Cloning and sequence analysis of dehydroascorbate reductase gene (dhar) from strawberry by SON-PCR
Hou Yanxia, *Tang Haoru, Zhang Yong and Chen Qing
*Corresponding author: Horticultural College, Sichuan Agricultural University, Ya’an 625014, Sichuan, China E-mail: email@example.com
Dehydroascorbate reductase (DHAR) play an important role in regenerateing ascorbic acid (AsA), since it can catalyzes ascorbic acid oxidized form dehydroascorbate (DHA) to AsA. In this study, we cloned dhar gene from strawberry by SON-PCR, named Fadhar. And Fadhar is 1374 bp length, including a full ORF of 887 bp, which encoding a deduced protein of 190 amino acids with a molecular weight of 20.987 kDa and a pI of 6.24. The coding region of Fadhar is interrupted with three introns of high AT content. Homology analysis of deduced amino acid sequence on the Fadhar showed that it shared high homology with different plants species, among which it shared the highest sequence homology with Malus×domestica and the lowest with Brassica oleracea.
Keywords: Strawberry, dehydroascorbate reductase, cloning, SON-PCR.
Ascorbic acid (AsA) or vitamin C is a major antioxidant in plant tissues, as well as one of the necessary nutrients for maintain growth, reproduction and health of humans and animals (Smirnoff, 1996; Davey et a1.,
microorganisms and most of the animals. However, humans lack the gene for L-gulono-1,4-lactone oxidase, which catalyzes the final step of AsA biosynthesis in mammals, so AsA must be obtained from food, mainly fresh vegetables and fruits (Nishikimi et al., 1994; Chen et al.,2003). AsA not only can provide more nutrition for humans, but also it can enhance the ability of plants to against environmental stress. Therefore, it is particularly important to accumulation and recycling of AsA in plants.
In plant, AsA is oxidized to dehydroascorbate (DHA) via successive reversible single electron transfers with monodehydroascorbate (MDHAR) as a free radical intermediate (Foyer and Mullineaux, 1998). However, dehydroascorbate reductase (DHAR; EC 188.8.131.52) can reduce DHA to AsA with glutathione (GSH) as the electron donor. Thus, it is a key enzyme to regenerate AsA. Zou (2005) found the AsA content of the leaves and fruits in transgenic dhar gene tomato plants increased respectively by 58% and 57%. Moreover, DHAR is very impormant for plant growth. It is reported that the lack of DHAR caused the loss of AsA quickly in plants, and as a consequence, affecting
plant growth and development (Ye et al., 2000). Chen and Gallie (2006) also found that suppression of dhar gene expression resulted in a slower rate of leaf expansion. DHAR can also enhance the level of plant resistance to heat, cold and saline environment (Chen and Gallie, 2004; Ushimarua et al., 2006). Therefore, the research of DHAR is very important.
Strawberry (Fragaria × ananassa) is one of the most economically important fruit trees and fruits are rich of AsA. Therefore, it is a good model plant to research AsA in fruit trees. At present, cloning of dhar gene in strawberry is still not been reported. In this study, we cloned the dhar gene from strawberry cultivar (Fragaria × ananassa cv. Toyonaka) by single oligonucleotide nested PCR (SON-PCR), and thus we make a great foundation for make further research of its structure, expression and function.
MATERIALS AND METHODS
Plant material and DNA isolation: Strawberry (Fragaria × ananassa cv. Toyonaka) was used in this study. Genomic DNA was extracted from strawberry fruits by the method of nuclear DNA (Hou et al., 2008).
Primer design: According to the conserved regions of dhar genes amino acid sequences in GenBank from NCBI web site (www.ncbi.nlm.nih.gov), a pair of gene-specific primers were designed as follows: