| Pest problems have a significant impact on global agricultural production and result in substantial economic losses.Bacillus thuringiensis(Bt),specifically the insecticidal crystal proteins it produces,has become an important genetic source for developing genetically modified insect-resistant crops due to its highly specific and effective pest control properties.However,challenges such as the development of insect resistance and limited genetic resources have prompted the exploration of alternative Bt insecticidal crystal proteins,such as the uncharacterized structures pesticidal proteins(Xpp-like proteins).Nonetheless,the lack of physical resources and limited availability of sequence information have impeded research on the activity of these novel insecticidal crystal proteins.Therefore,constructing a comprehensive library of novel insecticidal protein entities and conducting functional activity studies would significantly contribute to the development and utilization of Bacillus thuringiensis,while also acquiring gene resources with independent intellectual property rights.In this study,we have made the following advancements in the construction of the Bt insecticidal crystal protein entity library:Cloning of 79 novel Bt insecticidal protein genes: We successfully cloned 30 genes encoding Xpp-like novel insecticidal crystal proteins and 49 genes encoding nonXpp-like novel insecticidal proteins.These genes were inserted into p ET15 d and p ET21 b vectors,and subsequently transferred into Escherichia coli cells.Additionally,using Alpha Fold2,we performed three-dimensional structure predictions for 30 Xpplike insecticidal proteins,revealing diverse structural features such as "head + tail" and "jelly roll fold."Expression of 43 novel Bt insecticidal crystal proteins: Through expression tests,we confirmed the expression capability of 12 Xpp-like novel insecticidal crystal proteins and 31 non-Xpp-like novel insecticidal proteins within the p ET expression vector and the Escherichia coli expression system,achieving an overall expression rate of 54%.Identification of a highly potent Xpp-like insecticidal crystal protein against aphids: By standardizing the expression and conducting biological activity tests against aphids,we discovered that the newly identified Xpp-like insecticidal crystal protein,HZAU075,exhibited a mortality rate of 79.08% against aphids at a test concentration of 325 μg/m L.Notably,this protein is encoded by a newly discovered gene,highlighting its potential for effective aphid control applications.By establishing a library of novel insecticidal protein entities and conducting indepth functional activity studies,we aim to expand the insecticidal spectrum of Xpplike novel insecticidal crystal proteins.Furthermore,our research endeavors to advance the development of Bt formulations targeting piercing-sucking insect pests and contribute to the cultivation of genetically modified insect-resistant crops.This work also aims to reduce China’s reliance on foreign insecticidal protein genes while providing gene resources with independent intellectual property rights for sustainable and environmentally friendly pest control measures in the country. |
