Cloning And Expression Profiling Of Genes Involved In Ascorbate Biosynthesis And Recycling In Radish(Raphanus Sativus L.)

Radish(Raphanus sativus L.) is an important annual or biennial vegetable in the world, especially in East Asia. The fleshy taproot comprises the edible portion of the plant, and is rich in potassium, carbohydrate, folic acid and ascorbic acid. Ascorbic acid (AsA) is a vital antioxidant compound and has several essential functions in plant physiology. AsA also has an important role in the development of roots. It participates in the maintenance of the quiescent centre, and the development of lateral root, and also has the effects on the gravitropic response of root. On the contrary, AsA related researches on roots and radish remains limited. In this study, AsA content variations were analyzed in radish, and expression profiling of AsA related genes were also conducted. It provide new insights into AsA accumulation during root development in radish and facilitate identifying the key genes involved in the AsA biosynthesis and recycling, which would contribute investigation into the molecular mechanisms of AsA regulation in root vegetable crops. Main results are as follows:(1) Four ascorbic acid related genes were isolated from radish using using T-A clone, named as RsGMP, RsGME, RsGGP and RsDHAR respectively, and analyzed their sequences as well as the translated protein. The full-length cDNA and DNA sequences of RsGMP were2253bp and1086bp, containing a1086bp ORF which ecoded361amino acids. The full-length cDN A and DNA sequences of RsGME were2013bp and1252bp, containing a1140bp ORF which ecoded379amino acids. The full-length cDNA and DNA sequences of RsGGP were2256bp and1827bp, containing a1305bp ORF which ecoded434amino acids. The full-length cDNA and DNA sequences of RsDHAR were1531bp and1033bp, containing a1305bp ORF which ecoded257amino acids.(2) To investigate the molecular mechanisms of ascorbate accumulation during root development and reveal the key genes of the ascorbate biosynthesis and recycling pathways, the expression of16related genes together with ascorbate abundance were analyzed in the flesh and skin of radish(Raphanus sativus L.) fleshy root. The content of ascorbate decreased with root growth in both the flesh and skin Expression of GMP, GME and GalUR were also decreased and correlated with ascorbate levels in the flesh. In the skin, the expression of GMP and GalDH was correlated with ascorbate levels. These results suggested that ascorbate accumulation is affected mainly by biosynthesis rather than recycling in radish root, and the L-galactose pathway may be the major biosynthetic route of ascorbate, and moreover, the salvage pathway may also contribute to ascorbate accumulation. The data suggested that GMP could play an important role in the regulation of ascorbate accumulation during radish fleshy taproot development.(3) Compared the AsA contents of mature leaves, young leaves, root flesh and skin of radish under light treatment. At the same time, the research analyzed the expression of genes which involved in AsA ascorbate biosynthesis and recycling pathways under light treatment in the four different parts of radish useing RT-PCR and RT-PCR. AsA content in mature leaf was increased with light treatment and reduced with dark treatment, suggesting a correlation with light. However, this correlation is unclear in other parts of radish. The expression levels of PMI, GMP, GGP and GPP were correlatd with light treatment using RT-PCR, and similarly, the transcription levels of PMI, GMP, GGP, GPP, MIOX and GalUR expressed were also correlatd with light treatment using qRT-PCR.