Rt pcr system

The hairy roots of S. miltiorrhiza were dried and pulverized in liquid nitrogen with mortar and pestle for extraction of the total RNA. Total RNA was used to synthesize the first strand cDNA with primer AP (5′-GGCCACGCGTCGACTAGTAC(T)₁₆-3′) by using RT-PCR system (TaKaRa, Japan). A local S. miltiorrhiza transcription database was built up from public transcripts from NCBI as well as our own transcriptomic data sequenced by hairy roots. Conserved domains of ZIM and Jas were utilized to search our transcriptome database to identify putative JAZs from S. miltiorrhiza with Blastx and Blastn. ORF Finder was used to identify the length of the ORF. Among the candidate JAZ sequences, SmJAZ3 (lack of 3-terminal) was obtained with the specific forward primer SmJAZ3-F421 (5′-GCCGTTGGAACCACTGATTTTAG-3′) together with the primer AUAP. Primer pairs (SmJAZ9-F: 5′-ATGGAGAGAGATTTCATGGGGT-3′, SmJAZ9-R: 5′-TCAGTCATCCTTGCTGACGGA-3′) were synthesized to amplify the whole ORF of SmJAZ9. The PCR amplification conditions were as follows: initial denaturation at 95 °C for 8 min, followed by 35 cycles denaturation at 95 °C for 30 s, annealing at 58 °C for 1 min and extension at 72 °C for 1 min 30 s.

The total RNA of the cells was extracted with TRIzol (Invitrogen) in accordance with the manufacturer’s instructions. Thereafter, the mRNA was reverse-transcribed to single-stranded cDNAs using a reverse-transcription PCR (RT-PCR) system (TaKaRa). The primers were listed in Supplementary Table 2. Thereafter, real-time fluorescent quantitative PCR or semi-quantitative PCR was performed to analyze the gene expression levels.

Total RNA was extracted from cells with TRIzol (Invitrogen, USA). RT-PCR system (TaKaRa, Japan) was used to reverse RNA, and 1 ul cDNA sample was quantified using primers with SYBR Green PCR Master mix (TaKaRa, Japan) by real-time PCR. Let-7a specific primers were used as previously reported [11 (link)]. All analyses were performed in triplicate.