| Cassava is a perennial shrub cultivated in the tropical and sub-tropical regions of the world and is characterized by its ability to develop secondary roots of high starch storing capacity. This root crop shows certain advantages such as the capability of growing in marginal conditions of drought periods and poor and acidic soils. It also shows resistance to certain herbivore pests and can persist in the soil for 8--24 months without decaying. Certain African regions contribute to the main inputs in global cassava production, where it is mainly used as a food source.;Cassava is known to accumulate linamarin in all of its tissues. The enzymatic action of linamarase and HNL cause cyanide release from linamarin mainly after tissue injury, serving as chemical defense molecule against herbivores. In addition, it has been considered the implication of cyanoglycosides as storing and transportable forms of reduced nitrogen. ss-cyanoalanine synthase (ss-CAS) provides the first step in the cyanide detoxification pathway, allowing its incorporation into the amino acid biosyntheses pathways. This enzyme belongs to the ss-substituted alanine synthases (Bsas ) family of enzymes, being close-related to cysteine synthase (CS). Despite the importance of the ss-CAS detoxification pathway in cyanide metabolism in cassava, the enzymes involved in it have not been identified at the molecular level. This project aims to isolate the cassava Bsas genes and determine their role in cyanide metabolism.;Three cDNAs were isolated using RACE-PCR, which were first identified as Bsas-specific genes harboring sequences of different molecular weight. Sequencing information led to the conclusion that two of these contained identical sequences, each one of which encodes a short and a long version of the same cDNA. Sequence analysis tools allowed the prediction of the amino acid sequences encoded by these genes, being MANes;BsasA the protein encoded by the two identical sequences and MANes;BsasB the remaining gene. Sequence cluster analyses grouped both proteins among the Bsas enzymes targeted to the mitochondria, the cell compartment where ss-CAS is found. However, one of the sub-cellular location estimating tools identified MANes;BsasA as a possible plastidic enzyme, which together with the cytoplasm, comprise the two main organelles for cysteine biosynthesis.;Cloning and over-expression of these genes in a bacterium of Bsas mutant background provided concrete evidence of the in vitro kinetic properties of these genes. MANes;BsasB showed higher ss-CAS activity levels than MANes;BsasA and ARAth;Bsas3;1, which was used as a ss-CAS positive control. In the other hand, MANes;BsasA showed remarkably higher CS activity levels than ARAth;Bsas3;1, but more importantly than MANes;BsasB. This information clarifies that MANes;BsasA truly encodes a CS isoform, whose shorter version lacking the signal peptide might encode the cytoplasmic isoform. The longer cDNA could encode an enzyme of plastidic or mitochondrial sub-cellular location, since both compartments have been shown to have both enzymes. In the other hand MANes;BsasB encodes a mitochondrial ss-CAS enzyme, since no other evidence has been shown that a true ss-CAS protein is targeted outside this compartment.;An attempt of transformation was done by cloning these genes in a plant binary vector for transformation of cassava somatic embryos. A total of 2,806 and 2,513 explants were transformed harboring the ARAth;Bsas3;1 and MANes;BsasA genes, respectively, producing a total of 80 and 212 lines that endured antibiotic selection. However, no line showed a repetitive pattern of PCR amplification, when screening these lines using different primer combinations. Moreover, no line showed increased ss-CAS activity. Apparently the selection protocol employed did not guaranteed a proper selection of transgenic lines with the T-DNA integrated into its genome. |
