Study Of Region On Bacillus Thuringiensis Cry1Ba3Protein Related To Insecticidal Specificity

Bacillus thuringiensis (Bt) is one of insect pathogens that rely on insecticidal proteins to kill theirinsect larval hosts. It is the most widely used insecticidal microbe all over the world at present. Theparasporal crystal proteins produced in growth are also called insecticidal crystal proteins(ICPs). Thestructure and amino acid composition of ICPs are closely related to insecticidal activity, and they arealso heat spots for research domestically and overseas. Cry1Ba is the only insecticidal crystal proteinfound to be toxic to three orders of insects, including Lepidoptera, Coleoptera, Diptera. In order to findamino acid sites of Cry1Ba which are correlated to insecticidal specificity, some work has been done asfollows.Firstly,45amino acid residues, which were located on domain II of Cry1Ba3and influenced bypositive selection pressure, were predicted by bioinformatics method. Site-directed mutations wereintroduced to Cry1Ba3by site-directed mutation kit and the selected amino acids were substituted byalanine.45different mutants were constructed and mutant proteins were induced to express in E.colistrain BL21. Primary bioassays of Cry1Ba3against Plutella xylostella, Colaphellus bowringi, Bradysiaodoriphaga were conducted respectively. The result showed that Cry1Ba3had a high toxicity to P.xylostella with LC50of1.478μg/g and inhibited the growth of C. bowringi at the concentration of500μg/mL. Cry1Ba3was not toxic to B. odoriphaga.According to the assay results,0.8μg/g and8.0μg/g were set to be screening concentrations formutant toxicity against P. xylostell.100μg/mL and500μg/mL were set to be screening concentrationsfor mutant toxicity against C. bowringi. Then preliminary screening was conducted. The result showedthe insecticidal activity of mutants against both two insects increased or decreased variously to someextent. Six mutants which were obviously decreased in insecticidal activity against P. xylostella werepicked out. They were M339, M342, M348, M376, M379, M454. The proteins were diluted as eightconcentration from1.0μg/g to128.0μg/g respectively. Secondry screenings of selected mutants against P.xylostella were performed with different concentrations. The result showed LC50of the six mutantproteins were higher than that of Cry1Ba3, ranged from4.5times to19times. These mutant proteinsreally decreased in insecticidal activity against P. xylostella. Meanwhile, M338were found due to itsobvious decrease in toxicity against C. bowringi.On the basis of finding Cry1Ba3was tocix to Ostrinia furnacalis, Cry1Ba3protein was extractedand purified. Bioassay of Cry1Ba3against Ostrinia furnacalis was conducted. The result showed thatLC50of Cry1Ba3against O. furnacalis was5.294μg/g, and the toxicity of Cry1Ba3was lower than thatof activated Cry1Ac protein (LC50=0.833μg/g). BBMV was extracted from the midgut of O. furnacalis,then purified Cry1Ba3and Cry1Ac active fragment were used to perform competitive bindingexperiments against BBMV of O. furnacalis.The result showed Cry1Ba3and Cry1Ac had nocompetitive binding on BBMV of insects.Six amino acids located on Cry1Ba3protein were found to be related to insecticidal activity againstP. xylostella in this research. They were R339, N342, Y348, N376, T379, E454. S338was found to be related to insecticidal activity against C. bowringi. It was demonstrated that Cry1Ba3protein had goodbioactivity against O. furnacalis. Cry1Ba and Cry1Ac had no competitive binding on BBMV of O.furnacalis. These results provided theoretical foundations to elucidate specifically insecticidalmechanism of Cry toxins and guide protein-engineering for Cry toxins.