Clonagem da sequência codificante do gene ras de apis mellifera e modificação da mesma para criação da variante oncogênica V12

Abstract

Bees are social insects that group in colonies, originated in Africa with expansion in Europe and Asia, bees are present in almost every country, they pollinate 42% of the world's leading crops. Bees are important for the ecosystem, by preventing desertification and for the economy, by producing a variate of compounds such as beeswax, honey, and propolis. Apis mellifera represents the main economically used species in Brazil, indeed several factors have contributed to the disappearance and death of bees in the country and in the world. Some of these events can be explained by the extensive use of pesticides in crops that are close to apiaries, by nutritional deficiencies because of the lack of studies, by climate change e deforestation and also viral infections. In order to analyze the conditions that affect the wellbeing and the longevity of bees, in vitro studies are necessary for directly accessing the specificities of each species. Considering that the creation of immortalized lineage of Apis mellifera was established only through insertion of exogenous genes and that other studies are based on the use of already differentiated tissues, this work aims the induce mutation of the ras gene with cDNA from bee larva cells of Apis mellifera for the production of an expression plasmid that could induce indeterminate cell proliferation and posterior immortalization of bee larva cells. This could be possible once the ras gene is already present in Apis mellifera. The Ras protein has a range of mutations related to cancer, mainly from the inactivation of the p53 tumor suppressor, which controls the cell cycle. The HRAS protein variant with the V12 mutation, present in a large number of neoplasms, was recently used for the immortalization of Drosophila melanogaster cells. The ras V12 protein was then chosen as a potential inducer of cell proliferation in Apis mellifera. For the immortalization process, PCR with degenerate primer was used to exchange Guanine for Thymine at nucleotide 35. The generated amplicon was cloned into the Invitrogen company's pcDNA ™ 3.1/V5-His TOPO TA vector once it has the insect-compatible CMV promoter. After the amplification of the plasmid in Escherichia coli DH5α, the sequence was evaluated by Sanger sequencing, the plasmid contained the desired mutation and was named pcDNA3.1-RASV12, for future insertion in Apis mellifera and establishment of permanent lineage.