5’-RACE
The 5’modified RACE was performed using the FirstChoice RLM-RACE Kit
(Invitrogen). In brief, total RNA was isolated from young leaves ofB. napus . Then, 8–10 µg high-quality total RNA (A260/280≈2.00)
was directly ligated to the 5′-RACE adapter without calf intestinal
phosphatase and tobacco acid pyrophosphatase treatment. The first-strand
cDNA was synthesized using M-MLV reverse transcriptase according to the
manufacturer’s instructions. The PCR amplification was performed using
the 5′ outer primer and gene-specific outer primer, and 0.1 µL PCR
product was used as the template for nested PCR. Nested PCR
amplification was performed using the 5′ inner primer and gene-specific
inner primer. The PCR products were gel-purified and then cloned and
sequenced.