High fidelity thermostable dna polymerase

Total RNA samples of Vitis vinifera L. cv. Jingxiu leaves and fruits were extracted with a RNAprep Pure Plant Kit (Tiangen, Beijing, China), from which first-strand cDNA was synthesized using a PrimeScript 1st strand cDNA synthesis kit (TaKaRa, Dalian, China). The full-length cDNA of VvTIFY9 was then amplified using gene-specific primers (Table S1) and real-time quantitative PCR (qPCR) performed in a Bio-Rad IQ5 real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA). PCR amplifications used high-fidelity thermostable DNA polymerase (Takara, Dalian, China), in a total system volume of 50 µL. PCR reaction conditions were 94 °C for 5 min, 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 2 min in 30 cycles, followed by 5 min at 72 °C [29 (link)]. The relative expression of the target gene was calculated using the 2^−ΔΔCt method [30 (link)]. All reactions were performed in triplicate.

A gene that encodes a predicted protein with high sequence similarity to Arabidopsis GPX5 was obtained from the transcriptome unigene assembly database of R. crenulata (unpublished). This unigene was named RcGPX5 and has been confirmed to carry the entire open reading frame of a GPX. The RcGPX5 cDNA was isolated from RNA extracted from R. crenulata leaves. Total RNA extraction and first-strand cDNA synthesis were performed using the Easy RNA extraction kit and the First chain cDNA synthesis kit (Promega, Beijing China), respectively. PCR amplification of RcGPX5 was conducted using a high-fidelity thermostable DNA polymerase (Takara, Japan) and primers containing the restriction sites NcoI and Bst EII. The amplification product was inserted into the pEASY-T1 vector (TransGen Biotech, Beijing, China) and the DNA insert was sequenced using Sanger technology (Biotech, Shanghai, China). To construct the recombinant plant expression vector, the RcGPX5 cDNA fragment was cloned into the binary vector pCAMBIA3301 under control of the CaMV35S constitutive promoter by double digestion with NcoI and Bst EII. The resulting plasmid vectors were transformed into Escherichia coli DH5α and Agrobacterium tumefaciens EHA105 using a standard heat-shock method.