| Lepidoptera pests, such as cotton boll worm, beet army worm, and com borer worm, were very harmful to many crops and vegetables. Recently, resistance of cotton boll worm and beet army worm for chemicals was increasing quickly in the field, and it was very hard to control some pest by traditional methods. Leguminivora glycinivorella was the most important pest for soybean in north of China. But no formal report involved in the biocontrol using BtBt, the best microbial insecticide, has been reported for control of cotton boll worm, beet worm, but the narrow choice of cry genes, for instance only crylAb or cry1 Ac, may induce the pest resistance.In this study , different Cry protein expressed in E.coli. and the combinations between Cry proteins were screened against mentioned pests. It was very significant for construction of novel genetically engineered bacteria and transgenic plant for insect pest biocontrol.1. Biological characteristics of Bt Cry proteinThe solubility, digestion of Cry protein with trypsin and growth speed of E.coli. strain were researched respectively.The solubility of CrylCa7, CrylCb2, Crylla8, CrylIe1, Cry2Ab4 proteins, expressed in E.coli. strains, and CrylAc from HD-73 strain were demonstrated that they were more soluble in Na2CO3 than in Ethanoloanmine by SDS-PAGE analysis.Trypsin, the hydrdytic enzyme of insects, was crucial for digestion and toxicity of Cry protein. After reaction 6hrs, all the Cry protein were digested, and 55kDa proteins were obtain from CrylCa7 and Crylla8, 57kDa fragment were from CrylCb2, Cryllel and Cry2Ab4, but the 60kDa, peptide was maintained from CrylAc. If the concentration and reaction time were much more , all the Cry protein were degraded completely.It was the first report of the digestion of Cryllel and CrylIa8 in the LB broth, the growth speed of all the E.coli. strains harboring the recombinant plasmids inserted cry gene, were slower than their host BL21. The over expression of cry gene maybe affect on the growth and development of host cell.2. Purification of Bt Cry proteinsExpression time of cry protein in Ecoli strain was investigated. After induction by IPTG, 4~6hrs, the accumulation of Cry protein was the most, but prodution of CrylAc from HD-73 strain was the most after 28~32hrs. The result of FPLC showed that all of protoxins and toxins activated by tryspin could be well purified using elutin buffer at pH9.53. Bioassay of Cry protein against cotton boll worm larvaeThe bioassay result demonstrated that Cry 1 Ac and Cry2Ab4were higher toxicity than other four Cry proteins, and LC50 was 31.5ug/ml and 415ug/ml respectively. Furthermore, the co-toxicity coefficient of Cry lac and Cry2Ab4 combination was 121% according to the LC50 48.7ug/ml of protein combination. It was means that Cry 1 Ac and Cry2Ab4, but other four Cry toxin, could synergize each other.4. Bioassay of Cry protein against corn borer worm larvaeThe result of bioassay indicated that Cry1Ac, Crylla8 and CrylIe1 were high toxicity, and the combination between the three protein, illustrated that CrylIe1+CrylAc combination were synergistic against corn borer worm larvae. LC50 of Cry1Ac, CrylIa8, CrylIe1, CrylAc+CrylIe1 and CrylAc+CrylIa8 was 0.134, 0.500, 0.223, 0.046, and 0.198ug/g respectively. Cotoxicity coefficients of CrylIe1+CrylAc and Crylla8+CrylAc were 409 and 106 separately. Cry1Ac was the highest among the Cry protein on the level of LC90 and LC95. Those bioassay results showed that CrylAc+CrylIe1 combination could be used for construction of engineered bacteria and transgenic plant, and meanwhile Cry1Ac as an enhancer protein could be combinated with other Cry protein for Insecticidal research.5. Bioassay of Cry protein against beet army worm larvaeThe larvae could be inhibited by all the Cry protein, and Cry1Ac and CrylCa7 combination was more toxic.6. Bioassay of Cry protein against Leguminivora glycinivorella larvaeThe result of bioassay of Cry protein to Leguminivora glycinivorella larvae, by two time select...
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