| Bacillus thuringiensis is widely used in insect control because it shows strong toxicity against many insects, but no toxicity to non-target organisms and safe for the environment. The novel gene crylAhl, intellectual property right owned, was cloned by our lab and was successfully transformed into corn and rice because of encoding a protein with highly toxic against many lepidopteran insect.To evaluate the safety of crylAhl gene against non-target insect, the bioassay of CrylAh protein against important economic lepidopteran insect-Bomfyx mori was tested. The results showed that the LC50of CrylAh against B. mori was more than500μg/mL, which confirmed that CrylAh highly toxicity against lepidopteran insect such as Helicoverpa armigera (LC50=7.04μg/g) was safe to economic insect B. mori (LC50>500μg/mL), moreover, it provided theory foundation for the promotion and application of crylAhl gene.These characteristic of CrylAh protein were just the opposite with that of another intellectual property right owned gene crylAi gene encoding a protein with low toxicity against H. armigera (LC50>500μg/g), but strong toxicity against B. mori (LC50=5.25μg/mL). The amino acid identity between CrylAh protein and CrylAi protein is84%. They share a very similar structure. In this study, we aimed to explore the molecular mechanism of CrylAh toxin with strong toxicity against H. armigera, and safe to economic insect B. mori.CrylAh protein is chose to be the key object, and CrylAi as control. On one hand, we plan to determine the amino acid sites related to insecticidal on CrylAh, the active fragment of CrylAi and the loop region of specific insecticidal activity of CrylAh and CrylAi protein by construction of site-directed mutants, truncated fragments and Loop mutants. On another hand, we plan to search the specific binding protein in BBMVs using an improved receptor-fishing method.The results are shown as follows:(1) Seven mutants on Domain I and Domain II of CrylAh were constructed by site-directed mutagenesis methods in this study. According to the bioassay results of mutants against H. armigera, the amino acid residue of206on helix a-6of Domain I was changed to Ala make the reduction of toxicity, and more than one additional Asn inserting in463on loop3of Domain I may reduce the toxicity against H. armigera.(2) Twenty six truncated fragments were constructed in this study. The active fragment of CrylAi toxin against P. xylostella was exactly located between amino acid residues361and6051, according to the bioassay results against P. xylostella.(3) We analyze the binding protein on BBMVs from H. armigera and B. mori to explore the relationship between binding proteins and insecticidal specificity. The results showed that there was a special binding band-APN of CrylAh toxin on BBMVs from H. armigera compared with B, mori. This difference may cause CrylAh toxin to show insecticidal specificity against H. armigera. However, there were also some different binding proteins of CrylAi toxin on BBMVs from B. mori compared with H. armigera, although this part of work need to be repeated further.(4) To determine the reasons of specific binding between CrylAh toxin and APN, we compared the structure and amino acid sequences with CrylAh and CrylAi toxin, the results indicated that the major differences on receptor binding region between two toxin was located on Loop2and Loop3of Domain Ⅱ. By constructing some Loops exchange mutants and comparing the bioassay results of Loops mutants against H. armigera and B. mori, we found that exchange of Loop2made CrylAi toxic against H. armigera (Cry1Ai-hloop2: LC50=64.23μg/g) and CrylAh lose toxicity against H. armigera (CrylAh-iloop2: LC50>500ug/g); while exchange of Loop2didn’t make two toxin exchange the toxiciry against B. mori (CrylAi-hloop2: LC50=11.60μg/mL; CrylAh-iloop2: LC50>500μg/mL), which indicated that Loop2is related to insecticidal specificity against H. armigera, however, exchange of Loop3not only made Cry1Ai non-increasing on toxiciry against H. armigera (Cry1Ai-hloop3: LC50>500μg/g), but also caused CrylAh lost the toxiciry against H, armigera (CrylAh-iloop3: LC50>500μg/g), which indicated Loop3was important for keeping original toxiciry of CrylAh and CrylAi against H. armigera.The determination of the safety of CrylAh to B. mori not only lays theory basis for the promotion and application of cry1Ah1gene, but also provides screen and evaluation standards for discovery and application of novel cry genes; The molecular mechanism of insecticidal specificity of CrylAh toxin was revealed by determination of receptor and amino acid region related to insecticidal specificity. All of these research progresses will lay foundation for the promotion and application of cry1Ah1gene, and provide the theoretical reference for modification of other Cry toxins. |
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