The Exploration Of Insecticidal Activity And The Model Of Action Of Cry1I Toxin

Pyramiding of multiple Bt toxin genes recognizing distinct binding sites in themidgut of the target pest has been demonstrated to delay evolution of resistance intransgenic plants. In2014,30%(55.2hectares) of the corn planted globally weretransgenic Bt-corn. Among these Bt corn, cry1Ab and cry1Fa gene were used forcontrolling of corn borer. This high adoption of Bt-corn underlines a high probabilityfor the development of resistance to these gene in corn borer. Therefore, identificationof new Cry toxins which has no cross-resistance with Cry1A and Cry1Fa toxin, andexplore the mechanism of action has important theoretical significance and practicalvalue for developing a new generation of insect resistant Bt-corn.cry1Ie gene, intellectual property right owned, was cloned by our lab and wasalready used in the study of transgenic corn because of encoding a protein with highlytoxic against corn borer and has no cross resistance with commercialized Cry1Atoxins. This study aims to explore the insecticidal activity of different Cry1I toxinsand the mechanism of action of Cry1Ie toxin, which will provide good candidate fortransgenic corn and provided theory foundation for the promotion and application ofcry1Ie gene.The cry1Ie2gene encodes a81kDa protein which is processed to aprotease-resistant core form of approximately55kDa by trypsin digestion. The toxinpurification is performed by affinity purification and anion exchange. The relativeinsecticidal activity of purified Cry1Ie2protoxin was tested. Activity was testedagainst seven lepidopteran and one coleopteran species. Highest relative insecticidalactivity for Cry1Ie2was detected against larvae of O. furnacalis and Ostrinianubilalis, LC50are0.51μg/mL and18.49μg/mL respectively. In contrast, no toxicitywas detected against larvae of Sopdoptera exigua, Helicoverpa zea, or Tenebrio.molitor While no toxicity was detected for Helicoverpa armigera, Helicoverpavirescens and Sopdoptera frugiperda, a clear growth inhibition was observed,suggesting low toxicity. 125I-labeled Cry1Ab and biotinylated Cry1Ie2were used in competition bindingassay. Results from binding assays with125I-labeled Cry1Ab toxin and brush bordermembrane vesicles from O. nubilalis larvae demonstrated that Cry1Ie2does notrecognize Cry1Ab binding sites in that insect. Reciprocal competition binding assayswith biotin-labeled Cry1Ie2confirmed lack of shared sites with Cry1Ab or Cry1Fa inO. nubilalis BBMV. These data support cry1Ie2as a good candidate for pyramidingwithcry1Ab or cry1Fa in Bt corn to increase the control of O. nubilalis and reduce therisk of resistance evolution.Solubilized BBMV from O. nubilalis were fractionated by size-exclusion andseperated to Peak1, Peak2, Peak3, Peak4, Peak5and Peak6. All these fractions werechecked by dot blot using Bio-Cry1Ie1and Bio-Cry1Ab protein. The result revealedthat both Bio-Cry1Ie1and Bio-Cry1Ab can bind to BBMV proteins in fractions C8,C9and C10. But for BBMV proteins in fractions C6, C7and C11, only Bio-Cry1Ie1protein can bind to them, which means there are proteins in C6, C7and C11that canonly recognized by Bio-Cry1Ie1protein not Bio-Cry1Ab protein. Fractions C6, C7,C8, C9, C10and C11were used in affinity purification to identify the protein whichcan bind to Cry1Ie1. The samples from affinity purification were sent for MassSpectrometry (LC-MS/MS). Totally14protein fragments from o.nubilalis BBMVwere identified by Mass Spectrometry with molecular weight from22kDa to213kDa.Among these14proteins,4proteins can bind to both Cry1Ab and Cry1Ie1toxin,2proteins can bind to Cry1Ab, Cry1Ie1toxin and control,2proteins are special forCry1Ab and6proteins are special for Cry1Ie. V-type proton ATPase submit andAminopeptidase N3b can only bind to Cry1Ie1toxin, for further study we will test thefunction of these proteins.Ten cry1Ia genes were successfully expressed in Escherichia coli Rosetta (DE3)strain. cry1Ia20, cry1Ia21, cry1Ia23, cry1Ia25, cry1Ia26, cry1Ia27, cry1Ia28,cry1Ia29gene could be expressed normally with a molecular weight of approximately81kDa. A60kDa protein was also detected and expression level was similar with81kDa protein. More important, only60kDa protein was detected in cry1Ia22andcry1Ia24gene expression product. The60kDa proteins were sent for MassSpectrometry and result shown that this60kDa fragment is Cry1Ia protein, whichmay result by the degradation of81kDa protein.Bioassay results show that only one amino acid change can greatly affect thetoxicity of Cry1Ia protein. Compared the amino acid sequence and the toxicity of Cry1Ia, we can conclude that serine at141and377sites are important for the toxicityof Cry1Ia against Plutella xylostella and Ostrinia furnacalis. Glycine at80sites isimportant for the toxicity of Cry1Ia against Ostrinia furnacalis, but not important forthe toxicity against Plutella xylostella. Phylogenetic analysis revealed that these tencry1Ia genes can be divided into two clusters and the amino acid recidues at329,376and549sites are the key factor to determine these two different differentiation.