplification. The reaction was performed for 25 cycles using the ‘Easy-A High-Fidelity’ PCR master mix from Stratagene (USA). Products from six PCRs were pooled, purified, using a Gel Extraction kit (Omega, USA), and ligated into pMD19-T simple vectors (Takara, Japan). The hybrid vectors were cloned into Escherichia coli TOP10. Clone libraries were constructed by random selection of approximately 100 white colonies from a single plate for each library. All clone libraries were named on the basis of the crop cycle and the plant growth stage of soil sampling. S1 and M1 were libraries constructed respectively for the seedling stage and pod-maturing stage in the first crop cycle; S2 and M2 were libraries constructed respectively for the two stages in the second cycle and S3 and M3 were libraries constructed respectively for the two stages in the third cycle.

FcCHS1, FcCHI1, FcDFR1, FcMYB21, and FcMYB123 were cloned based on the putative ORFs of fig unigenes from our RNA-seq data. The specific primer pairs (Table S8) were designed to amplify the ORF sequences using KOD DNA polymerase (TOYOBO, Osaka, Japan) from the cDNA of red fig peels. The reaction conditions were as follows: 96 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min, with 10 min extension at 72 °C. The PCR products were cloned into the pMD19-T simple vectors (TaKaRa, Shiga, Japan). Afterwards, those T-vectors were transferred into DH5α competent cells (Transgen Biotech, Beijing, China) for amplification, and the products were sequenced by GENEWIZ (New Jersey, USA).
The overexpression vectors of FcMYB21 and FcMYB123 were constructed via linking their ORFs into the linearized plant transformation vector pBI121 using fast-digest restriction enzymes of XbaI and BamHI (Thermo Scientific, Waltham, USA) and T4 ligase (Transgen Biotech, Beijing, China). Then the 35S::FcMYB21 and 35S::FcMYB123 recombinant vectors were transformed into Agrobacterium tumefaciens EHA105 competent cells, respectively.

Cloning of LrbHLHs was based on putative ORFs of unigenes from the RNA-seq database. Primers (Supplementary Table 1) were synthesized for ORF sequence amplification using Tks GflexTM DNA Polymerase (Takara Bio, Dalian, China) from L. radiata petal cDNA. Reaction conditions were: 5 min of 95°C, 35 cycles for 30 s at 94°C, 30 s at 60°C, 1 min at 72°C, with extension at 72°C for 10 min. PCR products were cloned into pMD19-T simple vectors (Takara Bio, Dalian, China). Afterward, those T-vectors were transferred into DH5α competent cells (Takara Bio, Dalian, China) for colony PCR amplification and then validated by sequencing.

According to the BNoV sequence available in GenBank, 12 pairs of primers were designed to successfully amplify SMU-YAK-J1 (GenBank accession number OK032546). An Omega Gel kit (Omega, New York, NY, USA) was used to purify all the PCR products, which were then cloned into the pMD19-T simple vector (TaKaRa Bio Inc.) and transformed into DH5 competent Escherichia coli cells (Yeasen, Shanghai, China). Three to five colonies were chosen for each product and sequenced in both directions (Sangon, Shanghai, China). Another nine pairs of primers targeting various locations were constructed to amplify the given genomic sequence in order to validate it. Tables S2 and S3 provide the sequence information for the PCR primers used for genome amplification.

Vitamin D (1,25-dihydroxy vitamin D3) receptor (VDR) primers were designed according to predicted mRNA sequences available in NCBI for Capra hircus VDR (reference sequence XM_013963843.1) and Pantholops hodgsonii VDR (reference sequence XM_005981021.1). NHeI and BamHI sites were engineered on either side of the open reading frame. Primer sequences were as follows:
VDR-F: 5′-CTAGCTAGCATGGAGGCGACTGCGGCCAGCAGC-3′(NHeI);
VDR-R: 5′-CGCGGATCCTCAGGAGATCTCGTTGCCAAACAC-3′(BamHI).
Total RNA from skin tissues was extracted using RNAiso Plus (Takara, Japan) and a reverse transcription kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to synthesize cDNA. Using this template, the CDS region of VDR was amplified using PrimeSTAR GXL DNA Polymerase (Takara, Kusatsu, Japan). The total volume of PCRs was 50 μL. The reaction conditions were as follows: 2 min at 98 °C, 34 cycles of 30 s of denaturation at 98 °C, 15 s of annealing at 60 °C, and 2.5 min of elongation at 68 °C, followed by a 10 min hold at 68 °C. PCR products were purified from 1% agarose gels. Adenine was added to 1 μg of the purified product and VDR segments were ligated into the pMD19-T (Simple) vector (Takara). Products were identified by PCR and commercial sequencing.

The SiFBA5 gene sequence was obtained from SaussureaKBase (http://www.shengtingbiology.com/SaussureaKBase/index.jsp.). Putative full-length SiFBA5 cDNA and genomic DNA were amplified using gene-specific primers SiFBA5-F and -R (Supplementary Table S1). SiFBA5 cDNA was ligated into the pMD®19-T simple vector (TaKaRa, China) and E. coli DH5α competent cells (TransGen, China); successful cloning was verified via Sanger sequencing (BGI, Beijing, China). All subsequent constructs were made using this clone as a template. SiFBA5 gene homologs were identified using BlastP in NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple sequence alignments were performed in ClustalX2 with default parameters and DNAMAN. Alignments were then adjusted for constructing the phylogenetic tree in MEGA 5.0.