Cloning and Expression of IbCAT2 from Sweet Potato

Total RNAs were isolated from sweet potato samples using TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. RNA quality and purity were assessed by agarose gel electrophoresis. The total RNAs were then reversely transcribed with PrimeScriptTMRT Reagent Kit (TaKaRa, Japan) using Oligo(dT) as primer. Cloning primers were designed according to the assembled contig of IbCAT2 in the sweet potato transcriptome [32 (link), 34 (link)] using Primer Premier 5.0 (PREMIER Biosoft International, CA, USA). Polymerase Chain Reaction (PCR) was performed using KOD-Plus-Neo (TOYOBO, Japan) with IbCAT F1 (5′-GATATCATGGATCCTTATCAGCACCG-3′) and IbCAT R1 (5′-GGAATTCTCACATTGTTGGCCGCAC-3′; bp position: 1049–1069) as primers with 35 cycles of 2 min at 94°C, 10 s at 98°C, 30 s at 56°C, and 45 s at 68°C. The PCR product was separated by 1% agarose gel and purified by DNA gel extraction kit (OMEGA, USA). It was then double digested by EcoR I and EcoR V (Fermentas, USA). The pET-32a(+) was also double digested by EcoR I and EcoR V (Fermentas, USA). Restricted DNA products were then separated by 1% agarose gel, purified by DNA gel extraction kit (OMEGA, USA), and then ligated with pET-32a(+) by T4 DNA ligase (TaKaRa, Japan). The recombinant plasmid was transformed into the Escherichia coli host strain JM109. Positive clones were sequenced with an ABI 3730 instrument.