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Cloning and sequence analysis of dehydroascorbate reductase gene (dhar) from strawberry by SON-PCR - page 3 / 5

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255bp length. Sequence comparison revealed that the two flanking sequences were all dhar gene. Eventually, a 1374bp sequence was obtained by splicing the isolated flanking fragments with the conserved region fragment (Fig. 4 B).

Fig. 3 Identification of the flanking region of strawberry

dhar gene by SON-PCR Note: M indicates the molecular weight standard; lane 1and 2 indicates 3’ end SON-PCR primary and secondary reaction; lane 3 and 4 indicates 5’ end SON-PCR primary and secondary reaction.

Sequence analysis: Sequence analysis indicated that the obtained 1374bp strawberry dhar gene sequence had a full length ORF of 887 bp (1161002 bp). The start codon is ATG, stop codon is TGA, 5’ end non-coding region is 115 bp and 3’ end non-coding region is 372 bp. The coding region sequence encodes a putative protein of 190 amino acids (Fig. 4 B) with a predicted molecular weight of 20.987 kDa and a pI of 6.24. However, the ORF is interrupted by three introns (264374 bp 520615 bp and 734840 bp) (Fig. 4 A). And the three introns all have high AT content of 65.8%, 64.6% and 69.2%, respectively. In addition, the splice site of the second and third introns are consistent with the typical GT-AG rule, that is with conserved GT in the 5’ splice site and AG in the 3’ splice site. But the 5’ and 3’ splice site of the first intron are GG and TA, which is the same with other strawberry varieties dhar intron splice site. Maybe the mRNA of dhar gene have unique intron splice site.

A

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Agric. Biol. J. N. Am., 2010, 1(4): 726-730

B

Fig.4 Gene structure (A) and sequence of strawberry dhar gene DNA (B) Note: The lower-case characters indicate noncoding regions; the upper-case characters indicate coding regions; dashed area indicates the intron; ATG

indicates the start codon; TGA indicates the stop codon.

Similarity analysis of dhar gene: The similarity analysis of deduced amino acid sequences of dhar genes within different plants species (Table 1)

revealed that they shared high homology (57.4%

88.4%), which suggested that the obtained sequence was a real dhar gene of Fragaria × ananassa cv. Toyonaka and named Fadhar. The amino acid homology tree (Fig.5) showed that Fragaria × ananassa cv. Toyonaka and Malus × domestica fell into one cluster, they shared the highest sequence homology. And Fadhar shared the lowest homology with dhar gene of Brassica oleracea. In addition, Fadhar was also had a low homology with dhar3 of

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