Rapid amplification of cdna ends kit

cDNA from flower buds in different culture tissue lines in B. oldhamii was used as a templet for gene cloning. To isolate a SOC1-like gene from B. oldhamii, we compared the amino acid sequences of SOC1 homologs from grass family plants, including rice, Triticum aestivum, and Z. mays, and designed primers (S-F/R) for conserved domain regions. A specific SOC1-like cDNA fragment (~250 bp) was obtained, ligated into the pMD18-T vector and confirmed as a partial sequence of a SOC1-like gene of B. oldhamii. The 5′ and 3′ RACE systems were used to isolate the 5′ and 3′ end cDNA with the Rapid Amplification of cDNA Ends kit (Invitrogen, USA). A 330 bp 5′-RACE cDNA and 740 bp 3′-RACE cDNA fragment were spliced to obtain 1049 bp full-length cDNA. With this sequence as the template, the ORF of BoMADS50 was amplified with a high-fidelity enzyme (TaKaRa, Japan). Moreover, four transcripts of BoMADS50 were obtained from B. oldhamii, named BoMADS50-1, BoMADS50-2, BoMADS50-3, and BoMADS50-4. Finally, the nucleotide variations were further confirmed by PCR using DNA templates of different lines. The primers used for BoMADS50 cloning are shown in Table S1.

HMGR and CHS gene core fragments were used to design six 5′ Rapid Amplification of cDNA Ends (5’ RACE) specific primers (HMGR-GSP1, HMGR-GSP2, HMGR-GSP3, CHS-GSP, CHS-GSP2, and CHS-GSP3) and four 3′ Rapid Amplification of cDNA Ends (3’ RACE) specific primers (HMGR-1, HMGR-2, CHS-1, and CHS-2) (Table 1). Primer Premier software (version 5.0) was used for the primer design. The 5’ and 3’ sequences of PtHMGR and PtCHS were separately cloned using the Rapid Amplification of cDNA Ends kit (Version 2.0, Invitrogen, Shanghai) and the SMARTerTM RACE cDNA Amplification Kit (Clontech, USA). The target fragment was purified with the E.Z.N.A.® Gel Extraction Kit, and linked to the pMD®18-T vector (TaKaRa, China). Subsequently, it was transformed into DH5α Escherichia coli competent cells (Tiangen, China). The positive DH5α with target gene was screened and subsequently sent to Sangon Biotech Co., Ltd. (Shanghai, China) for sequencing.