5 full race kit with tap

Total RNA from MV4-11 cells was extracted using TRIzol according to the manufacturer’s guidelines. The 5′- and 3′-ends of cDNA were acquired using a 5′-FULL RACE Kit with TAP (Takara, Japan) and 3′-FULL RACE Core Set with PrimeScript RTase (Takara, Japan) respectively according to the manufacturer’s instructions. PCR products were obtained and then cloned into pEASY-T3 (TransGen Biotech, China) for further sequencing.

To determine 5′ end of a truncated trehalase gene MeTRE2 (cassava4.1_015916m.g), total RNA was extracted from leaves of cassava variety SC8 using a Plant RNA Extraction Kit (TaKaRa Biotechnologies, Dalian, China). mRNA was purified using a Dynabeads mRNA purification kit (Invitrogen, Beijing, China). First-strand cDNA was synthesized using a 5′ Full RACE kit with TAP (TaKaRa) accordingly to the manufacturer’s protocol. Two rounds of RACE PCR were performed using a gene specific primer and a universal primer each provided in the kit. For the first round PCR, the MeTRE2 gene specific primer was 5′-CAATTCGGGTTGCTTCCTGACTTCG-3′ that was designed with MacVector 14.0 (MacVector Inc., Apex, NC, USA). For the second round PCR, the MeTRE2 gene specific primer was 5′-CTTAGGCAAGAACCCATCAGGCTC-3′. The PCR reactions were carried out using the parameters suggested by the manufacturer.

Total RNA from THP1 cells was extracted using Trizol reagent (Invitrogen, CA, USA) according to the manufacturer’ s guidelines. The 5′- and 3′-ends of cDNA were acquired using a 5′-FULL RACE Kit with TAP (Takara, Japan) and 3′-FULL RACE Core Set with PrimeScript RTase (Takara, Japan) respectively according to the manufacturer’s instructions. PCR products were obtained and then cloned into pEASY-T3 (TransGen Biotech, China) for further sequencing. All of the primers for RACE experiments are listed in Additional file 1: Table S3.

The full-length BxATG1 and BxATG8 cDNA were obtained using the 3′-Full RACE CoreSet with PrimeScript™ RTase kit (TaKaRa Biotechnology, Dalian, China) and 5′-Full RACE Kit with TAP (TaKaRa Biotechnology). Gene-specific primers of BxATG1: GSP1-1 (3′-Full RACE first round of PCR) and GSP1-2 (3′-Full RACE second round of PCR), and GSP1-3 (5′-Full RACE first round of PCR), and GSP1-4 (5′-Full RACE second round of PCR) (Table 1). Gene-specific primers of BxATG8: GSP8-1 (3′-Full RACE first round of PCR) and GSP8-2 (3′-Full RACE first round of PCR), and GSP8-3 (5′-Full RACE first round of PCR) and GSP8-4 (5′-Full RACE second round of PCR) (Table 1). There were designed for 3′ and 5′ RACE amplification based on the two partial sequences of BxATG1 and BxATG8, which were obtained from the homology-based cloning results. The cycling profiles used were as follows: a cycle at 94 °C for 3 min, followed by 30 cycles, each consisting of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 2 min. The final extension step was at 72 °C for 10 min.

The complete amino acid sequences of 10 MKKs in A. thaliana were used as query sequences for BLAST homology comparisons with P. trichocarpa protein sequences within a database (Table S4), and the top five poplar protein sequences with the highest similarity to each A. thaliana MKK protein sequence were selected. Oligo7 software was used to design specific amplification primers and RACE primers (Table 1). According to the operation instructions, the 3′-Full RACE Core Set with PrimeScript^TM RTase (Takara, Dalian, China) and the 5′-Full RACE Kit with TAP (Takara, Dalian, China) were used to amplify the full-length PeMKK2a. The isolated fragment was ligated to a pMD-19T vector and transformed into TOP10. Positive detection and sequencing were performed to obtain the nucleotide sequence of the target fragment. The 3′ RACE and 5′ RACE sequences were compared and spliced, and the NCBI ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/, accessed on 10 October 2021) was used to predict open reading frame (ORF). Afterward, we designed specific primers targeting both ends of the potential ORF (Table 1), conducted PCR amplification, and performed sequencing verification.

Putative targets of miR156b were predicted using psRNATarget (Dai and Zhao, 2011). For the 5′ RACE mapping of miR156b cleavage sites, total RNAs were isolated from a mixture of different organs collected from 4‐week‐old Williams 82 plants using Plant RNA Reagent (Invitrogen) according to the manufacturer's recommendations. A 5′‐Full RACE Kit with TAP (Takara Bio Inc., Kusatsu, Japan) was used to process the total RNAs and to map the 5′ termini of the primary transcripts. The cDNA samples were amplified by nested PCR according to the manufacturer's protocols. Gene‐specific primers (Table S2) were designed by Invitrogen.

Four pairs of PCR primers that direct amplification of fragments that span the entire CVA genome (S3 Table) were designed based on the reference CVA sequence (GenBank X82547) and conservative rigions of available genomes in GenBank. The 3′-terminal region was amplified using an oligo (dT) primer and a sequence-specific primer, and the 5′-terminal region was amplified using the 5′-Full RACE Kit with TAP (TaKaRa, Beijing, China), according to the manufacturer’s instructions. RT-PCR was performed as described above, except that we used the 2X Long Taq MasterMix, annealing was for 30 s at 54–56°C, and extension was at 72°C for 150 s. All amplification products were cloned and sequenced as described above. To overcome problems linked to intra-isolate sequence diversity and to avoid mistakes in sequence assembly, adjacent amplicons were designed to overlap for >100 bp, and at least three clones of each PCR product were sequenced. The resulting sequences were then assembled into a single contiguous genomic ChYT52 CVA isolate sequence.