Quantitative Analysis of Anthocyanin Biosynthesis

Total RNA from the pericarps of litchi and tobacco was extracted and first strand cDNA was synthesized as described above. The transcription levels of both the litchi and tobacco anthocyanin biosynthetic genes were analyzed using quantitative real-time PCR (qRT-PCR),) with THUNDERBIRD qPCR Mix (TOYOBO, Japan) and ABI 7500 Real-Time PCR Systems (Applied Biosystems, USA) according to the manufacturers' instructions. The primers are shown in Table S1. Each reaction (20 μL final volume,) contained 9.96 μL 2×SYBR® qPCR Mix (TOYOBO), 0.04 μL 50×ROX reference dye, 1.0 μL of each the forward and reverse primers (0.25 μM), 2.0 μL of the cDNA template (corresponding to 50.0 ng of total RNA), and 7.0 μL of RNase-free water. The reaction mixtures were heated to 95°C for 30 s, followed by 40 cycles at 95°C for 10 s, 56°C for 15 s, and 72°C for 35 s. A melting curve was generated for each sample at the end of each run to ensure the purity of the amplified products. The specific qRT-PCR primers were designed using a Primer 5.0 program (PREMIER Biosoft International, Canada) (Table 2). Using these gene-specific primers, each assay amplified a single product of the correct size and demonstrated an acceptable PCR efficiency (approximately 90%). qRT-PCR reactions were normalized to the Ct values for LcActin (HQ615689) and NtACT (GQ281246) in litchi and tobacco, respectively. The relative expression levels of the target genes were calculated using the formula 2^−ΔΔCT[61] (link). All biological replicates were measured in triplicate.