Expression of an engineered synthetic cry2Aa (D42/K63F/K64P) gene of Bacillus thuringiensis in marker free transgenic tobacco facilitated full-protection from cotton leaf worm (S. littoralis) at very low concentration

Abstract

Emergence of resistant insects limits the sustainability of Bacillus thuringiensis (Bt) transgenic crop plants for insect management. Beside this, the presence of unwanted marker gene(s) in the transgenic crops is also a major environmental and health concern. Thus, development of marker free transgenic crop plants expressing a new class of toxin having a different mortality mechanism is necessary for resistance management. In a previous study, we generated an engineered Cry2Aa (D42/K63F/K64P) toxin which has a different mortality mechanism as compared to first generation Bt toxin Cry1A, and this engineered toxin was found to enhance 4.1-6.6-fold toxicity against major lepidopteran insect pests of crop plants. In the present study, we have tested the potency of this engineered synthetic Cry2Aa (D42/K63F/K64P) toxin as a candidate in the development of insect resistant transgenic tobacco plants. Simultaneously, we have eliminated the selectable marker gene from the Cry2Aa (D42/K63F/K64P) expressing tobacco plants by exploiting the Cre/lox mediated recombination methodology, and successfully developed marker free T2 transgenic tobacco plants expressing the engineered Cry2Aa toxin. Realtime and western blot analysis demonstrated the expression of engineered toxin gene in transgenic plants. Insect feeding assays revealed that the marker free T2 progeny of transgenic plants expressing Cry2Aa (D42/K63F/K64P) toxin showed 82-92 and 52-61 % mortality to cotton leaf worm (CLW) and cotton bollworm (CBW) respectively. Thus, this engineered Cry2Aa toxin could be useful for the generation of insect resistant transgenic Bt lines which will protect the crop damages caused by different insect pests such as CLW and CBW.

Factibilidad técnica de variedades de algodón expresando proteínas Cry tóxicas contra Anthonomus grandis en el Valle del Sinú, Colombia

“…Esto significa un uso mínimo de insecticidas y el más bajo costo posible de manejo de plagas (Tabla I). este escenario solo es posible creando una proteína sintética de alta especificidad para A. grandis y con suficiente efecto tóxico contra la población de S. frugiperda (25)(26)(27).…”

“…cry2Aa (D42/K63F/K64P) representa una nueva generación de proteínas transgénicas modificadas sintéticamente con alta actividad tóxica específica, lo que permite que, aunque sea expresada en bajos niveles dentro de la planta, estos sean efectivos para el control del insecto objetivo. la incorporación de esta proteína en plantas de tabaco otorga resistencia completa contra Spodopte-ra littoralis y niveles significativos de resistencia a Helicoverpa almigera, para los cuales la proteína original cry2A no tiene efecto alguno (27).…”

Designing Bt constructs for Brassicas, with minimal IP issues – A case study

“…However, a common problem associated with this control strategy is the development of insect resistance to the Bt toxin present in the transgenic plants [2,3]. Several approaches have been used to reduce or prevent the development of insect resistance including the use of refuge crops (providing a sufficiently high populations of susceptible insects to prevent resistance genes from becoming homozygous), high expression of Cry genes in plants, deploying different Cry genes in individual plants in a crop (seed mixtures), and combining multiple Cry genes (i.e., stacking) in the same plant [4][5][6][7][8]. Of these, high expression and stacking of Cry genes in the same plant are considered the most effective strategies [1,5,9,10].…”

Minimizing IP issues associated with gene constructs encoding the Bt toxin - a case study

“…However, a common problem associated with this control strategy is the development of insect resistance to the Bt toxin present in the transgenic plants [2,3]. Several approaches have been used to reduce or prevent the development of insect resistance including the use of refuge crops (providing sufficiently high populations of susceptible insects to prevent resistance genes from becoming homozygous), high expression of Cry genes in plants, deploying different Cry genes in individual plants in a crop (seed mixtures), and combining multiple Cry genes (i.e., stacking) in the same plant [4][5][6][7][8]. Of these, high expression and stacking of Cry genes in the same plant are considered the most practical effective strategies [1,5,9,10].…”