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Cloning and sequence analysis of dehydroascorbate reductase gene (dhar) from strawberry by SON-PCR - page 4 / 5

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Arabidopsis thaliana, but higher with dhar1 and dhar2

of Arabidopsis thaliana. different gene family.

Maybe they belong to

A

Fragaria

×

G

Arabidopsis

ananassa

cv.

thaliana (dhar2)

Table 1. The dhar genes of different plants species in

H

Arabidopsis thaliana (dhar1)

I J

Oryza sativa

Triticum aestivum

K

Brassica oleracea

L

Arabidopsis thaliana (dhar3)

Code

this study

Scientific name

Code

Scientific name

×

B

C D

E

F

Toyonaka Malus domestica Vitis vinifera Lycopersicon esculentum Solanum tuberosum Nicotiana tabacum

Fig.5 Homology tree of deduced amino acid sequences of the dhar gene from different plants (as in Table 1)

DISCUSSIONS

AsA is an essential mineral elements to maintain health of humans. It is reported that althought the current recommended dietary allowance for vitamin C is 75-90mg, an increase to 200mg has been suggested (Levine et al., 1996). Such an increase would require foodstuffs rich in AsA. However, DHAR play an important role in recycling of AsA in plants, since it can catalyzes ascorbic acid oxidized form DHA to AsA, thereby regulating redox state of AsA. Therefore, cloning dhar gene is very important to increase the content of AsA and have a better understanding of the gene structure and functions.

729

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

SON-PCR can isolate DNA regions located on either side of a known DNA sequence and have high specificity, relying on only two amplification reactions with two of three nested sequence-specific primers. Therefore, it seems a very efficient and widely application technique for the rapid identification of large unknown DNA regions (Antal et al., 2004). In this study, we firstly cloned a fragment of strawberry dhar gene conserved region. Then based on the obtained conserved regions sequences, we designed two pairs of nested primers to isolate strawberry dhar gene flanking sequences by SON-PCR. At last, we obtained a 1374bp sequence by splicing the isolated flanking fragments with the conserved region fragment. Homology analysis of deduced amino acid sequence on the Fadhar showed that it shared high homology with different plants species, indicating that it is a relatively conservative gene in plants. In addition, the amino acid homology tree showed that three of Arabidopsis thaliana dhar gene did not fell into one cluster, indicating that dhar gene may be encoded by a multi-gene family. At present, only a few genes of dhar gene family in plants have been cloned. So it is very important to clone all the genes of the family, which will help to undetstand the composition and function of the gene family.

ACKNOWLEDGMENTS The present research is supported by the Foundation for the Educational Commission of Sichuan Province, China (07ZZ023; 2006ZD004).

REFETENCES

Antal, Z., Rascle, C., Fevre, M. and Bruel, C. (2004). Single oligonucleotide nested PCR: a rapid method for the isolation of genes and their flanking regions from expressed sequence tags. Curr Genet. 46: 240-246

Chen, Z. and Gallie, D.R. (2004). The ascorbic acid redox state controls guard cell signaling and stomatal movement. Plant Cell. 16: 1143-1162

Chen, Z. and Gallie, D.R. (2006). Dehydroascorbate reductase affects leaf growth, development, and function. Plant Physiology. 142: 775-787

Chen, Z., Young, T.E., Ling, J., Chang, S.C. and Gallie, D.R. (2003). Increase vitamin C content of plant through

enhanced 3525-3530

ascorbate

recycling.

PNAS.

100(6):

Davey, M.W., Montagu, M.V., Inze, D., Sanmartin, M., Kanellis, A., Smirnoff, N., Benzie, I.J.J., Strain, J.J., Favell, D. and Fletcher, J. (2000). Plant L-ascorbic acid:

chemistry,

function,metabolism,bioavailability

and

effects of processing. Journal of the science of food and

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