| Bacillus thuringiensis (Bt) is a gram-positive, spore-forming bacterium that exists worldwide in soil, dust, plants, water and insect cadavers. Cry proteins span a wide range of insect orders (Lepidoptera, Diptera, Coleoptera, Hymenoptera, and Orthoptera). Many mutation techniques have been used to study Cry toxins. Although valuable, the mutation techniques discussed in the previous paragraphs have important limitations. In the present study, DNA shuffling was used to create a library including large number different mutational proteins.An Escherichia coli-expressing mutant library containing 1000 cry mutant genes was constructed by DNA family shuffling method with five initial cry genes crylAb, cry1Ac, cry1C*, cry2A*, and cry9C*. All clones were inducibly expressed in Escherichia coli, and their toxicity was then tested by using Helicoverpa armigera (cotton bollworm) larva. All 1000 cry mutant genes in the library were sequenced, and amino acid sequence homology of all cry mutant genes was analyzed by multiple comparisons. The results showed that there were 235,186,16,420 and 88 clones homologues to crylAb, cry1Ac, cry1C*, cry2A*, and cry9C* respectively, and there were 55 clones unhomologues to initial five cry genes.Five cry1Ac-derived mutant toxins were found to have significantly enhanced toxicity compared to the parent cry1Ac gene product. Two were of similar toxicity,63 had decreased toxicity, and 116 did not show any toxicity. Compared to the parental Cry2A* toxin, the toxicity of 132 cry2A*-derived clones was significantly enhanced. Six cry2A*-derived clones had similar toxicity to the parental clone,282 had diminished toxicity, and 116 were devoid of detectable toxicity. There were 235 clones homologus to crylAb, which was the second amounts in the 1000 mutant clones. Only clone 977 which increased toxicity was in the top 10 toxicity clones, which was similar to cry1Ac-derived mutant toxins. Only 16 clones were homologus to cry1C*, for larger diferient disparation between it and other four parent genes. Mutant clones came from cry9C*, which was 8.8% in the 1000 clones and the amount was fewer than other resource clones. The cry9C*-derived mutant toxins were found that most of them lost or decreased their toxicity.Frame shift mutation was the typical type in clones with lost or decreased toxicity. Termination codon and frame shift mutations were appeared in part mutantional clones. The clones with same as parent proteins didn't found mutation in amino acid sequence or mutational site located in outside of structure. Few mutational sites in clones with increased toxicity and they distributed in variable region.Although amino acid substitutions were found in all three domains of mutant proteins with increased toxicity, most mutants were located in domains 1 and 2. Clones with increased toxicity contained single or few amino acid mutations per clone and distributed in different helix and foldback. R127L,R127F in clones with increased toxicity also increases hydrophbicity and might also lasting protein structure and facilitate protein insertion into the insect membrane. The T296H mutation in domain 2 of clones 305 and 323 with enhanced toxicity increases the polarity of this region and could contribute to receptor binding. Some mutational hot spots such as 124,127,296 amino acid sites were observed in clones from cry1Ac, and 12-14,25-28,140-146 amino acid sites in clones from cry2A*.We report that DNA shuffling allows the identification of rare substitutions that can improve the functional properties of a target polypeptide, and permit the relationship between structure and function to be investigated by highlighting sites at which amino acid substitutions have significant effects on protein function. DNA sequencing identified key amino acid substitutions affecting toxicity, casting light on the structure-function relationship of this class of polypeptide toxins. New high toxic proteins were produced for breeding for pest resistance in our research. |
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