Gene stacking as a strategy to confer characteristics of agronomic importance in plants by genetic engineering / Empilhamento gênico como estratégia para conferir características de importância agronômica em plantas via engenharia genética

ABSTRACT: Gene stacking refers to the introduction of two or more transgenes of agronomic interest in the same plant. The main methods for genetically engineering plants with gene stacking involve (i) the simultaneous introduction, by the co-transformation process, and (ii) the sequential introduction of genes using the re-transformation processes or the sexual crossing between separate transgenic events. In general, the choice of the best method varies according to the species of interest and the availability of genetic constructions and preexisting transgenic events. We also present here the use of minichromosome technology as a potential future gene stacking technology. The purpose of this review was to discuss aspects related to the methodology for gene stacking and trait stacking (a gene stacking strategy to combine characteristics of agronomical importance) by genetic engineering. In addition, we presented a list of crops and genes approved commercially that have been used in stacking strategies for combined characteristics and a discussion about the regulatory standards. An increased number of approved and released gene stacking events reached the market in the last decade. Initially, the most common combined characteristics were herbicide tolerance and insect resistance in soybean and maize. Recently, commercially available varieties were released combining these traits with drought tolerance in these commodities. New traits combinations are reaching the farmer's fields, including higher quality, disease resistant and nutritional value improved. In other words, gene stacking is growing as a strategy to contribute to food safety and sustainability.

RESUMO: O empilhamento gênico se refere a introdução de dois ou mais transgenes de interesse agronômico na mesma planta. Os principais métodos de produção de plantas geneticamente modificadas com empilhamento gênico envolvem (i) a introdução simultânea, pelo processo de co-transformação, e (ii) a introdução sequencial de genes, pelos processos de re-transformação ou por cruzamento entre eventos transgênicos. Em geral, a escolha do melhor método varia de acordo com a espécie de interesse e a disponibilidade de construções genéticas e eventos transgênicos preexistentes. Também é apresentado aqui o uso da tecnologia de minicromossomos como tecnologia potencial de empilhamento gênico. O objetivo desta revisão é discutir aspectos relacionados à metodologia para o empilhamento de genes a combinação de características (obtida via empilhamento de genes de interesse agronômico) via engenharia genética. Além de discutir, é apresentado uma lista de culturas e genes aprovados comercialmente que tem sido usado em estratégias de empilhamento e uma discussão sobre normas regulatórias. Um número maior de eventos com empilhamento de genes foi aprovado e liberado no mercado na última década. Inicialmente, a combinação das características de tolerância a herbicidas e resistência a insetos era a mais popular, principalmente em soja e milho. Recentemente, estas características combinadas com tolerância a seca nessas culturas foram liberadas comercialmente. Novas características combinadas estão entrando na lavoura, incluindo aumento da qualidade, resistência a doenças e aumento do valor nutricional. Em outras palavras, o empilhamento gênico está crescendo como tecnologia para contribuir para a segurança alimentar e sustentabilidade.

Agrobacterium-Mediated Co-transformation of Multiple Genes in Metarhizium robertsii

Fungi of the Metarhizium genus are a very versatile model for understanding pathogenicity in insects and their symbiotic relationship with plants. To establish a co-transformation system for the transformation of multiple M. robertsii genes using Agrobacterium tumefaciens, we evaluated whether the antibiotic nourseothricin has the same marker selection efficiency as phosphinothricin using separate vectors. Subsequently, in the two vectors containing the nourseothricin and phosphinothricin resistance cassettes were inserted eGFP and mCherry expression cassettes, respectively. These new vectors were then introduced independently into A. tumefaciens and used to transform M. robertsii either in independent events or in one single co-transformation event using an equimolar mixture of A. tumefaciens cultures. The number of transformants obtained by co-transformation was similar to that obtained by the individual transformation events. This method provides an additional strategy for the simultaneous insertion of multiple genes into M. robertsii.

Co-expression of human malaria parasite Plasmodium falciparum orotate phosphoribosyltransferase and orotidine 5?-monophosphate decarboxylase as enzyme complex in Escherichia coli: a novel strategy for drug development

Background: Human malaria parasite Plasmodium falciparum operates de novo pyrimidine biosynthetic pathway. The fifth and sixth enzymes of the pathway form a heterotetrameric complex, containing two molecules each of orotate phosphoribosyltransferase (OPRT) and orotidine 5’-monophosphate decarboxylase (OMPDC). Objective: Define the function of OPRT-OMPDC enzyme complex of P. falciparum by co-expressing the enzymes in Escherichia coli. Methods: The constructed plasmids containing either P. falciparum OPRT or OMPDC were cloned in E. coli by co-transformation. Both genes were co-expressed as OPRT-OMPDC enzyme complex and the complex was purified by chromatographic techniques, including N2+-NTA affinity, Hi Trap Q HP anion-exchange, uridine 5’- monophosphate affinity, and Superose 12 gel-filtration columns. Physical and kinetic properties of the enzyme complex were analyzed for its molecular mass. Results: Co-transformation of PfOPRT and PfOMPDC plasmids in E. coli were achieved with a clone containing DNA ratio of 1:2, respectively. Both plasmids remained stable and were functionally expressed in the E. coli cell for at least 20 weeks. The P. falciparum OPRT-OMPDC enzyme complex were co-expressed and the complex was co-eluted in all chromatographic columns during purification and physical analysis. The molecular mass of the complex was 130 kDa, whereas the PfOPRT and PfOMPDC component were 35.6 and 41.5 kDa, respectively. The enzymatic activities of the complex were competitively inhibited by their products of each enzyme component. Conclusion: P. falciparum OPRT and OMPDC in E. coli as an enzyme complex were co-transformed and functionally co-expressed. These have similar properties to the native enzyme purified directly from P. falciparum, and this character is different from that of the human host organism. The enzyme complex would be suitable as new target to research selective inhibitors as suitable drugs to better control this disease.