Screening and Characterization of Transgenic Arabidopsis Plants Expressing AnAFP Variants

As described by Sun et al.^{36 (link)}, T₁ seeds were surface-sterilized with 75% ethanol for 1 min and 10% NaClO for 10 min, and plated onto 1/2 MS plates with 50 mg/L kanamycin (Sigma, USA) for screening of transgenic plants, which were used to produce T₂ generation. The T₂ plants with 3:1 segregating-ratio to resistance/susceptibility of kanamycin were self-pollinated to generate T₃. The homozygous lines without segregation were collected from T₃, and were identified by PCR amplification using the primers (Table S4) for the specific fragments of AnAFP, AnAFPΔA, AnAFPΔK, AnAFPΔN and AnAFPΔS. The total RNA of every line was extracted by RNA extractor kit (Sangon, China), reacted with RNase-free DNase I, and used to reverse transcribed into cDNA using PrimeScript RT Reagent Kit (TaKaRa, Dalian). The ectopic expression of the transformed genes was identified by reverse transcription PCR (RT-PCR) using the above primers. The AtActin gene was amplified and used as reference. Five T₃ lines were planted in pots, and grown in green house at 22 ℃ and 60–70% relative humidity under a 10 h light/14 h dark photoperiod. One-month-old seedlings were used for heat-shock treatment at 46 °C for 3 h, and recovered for 2 weeks at 22 ℃, and investigated for phenotype.