Functional Analysis Of Two Dihydroorotate Dehydrogenase Genes From Rice (Oryza Sativa L.)

Pyrimidine nucleotides are abundant molecules with essential functions in a multitude of biochemical processes. They are required not only the precursors of RNA and DNA, but also required for energy metabolism and for the continued synthesis of many biosynthetic products such as phospholipids, polysaccharides and glycoproteins. The metabolism of pyrimidine nucleotides and their intracellular pool sizes influence vast areas of normal cellular metabolism. The first pyrimidine, UMP, is synthesized by de novo pathway and salvage pathway. The de novo biosynthetic pathway consists of six enzymatic steps. Dihydroorotate dehydrogenase (DHODH) is the fourth step of de novo catalyzing the oxidation of dehydroorotate to orotate.In the previous research OsDHODH1 and OsDHODH2 genes were isolated from rice, the deduced anim acids catalyze the fourth step of de novo catalyzing the oxidation of dehydroorotate to orotate. Expression analysis indicated that OsDHODH1 and OsDHODH2 are up-regulated by abiotic stress. Expression of OsDHODHl and OsDHODH2 in E. coli improved the tolerance to abiotic stress. For further research the function of OsDHODH1 and OsDHODH2, these two genes were transferred into yeast. The enzyme assay showed that the DHODH activity of transgenic OsDHODHl and OsDHODH2 yeast was significantly increased. The cell numbers of 1 ml yeast liguid culture in recombinant OsDHODH1 and OsDHODH2 yeast were more than that in transgenic pFL61 vector yeast in the third and forth days. This indicated that OsDHODHl and OsDHODH2 might be relevant with senescence.The overexpression and repression vectors of OsDHODHl and OsDHODH2 were constructed. The transgenic rice plants were obtained by Agrobacterium-mediated transformation. The expression of OsDHODH1 and OsDHODH2 in transgenic lines and WT were analyzed by semi-quantitative RT-PCR. RT-PCR showed that the expression levels of OsDHODHl in three independent OsDHODH1 overexpression lines (A1, A2 and A3) were significantly higher than those in WT plants, and in OsDHODHl repression lines (B1, B2 and B3) and OsDHODH2 repression lines (C1, C2 and C3) were significantly lower than those in WT plants. The DHODH activities were compared among the WT and transgenic lines. The enzymatic activity of DHODH in the OsDHODH1 overexpression lines were 1.9 times of WT. However, the enzymatic activity of DHODH did not show a significant reduction from the OsDHODH1 repression and OsDHODH2 repression lines. OsDHODH1 overexpression increase the DHODH activity, this indicated that OsDHODH have the function of dihydroorotate dehydrogenase. The agronomic traits of T2 transgenic rice were compared with type plants. There were no significant difference between WT and transgenic lines, but in OsDHODH2 repression lines, the size of seeds were smaller than WT. This indicated that OsDHODH2 repression or the transgenic insert sites may influence the normal growth of seeds.For further identification the function of OsDHODH1 and OsDHODH2 under salt and drought stress, salt and drought tolerance were tested in OsDHODH1 overexpression, OsDHODH1 repression and OsDHODH2 repression transgenic rice. Under salt and drought stress, OsDHODH1 overexpression lines showed improved tolerance to stress treatment, OsDHODH1 respression and OsDHODH2 repression lines were more sensitive. After salt and drought treatment, the chlorophyll and proline contents in OsDHODH1 overexpression lines were higher than in WT, and in OsDHODH1 and OsDHODH2 repression lines were lower than in WT. The relative electrolyte leakages in OsDHODH1 overexpression were lower than in WT, and in OsDHODH1 and OsDHODH2 repression lines were higher than in WT. Thus, we conclude that OsDHODH1 and OsDHODH2 may play important roles in salt and drought tolerance response in rice.