Construcción de un vector para modificación genética de cloroplastos de alfalfa y estudios para la regeneración in vitro de la planta

Abstract

There are several examples of situations in which systems would be cheaper, profitable and efficient for biopharmaceuticals production, due to its high demand in society. In this regard, chloroplasts genetic modification represents a valuable alternative in the production of proteins with therapeutic applications, significantly reducing the production costs that are inherent to fermentation, purification and conventional cold storage processes. As semi-autonomous organelles, chloroplasts contain the necessary metabolic machinery to carry out replication, transcription and translation of genes encoded in its genome and exogenous genes, which can be introduced by a modification technique like biolistic. However, most research on genetic modification of chloroplasts has been made in the tobacco plant (Nicotiana tabacum), which contains high concentrations of alkaloids and toxins. In this work, it was completed the design and construction of a specific vector for alfalfa genetic modification (M. sativa) and the establishment of a tissue regeneration protocol for this plant. Alfalfa is a safe plant species for humans and animals, so that the expression of therapeutic proteins within it represents a considerable improvement over the conventional tobacco model. For the construction of the specific transformation vector it was used the alfalfa (M. truncatula) psbA – trnH region, which was inserted into a multiple cloning site (MCS) with 19 unique restriction sites. Of these sites, EcoRV was used to insert the aadA gene that confers resistance to spectinomycin and / or streptomycin to susceptible species to these antibiotics. PsbA – trnH is highly conserved among different legume species and could be effectively used in genetic modification of many of them. The vector would also have the versatility to be used as a extension vector, making it possible that a group or set of exogenous genes be expressed by simply inserting the codon downstream end of the psbA gene without the need for an additional psbA gene promoter. The regeneration protocol could also be used to grow callus on MS medium with 2,4- dichlorophenoxyacetic acid (2.4-D) and benzylaminopurine (BAP) from non modified tissue, and to acquire somatic embryos in MS medium with 2.4-D and kinetin to obtain genetically modified non chimeric plants.