Cloning of 3 ’ End cDNA of Ascorbate Peroxidase Gene from Fragaria × ananassa cv . Toyonaka

A fragment of 3’ end cDNA of apx in Fragaria × ananassa cv. Toyonaka was cloned by rapid amplification of cDNA 3’ end (3’RACE) PCR. By joining the sequence with the known fragment sequence which we had submitted to GenBank (FJ896040), a 3’-end partial cDNA of 672bp was obtained, encoding 140 amino acids. The sequence contained the corresponding coding sequence of 424bp long including a stop codon TAA, and the 3’-untranslated region(3’UTR) of 248bp including a potential polyadenylation signal (AAATAA) and poly(A) of 28bp long. Homology analysis of nucleotide sequence and deduced amino acid sequence on the apx gene showed that it shared high homology with different plants species, among which it shared the highest sequence homology with Fragaria × ananassa cv. Yoho.


Introduction
Ascorbate peroxidase (APX;EC 1.11.1.11)is a hydrogen peroxide-scavenging enzyme, found in higher plants, algae, and some cyanobacteria (Asada, 1992).It catalyses the conversion of H 2 O 2 to H 2 O and O 2 using ascorbate as the specific electron donor (Asada, 1999).APX has a presumed function of protecting cells from hydrogen peroxide accumulation, particularly under stressful conditions (Lin et al., 2007).Researchers have reported that the activities and the mRNA expression levels of APX would increase when plants encounter environmental stresses such as high temperatures, drought, light, and so on (Tanaka et al., 1990;Mittler et al., 1994).
APX comprises a family of isoenzymes with different characteristics in higher plants (Chen and Asada, 1989;Bunkelmann and Trelease, 1996;Shigeru et al., 2002).To date many researchers have cloned apx isozyme genes from a variety of plants.Strawberry is one of the most economically important fruit trees.Although apx gene fragment has been isolated from different strawberry cultivars, the full-length sequences of the genes have not been reported.However, the entire 3' sequence of a cDNA is very important because the non-coding terminal region often contains signals that regulate the stability or subcellular localization of mRNAs, and sometimes alternative genomic sites are used for cleavage and polyadenylation, which can alter these aspects or change the encoded protein.In this study, we obtained the 3' end cDNA sequence of apx gene from Fragaria × ananassa cv.Toyonaka by rapid amplification of cDNA ends (RACE) PCR.

Plant material and RNA isolation
Strawberry (Fragaria × ananassa) cv.Toyonaka was used in this study.Total RNA was extracted from strawberry fruit using the Plant RNA OUT kit, as per the manufacturer's instructions(TIANDZ, China).

cDNA synthesis
The first strand cDNA was synthesized from 2µg of the total RNA by reverse transcriptase with Q T primer according to the instructions of the Easy-Go TM RT PreMix kit (SBS Genetech, China).

3'-RACE PCR amplification
The first round PCR was performed with a 20 µl reaction mixture containing 1 µl of first-strand cDNA(50ng/µl), 2.0µl of 10×PCR buffer without Mg 2+ , 2.0mM MgCl 2 , 200µM dNTPs, 0.5µM Q O , 0.5µM GSP1 and 1.0 unit of Taq DNA polymerase.The second round PCR template was 1/50 of the product from the first round PCR and PCR performed with the primer pair Q I / GSP2.Others were the same with the first round PCR.The PCR procedure were all strarted with 94 o C for 3min, then 35 cycles of 94 o C for 1min, 64 o C for 1min, 70 o C for 2min, and finally 72 o C for 7 min.The PCR products were analyzed on a 1.0% agarose/EtBr gel and the corresponding DNA band was recovered, then cloned into the pMD19-T Vector (TaKaRa, China) for sequencing.Sequence analysis was performed using the software DNAMAN (Version 3.0, Lynnon BioSoft).

The cloning of 3' RACE
The agarose gel electrophoresis showed that no specific band but a smear was found for the first PCR product (Fig. 1, Lane 1).However, a very specific band of about 500bp was obtained in the second PCR product using inner primer pair Q I / GSP2 (Fig. 1, Lane 2).After the RACE-PCR product was recovered and cloned into pMD19-T vector, plasmid PCR was then performed for rapidly screening APX cDNA clone with primers Q I and GSP2.Finally, positive cDNA clones were picked out and sequenced.

Sequence analysis of 3'-end partial cDNA
Sequence analysis revealed that 3'RACE product was 506bp long, and the sequence showed a high sequence homology with apx of many kinds of plants.Therefore, we presumed that it was the target fragment.By joining the sequence with the known fragment sequence which we have submitted to GenBank (FJ896040), a 3'-end partial cDNA of 672bp was obtained, encoding 140 amino acids, named Faapx.The sequence contained the corresponding coding sequence of 424bp long including a stop codon TAA, and the entire 3'-untranslated region(3'UTR) of 248bp including a potential polyadenylation signal (AAATAA) and poly(A) of 28bp long (Fig. 2).

Similarity analysis of apx gene
The similarity analysis of nucleotide sequence and deduced amino acid sequences of apx genes within different plants species (Table 1) revealed that they shared high homology (Fig. 3).Faapx shared a similarity of the nucleotide (62.8~98.1%)and deduced amino acid sequences (79.9~98.6%)with apx genes of other plants, among which it shared the highest sequence homology with Fragaria × ananassa cv.Yoho.Both nucleotide phylogenetic tree and amino acid homology tree showed that the two strawberry cultivars fell into one cluster.The nucleotide homology tree clustering analysis revealed that strawberries and Malus × domestica then clustered together, but they did not cluster directly in the amino acid homology tree.Although there were some differences in the cluster results between nucleotide sequence and amino acid sequence, the homology of them were respectively over 65%.These results indicate that apx genes are relatively conserved in different plants.

Discussions
As a key enzyme in decomposing H 2 O 2 , APX is not only closely related to plant growth and resistance to environmental stress, but also one of the enzymes in the metabolism of vitamin C (Schantz et al., 1995).Therefore, cloning full length cDNA encoding APX is very important to have a better understanding of its functions.RACE technology has become a common strategy to cloning full cDNA sequence of unknown genes, from reverse transcription through to amplification, and took less a day to perform.In this study, we used a classic RACE technique to clone 3' end cDNA encoding APX.The result showed that the nucleotide and deduced amino acid sequence shared high homology with those of other plants APX reported to date.Thus, the 3' end cDNA fragment obtained is the target gene which we needed.However, the technique is particularly sensitive to amount of cDNA template used in the research.Very little substrate is required for the PCR, because too much starting material will lead to the production of large amounts of non-specific product.We obtained successfully the 3' end cDNA sequence of apx from Fragaria × ananassa cv.Toyonaka by the means, only using 1µl of first-strand cDNA(50ng/µl).Now we are cloning 5' end cDNA encoding APX of Fragaria × ananassa cv.Toyonaka in order to make a great foundation for researching of its structure and function.A   Table 1)

Figure 1 .
Figure 1.Agarose gel electrophoresis of PCR products and plasmid PCR product M = DNA ladder; lane 1 indicates the first PCR product; lane 2 indicates the second PCR product; lane 3 indicates the plasmid PCR product

Figure 2 .
Figure 2. Nucleotide sequence and deduced amino acid sequence of apx gene The lower-case characters indicate noncoding regions; arrow indicates primer (GSP1 and GSP2) locations; single underline indicates poly(A); double underline indicates potential polyadenylation signal; TAA indicates the stop codon