Quantitative Real-Time PCR
The expression of Sh2 was measured in the fifth leaf, three days
after emergence. The first, third and eighth centimeter from the leaf
base were harvested in sh2 as meristem, elongation and mature
tissues, respectively, based on the cell length profile. In the wild
type, these zones were harvested as the first, fifth and tenth
centimeter from the leaf base, respectively. Total RNA was diluted to
the same concentration (0.4 μg. μL-1) for all samples and first strand
cDNA synthesis was performed with superscript II Reverse Transcriptase
according to the manufacturer’s protocol (Thermo Fisher Scientific,
USA). Quantitative Real-Time PCR was performed with the Takyon qPCR Kits
for SYBR Assay (Eurogentec, Belgium) using the UBCP gene
(Zm00001d001913) as a housekeeping gene (Manoli et al. , 2012).
The primers 5’-GTAAGGGCATCCAAGAGG-3’ and 5’ -AAGCGGCTCTT ACCATAC-3’ were
designed to amplify the Sh2 gene.