Comparative Proteomic Study On Insecticidical Crystal Proteins From Three Bacillus Thuringiensis Ssp. And Two Engineering Strains

Bacillus thuringiensis(Bt) is a gram-positive and spore-forming entomopathogenic bacterium that produces one or more insecticidal crystal proteins (ICPs,δ-endotoxin) during sporulation. Here, we used proteomic technology to analyze ICPs comparatively from three Bt ssp. and two engineering strains.The ICPs were separated by 2-DE at pH ranges of 3-10 or 4-7. The ICPs patterns at pH range of 3-10 revealed significant similar among five strains, that is almost all ICPs were at pH range of 4-7 on the 2-DE gels. PDQUEST 2-DE software analysis detect 159±21 spots in Bt ssp. kurstaki 4.0718 strain, 184±6 spots in Bt ssp. Wuhanensis 140 strain and 366±11 spots in Bt ssp. israelensis H14 strain, 370±4 spots in Bt engineering strain XL004, 240±4 spots in engineering strain XL002. There are 62 matched spots (the matching rate was 38%) between Bt 4.0718 and Bt XL004 maps, and only 17 matched spots (the matching rate was 10%) between Bt 4.0718 and Bt 140 maps.The dominating and differential ICPs between the strains were identified using MALDI-TOF MS and MALDI-TOF/TOF MS. Variability of some ICPs and the level of the expression have been evaluated. We also identified some proteins related to the formation of ICPs, we name them ICPs-associated proteins. The dominating proteins in Bt 4.0718 and Bt XL004 were Cry1Ac, Cry2Aa, HSP-60, Translation elongation factor Tu and so on, in Bt ssp. Wuhanensis 140 were delta-endotoxin CryIG protoxin, insecticidal crystal protein CryH2, Cry1Ab16, translation elongation factor Tu, Cry2Ac and so on. In Bt israelensis H14, there were translation elongation factor G, HSP60, Trigger factor, ppiase, Translation Elongation Factor Tu, ABC transporter ATP-binding protein. Comparative to ICPs of Bt4.0718, the dominating ICPs of recombined strain XL004 were similar, But significantly different to the ICPs of Bt ssp. Wuhanensis 140.The three dimensional structure of the actived Cry2Ac is similar to the actived Cry2Aa, in spite of the distinct difference in the loop 2 of Domainâ…¡(³²²IGGLPVYHNSTLH³³²), and electrostatic potential surface of loop 2 of Domainâ…¡is positive, it may bind receptor proteins through electrostatic interaction, but the affinity of the actived Cry2Ac is different from the actived Cry2Aa.We also found out that HSP-60 and translation elongation Tu are exist in every Bt strains. So we suspected that they were necessary to the formation of the iCPs. We also identified some specified proteins except ICPs in each strain. The results revealed that the virulence of Bt strains was closely related with the variety and the level of the expression of ICPs, and introducing of the exogenous foreign gene had a great significance to the expression of ICPs, so had great effect on the virulence of Bt.