title: 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.
authors.affiliation: Department of Human Genetics, University of Michigan, Ann Arbor, MI, 48109, USA. sgayen@umich.edu.
authors.forename: Srimonta
authors.initials: S
authors.lastname: Gayen
authors.affiliation: Department of Biochemistry, School of Life Sciences, Central University of Rajasthan, Ajmer, 305817, India.
authors.forename: Chandi Charan
authors.initials: CC
authors.lastname: Mandal
authors.affiliation: Advanced Laboratory for Plant Genetic Engineering, Indian Institute of Technology, Kharagpur, 721302, India.
authors.forename: Milan Kumar
authors.initials: MK
authors.lastname: Samanta
authors.affiliation: Advanced Laboratory for Plant Genetic Engineering, Indian Institute of Technology, Kharagpur, 721302, India.
authors.forename: Avishek
authors.initials: A
authors.lastname: Dey
authors.affiliation: Advanced Laboratory for Plant Genetic Engineering, Indian Institute of Technology, Kharagpur, 721302, India.
authors.forename: Soumitra Kumar
authors.initials: SK
authors.lastname: Sen
mesh_terms: D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics* / Q000378:metabolism; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D060467:Disease Resistance; D004731:Endotoxins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D007915:Lepidoptera / Q000187:drug effects; D010572:Pest Control, Biological; D010935:Plant Diseases / Q000517:prevention & control*; D018515:Plant Leaves / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics / Q000254:growth & development* / Q000469:parasitology; D015202:Protein Engineering; D014026:Nicotiana / Q000235:genetics / Q000254:growth & development* / Q000469:parasitology
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis
keywords: Bacillus thuringiensis; Cre/lox recombination; Cry2Aa; GM crops; Insect resistance; Lepidopteran pests; Selectable marker gene