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Abstract Receptor binding is a key step in the mechanism of Bacillus thuringiensis Cry toxins and mainly determined by 3 loops locate on the apex of β-sheet in domainⅡ. The important role of these 3 loops, function in binding with specific receptors in the midgut of target insect, has currently been widely reported. However, the importance of 3 loops to influence the toxicity of Cry is different. To determine the important role of these 3 loops in insecticidal activity of Cry1A toxins, in this research, overlap PCR was conducted by using a pea aphid (Acyrthosiphon pisum) gut APN (amino peptidase N) receptor binding peptide GBP3.1 to replace 3 loops in domainⅡ. Then 3 plasmids carrying each modified cry1Ab gene were constructed. After that, prokaryotic expression and purification of 3 modified Cry toxins were conducted. Leaf residue bioassay was conducted by feeding each modified Cry toxin to Plutella xylotella lavae to test the impact of loop substitution on insecticidal activity. Bioassay results indicated that substitution of each loop decreased the toxicity against P. xylotella larvae at different degree, with the LC50 of 12.17, 32.25 and 23.00 μg/mL for the substitution of loop 1, 2 and 3, respectively, compared with wild-type toxin (LC50=0.88 μg/mL). It suggested that compared with loop 1 and 3, loop 2 played the most important role in exhibition of insecticidal activity of Cry1Ab toxin. Prediction of 3-dimensional structure of Cry1Ab toxin and 3 modified Cry toxins showed that replacement of each loop by GBP3.1 did not change the main structure of Cry1Ab protoxin, which meaned that the variety of toxicity against P. xylatella was mainly attributed to the substitution of each receptor binding loop. Moreover, analysis of 3-dimensional structure of Cry1Ab protoxin indicated that loop 2 was the most exposed receptor binding loop compared with loop 1 and 3. These results will be helpful for the understanding of mechanism of Cry1A toxins in the midgut of target insects and benefit further research on gene modification in receptor binding regions of Bt Cry toxins.
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Received: 15 May 2014
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NI He-Jia, CHENG Xue, WANG Jing, GAO Ze-Ping, WEI Pan-Pan, TAN Jia-Li, SUN Jing, LI Hai-Tao, GAO Ji-Guo. Isolation and Screening of High Virulent Bacillus thuringiensis Strains Against Lepidopteran Pests in Some Areas of Liaoning Province[J]. 农业生物技术学报, 2019, 27(8): 1513-1520. |
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