Desenvolvimento e otimização de sistemas de expressão de proteínas recombinantes em capim-elefante e em tabaco

Abstract

Advancements in biotechnology combined with genetic improvement have allowed more precise and controlled genetic modifications to obtain desirable traits in organisms. Transgenic plants are an attractive option in agriculture and the pharmaceutical industry, providing resistance to diseases and environmental stresses, as well as enabling the production of recombinant proteins and therapeutically relevant secondary metabolites. Elephant grass (Pennisetum purpureum Schumach syn. Cenchrus purpureus Schumach) is a widely used grass with high potential for dry matter production in dairy and beef cattle farming. However, insects with a high potential for crop damage, such as pasture spittlebugs, can impact the grass's productivity. The development of transgenic plants resistant to pests is a viable alternative to overcome this issue. In this study, we aimed to obtain a regeneration protocol for the transformation of elephant grass BRS Capiaçu for further application in genetic transformation via Agrobacterium tumefaciens. Meristem induction was performed using different combinations of growth regulators 2,4-D, BAP, and TDZ, resulting in 93% regeneration of multiple shoots induced over 28 days with a treatment containing 0.1 mg.L-1 2.4-D and 2 mg.L-1 BAP, with a selection curve determined at 50 mg.L-1 kanamycin and a three-day pre-culture period. The regenerated shoots showed suitable root and shoot development, linked to a kanamycin-based determination of the selection curve. The second chapter of this document, our aim was to characterize the transgene expression for the production of a recombinant single-chain antibody scFAB-anti-IL1ß by obtaining stable homozygous lineages of Nicotiana benthamiana. This antibody was synthesized due to its biological importance in inflammatory diseases related to prolonged cytokine IL-1ß secretion. Thus, we intended to select N. benthamiana lineages for stable production of scFAB-anti-IL-1ß. Seeds were sown in plates containing MS medium with kanamycin and the presence of the scFAB-anti-IL1ß was assessed, with lines showing transgene rates between 48.59% for IL- 1ßT0P122 and 95.55% for IL-1ßT0P20. T1 seedlings from IL-1ßT0P20 plants were subcultured and (i) compared with hemizygous T0 plants by the 2−ΔΔCt method, with values normalized with the 18S endogenous gene, and (ii) subjected to protein extraction and purification. The production of scFAB-anti-IL1ß by the T1 regenerants showed an approximate yield of 474 µg per gram of fresh leaf mass. Analysis of qPCR and RT-qPCR data showed the presence of lines with estimated values for two copies of the transgene in the expected 2:1 ratio. However, homozygous individuals tended to exhibit genetic silencing, with reduced transgene expression.