Directed Screening And Modification For Bt Cry Toxin Against Rice Planthopper, Nilaparvata Lugens

Cry toxins are the most important toxin extracted from Bacillus thuringiensis (Bt),which have been widely used in the management of lepidopteran, coleopteran,dipteran pests and nematode. Alone with the widely planting of transgenic cropscarrying Bt cry genes, target pest which is susceptive to Cry toxins will be effectivelycontrolled and the use of classical chemical pesticides which may cause risks to theenvironment will be decreased。However, insecticidal spectrum of Cry toxins are limited. Only Lepidoptera,Coleoptera, Diptera are susceptible to Cry toxins. Currently, seldom reports concernedto the toxicity from Cry toxins against hemipteran pests such as aphids, planhoppersand leafhoppers. As a result, after the planting of Bt transgenic crops and widely usedof Bt insecticide, lepidopteran, coleopteran, dipteran pests will be efficientlycontrolled, but hemipteran pests with sucking and piercing mouth part like aphids,planthoppers and leafhoppers will only be managed by the use of traditional chemicalpesticides. The deterioration of these Bt nontarget pests will increase and even lead toan explosion. Thus, it is emergency to search or create a novel Cry toxin that showsconsiderable toxicity against these hemipteran pests.Our research, firstly, prepared purified and soluble Cry1Ab pro-toxin. ThenCry1Ab antiserum was produced by immuning Cry1Ab pro-toxin to Newzealandwhite rabbit.The reason of low toxicity from Cry1Ab against N. lugens, an importantpest in rice production, was analyzed by the use of membrane feeding, pull-downtechnology and in vitro hydrolysis. Results showed that Cry1Ab pro-toxin can beefficiently activated into~60kDa peptide by proteaeses exist in N. lugens gut andthese activated mature peptide can bind to N. lugens BBMV (brash border membranevesicle). According to the reported mechanism of Cry1Ab toxin, activated protein cannot bind to proper receotros is the most likely reason for the low toxicity of Cry1Abagainst N. lugens.On the other hand, three receptor binding loops in domainⅡ of Cry1Ab toxinwas replaced by an aphid gut inner membrane binding peptide GBP3.1respectively.To find out which loop is the most important receptor binding site, LC50of three modified Cry toxin, with each loop be substituded by GBP3.1, against diamond backmoth (Plutella xylostella) was tested. Results showed that toxicity of Cry1Ab toxindroped sharply when loop2was replaced, indicating that loop2is more importantthan loop1and loop3in receptor binding and toxicity excerting.To develop a novel Cry toxin which will show toxicity to N. lugens, our researchscreened a peptide which could bind to N. lugens gut inner membrane. After fusingP2S with egfp (enhanced green fluorescent protein) gene, P2S-EGFP fusion proteinwas prepared to confirm the binding acitivy between P2S and N. lugens gut BBMV orgut inner membrane. After that, three receptor binding loops of Cry1Ab domainⅡwas replaced by P2S or P1Z (a peptide screened from N. lugens BBMV bindingphage) respectively to produce six novel Cry proteins. With the use of in vitrohydrolysis, pull-down technology and membrane feeding, one novel Cry toxinCry1Ab-2S with loop2be replaced by P2S was selected. Cry1Ab-2S was the moststable novel toxin after the treatment by N. lugens gut proteases and had the strongestbinding activity to N. lugens BBMV. Bioassay results demonstrated that toxicity ofCry1Ab-2S against N. lugens nymphs were nearly13folds more than Cry1Ab toxin.By observing gut ultrastructure of N. lugens though electron microscope, gutmicrovilli of N. lugens fed by Cry1Ab-2S was obviously hydrolyzed.This research provides a new method for molecular modification of Cry toxinstarget on specific pests. Furthermore, result of this research will help theunderstanding of mechanism of Cry toxins in hemipteran insects. In summary, thisresearch work was focused on the following:1. Analyzed the probable reseaon of low toxicity of Cry toxin against N. lugens andother hemipteran pest with sucking and piercing mouth part;2. Determined the importance of three loops in domainⅡ in excerting toxicity ofCry1Ab toxin;3. Screened a peptide which can bind to N. lugens gut inner membrane by phagedisplay library;4. Obtained a novel Cry toxin showed high toxicity against N. lugens by directedmodification.