Expressão heteróloga da proteína FEM de abelha Melipona interrupta (Hymenoptera, Apidae) em bactéria Escherichia coli

Abstract

In all planet, the bees' population has fallen, which has led scientists from all over the world to find ways to help preserve these animals. Population reduction has an extremely negative effect on these insects due to decreased alleles of sex determination genes. Therefore studies that seek to understand the role of these genes may help to preserve these insects. The fem gene plays an important role in determining sex and bees castes. The contribution of the fem gene to these processes in Mellipona interrupta, a bee native to the Amazon region, has been investigated by transcriptomic methods. To contribute to the results and hypotheses of other authors on the participation of this gene in sex and caste determination of M.interrupta. This work aimed to express, purify and renature the rMiFEM protein. The synthetic Fem gene was cloned into the expression vector pGS-21a and expressed in different strains of Escherichia coli, with the best results obtained in the Rosetta (DE3) strain. The clones expressed a 50 kDa protein (rMiFEM) in inclusion corpuscle form followed by in vitro renaturation using three different protocols. Of these, the one that provided the best results was a recommended for renaturation of SR-proteins. When analyzing the peptide sequence of the MiFEM protein with bioinformatics tools, an RNA binding domain was identified that had not yet been described for this protein, as well as two regions rich in arginine and serine (RS Domains that had already been described previously) and several possible phosphorylation sites. Using an SR-protein enrichment protocol a different pattern was obtained in the proteins of males, workers and queens, indicating that different SR-proteins are differentially expressed between sexes and castes. Although it has not been possible to detect Fem protein in the SR-protein enriched extract, it does not exclude the hypothesis that Fem of M. interrupta is in the precipitate, although it is not an SR-protein, but having the necessary domains for precipitation. The rMiFEM protein will subsequently be used to produce specific anti-Fem antibodies, which will help in understanding the participation of Fem protein in the determination of sexes and casts in M. interrupta.