Development of a hybrid delta-endotoxin and its expression in tobacco and cotton for control of a polyphagous pest Spodoptera litura.
A hybrid delta-endotoxin protein was designed against a polyphagous lepidopteran insect pest Spodoptera litura, which is tolerant to most of the known delta-endotoxins. The hybrid delta-endotoxin was created by replacing amino acid residues 530-587 in a poorly active natural Cry1Ea protein, with a highly homologous 70 amino acid region of Cry1Ca in domain III. The truncated delta-endotoxins Cry1Ea, Cry1Ca and the hybrid protein Cry1EC accumulated in Escherichia coli to form inclusion bodies. The solubilised Cry1EC made from E. coli was 4- fold more toxic to the larvae of S. litura than Cry1Ca, the best known delta-endotoxin against Spodoptera sp. None of the two truncated toxins, solubilised from E. coli caused larval mortality. However, trypsinised Cry1Ca protoxin obtained from E. coli and solubilised from inclusion bodies caused mortality of S. litura with LC50 513 ng/ml semi synthetic diet. A synthetic gene coding for the hybrid delta-endotoxin Cry1EC was designed for high level expression in plants, taking into consideration several features found in the highly expressed plant genes. Transgenic, single copy plants of tobacco as well as cotton were developed. The selected lines expressed Cry1EC at 0.1-0.7% of soluble leaf protein. Such plants were completely resistant to S. litura and caused 100% mortality in all stages of larval development. Hence, unlike in E. coli, the hybrid delta-endotoxin folded into a functionally active conformation in both tobacco and cotton leaves. The truncated Cry1EC expressed in tobacco leaves was about 8-fold more toxic (LC50 58 ng/ml diet) compared to expression in E. coli.
Singh
P K
PK
National Botanical Research Institute, Rana Pratap Marg, Lucknow 226001, India.
Kumar
Mithilesh
M
Chaturvedi
C P
CP
Yadav
Dinesh
D
Tuli
Rakesh
R
D000595:Amino Acid Sequence; D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics*; D001427:Bacterial Toxins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004731:Endotoxins / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005813:Genes, Synthetic; D003368:Gossypium / Q000235:genetics*; D006460:Hemolysin Proteins; D008969:Molecular Sequence Data; D010572:Pest Control, Biological; D030821:Plants, Genetically Modified / Q000235:genetics*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera / Q000472:pathogenicity*; D014026:Nicotiana / Q000235:genetics*
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D011994:Recombinant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis