DHAR-1R: 5’-TGGGATCCTTGCTCTTGAGG-3’. Then based on the obtained conserved region sequences, two pairs of nested primers used for isolating Fragaria × ananassa cv. Toyonaka dhar gene flanking sequences were designed (Fig.1). The 3’end nested primers sequences were 3’-DHAR1 (5’-GAAAGGTGCCTGTGGTGAAG-3’)and 3’-DHAR2 (5’-GGGTCGCTGATTCTGATGTG-3’); The 5’ end nested primers sequences were 5’-DHAR3 (5’-GGAG TTCGGTGACAAGGTTCA-3’) and 5’-DHAR4 (5’-TGT CAGCCAAGTTGATGAGG-3’).
Fig.1 Position and orientation of nested primers designed for SON-PCR
PCR and SON-PCR amplification: The conserved region of dhar gene fragment was amplified with a 20 µl reaction mixture containing DNA 100ng, 2.0 µl of 10×PCR Buffer without Mg2+, 2.0mmol MgCl2, 200µmol dNTPs, 0.5µmol of DHAR-1F and DHAR-1F each, and 1.0 unit of Taq DNA polymerase (TIANGEN,
China). The PCR procedure strarted with 94℃for 3min, then 35 cycles of 94℃ for 1min, 57℃ for 1min,
72℃ for 2min, and finally 72℃ for 7 min. The first round SON-PCR was performed with a 50 µl reaction mixture containing DNA 100ng, 2.0µl of 10×PCR buffer without Mg2+, 2.0mmol MgCl2, 200µmol dNTPs, 1.0µmol primer 3’-DHAR1 or 5’-DHAR3 , and 2.5 unit of Taq DNA polymerase. The second round SON-PCR template was 1/100 of the product from the first round SON-PCR and PCR performed with 2.0µmol primer 3’-DHAR2 or 5’-DHAR4. Others were the same with the first round SON-PCR. The SON-PCR procedure were all strarted with one denaturation step (3min at
94℃), the reactions consisted of five cycles of amplification [30s at 94℃, 1min at 58℃ (The first
round SON-PCR reaction) or 60℃ (The second round SON-PCR reaction), 2.5min at 72℃] followed by one ramping step (30s at 94℃, 3min at 29℃, ramp to 72℃ in 3min, 2.5min at 72℃) and 60 (The first round SON-PCR reaction) or 30 (The second round SON-PCR reaction) new amplification cycles [10s at 94℃, 1min at 58℃ (The first round SON-PCR reaction) or 60℃ (The second round SON-PCR reaction), 2.5min at 72℃]. The reaction ended with a final elongation step of 7min at 72℃.The PCR products were analyzed on a 1.0% agarose/EtBr gel and the corresponding DNA bands were recovered,
Agric. Biol. J. N. Am., 2010, 1(4): 726-730
then cloned into the pMD19-T Vector (TaKaRa, China) for sequencing. Sequence analysis was performed using the software DNAMAN (Version 3.0, Lynnon BioSoft).
RESULTS AND ANALYSIS The cloning and sequence
conserved region fragment: In this study, a pair of specific primers DHAR-1F and DHAR-1R were used to amplify dehydroascorbate reductase gene conserved region fragment from Fragaria × ananassa cv. Toyonaka. PCR amplification generated a expected 546 bp DNA fragment (Fig. 2). The homology analysis by NCBI showed that the sequences was 94%～98% identical to the sequences
with that of Malus×domestica , Nicotiana tabacum (AY074787), Vitis
vinifera (EU280162) and Lycopersicon esculentum (DQ521269) dhar gene from the GenBank. Therefore,
fragment from Fragaria × ananassa cv. Toyonaka.
Fig. 2 The electrophoresis result of the RT-PCR of dhar gene conserved region Note: M indicates the molecular weight standard; lane 1 indicates the PCR product. The cloning of dhar gene flanking sequences: The SON-PCR technique was used to isolate the flanking regions of strawberry dhar gene. As showed in Figure 3, the two rounds of SON-PCR reaction all had a specific band between 900～1000 bp in the 3’ end amplification; In the 5’ end amplification, the second round of SON-PCR reaction had three bands, but the band of about 250bp was presumed target fragment. Then the two presumed target bands were recovered and sequenced. The sequenced results showed that 3’ and 5’ end fragments were respectively 922bp and