Southern Blot and Gene Expression Analysis

For southern blot, leaf genomic DNA was extracted using the CTAB method and purified with the EasyPure® Plant Genomic DNA Kit (Transgenbiotech, #EE111-01, China). Thereafter, 5 μg of purified genomic DNA was digested with BamHI and separated by electrophoresis in a 1% agarose gel and then transferred to a Hybond N+ nylon membrane (Amersham, #RPN303B, UK). The pSAG12-specific probe (950 bp) was labeled with DIG-dUTP in PCR, using the PCR DIG Probe Synthesis Kit (Roche, # 11636090910, Switzerland). Subsequent hybridization was performed using the DIG High Prime DNA Labeling and Detection Starter Kit I (Roche, # 11745832910, Switzerland) according to standard protocols.

Gene expression level was detected using a q-RT PCR assay. Total RNA was extracted using the TriZol reagent (Invitrogen, #15596026, Germany) and treated with DNase I (Thermo, #EN0521, USA) to avoid genomic DNA contamination. First-strand cDNA was synthesized from each preparation of total RNA (1 μg/reaction) using the RevertAid First-Strand cDNA Synthesis Kit (Thermo Fisher Scientific, #K1622, USA) and an oligo-dT primer. Real-time quantitative PCR was performed using a LightCycler® 480 II system (Roche) with PowerUp™ SYBR® Green Master Mix (Thermo Fisher Scientific, #A25742, USA) in accordance with the manufacturer's instructions. UBQ5 was used as the internal control gene with the forward primers 5′-GTATTCCACCTGTTCAGC-3′ and the reverse primers 5′-ACCTTCAATGTTGTAATCCTT-3′. The forward and reverse primers for the RAmy1A gene were 5′-TCCTCATCGTCCTCCTTG-3′ and 5′-ACTCCCAGTTGAATCCCT-3′, respectively.