Contribución al Estudio de Dihidropirimidina Deshidrogenasa (DHPD) de la Ruta Catalítica de Pirimidinas en Oryza Sativa L.

Abstract

Rice is an essential food in the family basket, which makes it one of the most consumed cereals, despite the fact that in recent years there have been severe climatic changes such as high temperatures and continuous rainfall that affect growth, reproduction and cycles (Peñuelas, 2004), which leads to the fact that these abiotic factors represent a major problem, either in the structure of the plant or in the different metabolic pathways Resulting in very high or very low expression of certain regulated genes involved in tolerance to abiotic stress as a defense mechanism in the rice plant (Soren, 2010). One of the routes present in tolerance to abiotic factors such as drought and salinity could be the pathway of pyrimidines, since an overexpression against this type of stress has been reported in the first pyrimidine degradation enzyme Dihydropyrimidine Dehydrogenase ( DHPD) (Liu et al., 2009). The protein has been studied in pig liver showing that it is made up of 5 domains, one of them being a subunit where FMN and NADPH are coupled (Dobritzsch, et al., 2002), and in plants is unknown, so DHPD has Reported only half the size compared to mammalian DHPD, lacking the subunit in which NADPH is assembled and the electron transport from the latter to the substrate occurs (Zrenner et al., 2006). This suggests that in plants an interaction between DHPD and the protein that is in charge of the transfer of electrons is required for an optimal operation in the degradation pathway of Uracil or Thymine; However, this has not been reported for any plant species. A possible candidate of the missing fragment is the β-subunit of Nicotinamide Adenine Dinucleotide Phosphate-Glutamate Synthase (NADPH-GltS) for its homology reported in sequence banks with the missing DHPD segment (Vanoni et al. From this, the work was done to obtain the genes that are possibly part of the subunit complementary to the DHPD protein domain of the pyrimidine degradation pathway in Oryza sativa L. and thus contribute to future studies with this enzyme and Its importance in tolerance to the abiotic stress caused by salinity and high droughts that brings with it the current climatic problems. For this, the two NADH-GltS β-subunit isoforms were cloned into the pET19b expression vector for future complementation assays between the NADH-GltS β-subunit isoforms and the DHPD. Cloning was performed by extracting RNA in rice plants and subsequently obtained ANDc, from which primers were designed to function as annealing for the amplification of the two isoforms of the NADH-GltS β-subunit isoforms. The β-subunit of NADH-GltS 1 was cloned into pET-19b expression vector using both the NADH-GltS 1 and the restriction enzyme vector XhoI. The fragments were purified with QIAquick Gel Extraction Kit from QIGEN, for subsequent ligation and transformation into DH5α chemically-responsive cells. In the case of the β-subunit of NADH-GltS 2 the same procedure was performed but changing the restriction enzymes, using XhoI and BamHI for the sequence and vector pET-19b. Finally, cloning of the NADH-GltS 1 β-subunit was obtained by PCR, enzymatic restriction and sequencing of the clones. However, cloning of the β-subunit of NADH-GltS 2 was not achieved due to the similarity between the isoforms posing possible strategies from the primers for upcoming studies. Finally, the transformed bacteria of the isoform 1 were allowed to grow in LB medium to carry out frozen of the same in order to preserve the sequences of interest. As a conclusion of this investigation, cells transformed with the β-subunit of NADH-GltS 1 were obtained, which will contribute to studies of interaction between the possible electron transfer domain and the DHPD enzyme corresponding to the first degradation pathway of pyrimidines in Oryza sativa L , Providing tools for investigating the route and expression of the enzyme when subjected to abiotic stress factors such as drought and salinity. As a conclusion of this investigation, cells transformed with the β-subunit of NADH-GltS 1 were obtained, which will contribute to studies of interaction between the possible electron transfer domain and the DHPD enzyme corresponding to the first degradation pathway of pyrimidines in Oryza sativa L , Providing tools for investigating the route and expression of the enzyme when subjected to abiotic stress factors such as drought and salinity.