Chloroplast-targeted expression of recombinant crystal-protein gene in cotton: An unconventional combat with resistant pests

•Hybrid Bt-RB protein having more than double (FIVE) lectin-binding domains than (TWO) in normal Cry1Ac.•Enhanced chloroplast-targeted expression of transgene after nuclear transformation in cotton.•Killing of insect pests that have developed resistance against Bt toxin.•Increased yield because of l...

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Bibliographic Details
Published in:Journal of biotechnology 2013-07, Vol.166 (3), p.88-96
Main Authors: Kiani, Sarfraz, Mohamed, Bahaeldeen Babiker, Shehzad, Kamran, Jamal, Adil, Shahid, Muhammad Naveed, Shahid, Ahmad Ali, Husnain, Tayyab
Format: Article
Language:English
Subjects:
Agrobacterium - genetics
Animals
Bacterial Proteins - genetics
Bacterial Proteins - metabolism
Bacterial Toxins - genetics
Bacterial Toxins - metabolism
Bollworms
Chloroplast expression
Chloroplast transit peptide
Chloroplasts - metabolism
Confocal microscopy
Cotton
Cotton transformation
Endotoxins - genetics
Endotoxins - metabolism
Genes
Gossypium - genetics
Gossypium - metabolism
Gossypium - microbiology
Gossypium hirsutum
Heliothis armigera
Hemolysin Proteins - genetics
Hemolysin Proteins - metabolism
Hybrid-Bt protein
Insect Control - methods
Insecta - metabolism
Insecticide Resistance - drug effects
Insecticide Resistance - genetics
Insecticides - metabolism
Insecticides - pharmacology
Insects
Larva - metabolism
Pest Control, Biological
Plant Leaves - metabolism
Plants (organisms)
Plants, Genetically Modified
Recombinant
Ricin B-chain
Spodoptera frugiperda
Toxins
Transformation, Genetic
Transgenic
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Summary:•Hybrid Bt-RB protein having more than double (FIVE) lectin-binding domains than (TWO) in normal Cry1Ac.•Enhanced chloroplast-targeted expression of transgene after nuclear transformation in cotton.•Killing of insect pests that have developed resistance against Bt toxin.•Increased yield because of less pest damage. Plants transformed with single Bt gene are liable to develop insect resistance and this has already been reported in a number of studies carried out around the world where Bt cotton was cultivated on commercial scale. Later, it was envisaged to transform plants with more than one Bt genes in order to combat with resistant larvae. This approach seems valid as various Bt genes possess different binding domains which could delay the likely hazards of insect resistance against a particular Bt toxin. But it is difficult under field conditions to develop homozygous plants expressing all Bt genes equally after many generations without undergoing recombination effects. A number of researches claiming to transform plants from three to seven transgenes in a single plant were reported during the last decade but none has yet applied for patent of homozygous transgenic lines. A better strategy might be to use hybrid-Bt gene(s) modified for improved lectin-binding domains to boost Bt receptor sites in insect midgut. These recombinant-Bt gene(s) would express different lectin domains in a single polypeptide and it is relatively easy to develop homozygous transgenic lines under field conditions. Enhanced chloroplast-localized expression of hybrid-Bt gene would leave no room for insects to develop resistance. We devised and successfully applied this strategy in cotton (Gossypium hirsutum) and data up to T3 generation showed that our transgenic cotton plants were displaying enhanced chloroplast-targeted Cry1Ac-RB expression. Laboratory and field bioassays gave promising results against American bollworm (Heliothis armigera), pink bollworm (Pictinophora scutigera) and fall armyworm (Spodoptera frugiperda) that otherwise, were reported to have evolved resistance against Cry1Ac toxin. Elevated levels of hybrid-Bt toxin were confirmed by ELISA of chloroplast-enriched protein samples extracted from leaves of transgenic cotton lines. While, localization of recombinant Cry1Ac-RB protein in chloroplast was established through confocal laser scanning microscopy.
ISSN:0168-1656
1873-4863
DOI:10.1016/j.jbiotec.2013.04.011