Construction of Modified Bacillus thuringiensis cry1Ac Genes for Transgenic Crop Through Multi Site-directed Mutagenesis
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초록
The newly cloned Bacillus thuringiensis cry1-5 geneshowed high activity to both Plutella xylostella andSpodoptera exigua, while cry1Ac only showed highactivity against P. xylostella but low to S. exigua. Through the alignment of amino acid sequencesbetween Cry1Ac and Cry1-5, we found 12 differentresidues in domain I (6 residues) and domain II(6 residues). In this study, the modified cry1Ac gene,which is constructed according to a crop-preferringcodon usage, was used as a template to constructmutant B. thuringiensis cry1Ac genes based on cry1-5gene through multi site-directed mutagenesis. Total 63various mutant cry genes were obtained at 12 positionsrandomly. Among them, ten mutant cry genes, whosedomain I was totally converted and domain II wasrandomly, were selected to express in baculovirusexpression system as a polyhedrin fusion form. Therecombinant proteins were 95 kDa in size and werestably activated as 65 kDa by trypsin. The expressedmutant Cry proteins were applied to bioassays againstP. xylostella and S. exigua. All mutants showed highinsecticidal activity both to P. xylostella and S. exiguasimilar to cry1-5. These results suggest that thesemutant cry genes might be expected of desirable crygenes for introduction to transgenic crops.
Abstract
The newly cloned Bacillus thuringiensis cry1-5 geneshowed high activity to both Plutella xylostella andSpodoptera exigua, while cry1Ac only showed highactivity against P. xylostella but low to S. exigua. Through the alignment of amino acid sequencesbetween Cry1Ac and Cry1-5, we found 12 differentresidues in domain I (6 residues) and domain II(6 residues). In this study, the modified cry1Ac gene,which is constructed according to a crop-preferringcodon usage, was used as a template to constructmutant B. thuringiensis cry1Ac genes based on cry1-5gene through multi site-directed mutagenesis. Total 63various mutant cry genes were obtained at 12 positionsrandomly. Among them, ten mutant cry genes, whosedomain I was totally converted and domain II wasrandomly, were selected to express in baculovirusexpression system as a polyhedrin fusion form. Therecombinant proteins were 95 kDa in size and werestably activated as 65 kDa by trypsin. The expressedmutant Cry proteins were applied to bioassays againstP. xylostella and S. exigua. All mutants showed highinsecticidal activity both to P. xylostella and S. exiguasimilar to cry1-5. These results suggest that thesemutant cry genes might be expected of desirable crygenes for introduction to transgenic crops.
참고문헌 (13)
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