Influenlence Of The C-terminal Half Of Bacillus Thuringiensis On Inclusion Bodies Formation Of Vip3Aa7 And Cry7Ba1 Proteins

Bacillus thuringiensis has unique capability of high expression and crystal formation of these ICPs depended on various Regulated mechanisms.Here we established a high expression system to optimize the yield of Vip3Aa7 through relocating it into the mother cell of B.thuringiensis in the form of inclusion bodies using the regulated mechanism and the C-terminus,of Cry1C,Cry1Ac and Cry7Ba1.And this overexpression system has also been used to increase the yields of other B.thuringiensis proteins which have no C-terminus such as Cry2Aa and Cry3Aa.Then,In order to demonstrate the possibility of exploiting the latent insecticidal properties of crystals like Cry7Ba1,crystals vitro-solubilized were toxic to Plutella xylostella,we constructed a series of plasmids encoding recombinant forms of Cry7Ba1 in which the C-terminal domain was replaced by that of Cry1C and Cry1Ac,leading to improved solubility and effective toxicity to Pluetella xylostella.The following are the major results:1.The carboxy-terminal half of ICPs can markedly increased the yields of Vip3Aa7 through forming inclusion bodies in the mother cell of Bacillus thuringiensis.1.1 The C-terminus of the Cry1C could not markedly increase Vip3Aa7 yields 9.8-fold than that of wild type strain BMB8901 but also help it form the irregular inclusion bodies in B.thuringiensis.1.2 The C-terminal halves of Cry1Ac and Cry7Ba1 have the same function with that of Cry1C,could not largely increase Vip3Aa7 yields 9.4 and 9.0-fold recepectively than that of wild type strain BMB8901 but also help it form the irregular inclusion bodies in B. thuringiensis.1.3 Bioassays showed that all proteins produced by recombinant strains had similar toxicities with against H.armigera,S.exigua,and P.xylostella,which were evidently lower than that of wild-type Vip3Aa7 protein2.Helper protein p20 increased the yield of Vip3Aa7 fusion proteinsA searious of recombinant strains were obstained through transforming plasmid pBMB0177 containing the p20 and the expression vectors described above simultaneously.We found that the p20 could increase the yields of Vip3Aa7 protein but could not affect the forming of the inclusion bodies and the toxicities.In conclusion,we established an overexpression system to optimize the yield of Vip3Aa7 and relocated it into the mother cell of B.thuringiensis in the form of inclusion bodies.Alterations comprising Btâ… -Btâ…¡promoters,the STAB-SD sequence,the C-terminal,and the terminator of Cry1C affected the expression of Vip3Aa7.3.The C-terminus of Cry1C and Cry1Ac increased the yields of proteins of Cry2Aa and Cry3Aa In order to know whether the C-terminal of Cry1C can be used to increase the yields of other B.thuringiensis proteins without C-terminus,four recombinants,BMB0192, BMB0193,BMB0194,BMB0195,were gained by replaced the Vip code region with cry2Aa and cry3Aa.The result showed that this high expression system could increase the yields of Cry2Aa and Cry3Aa proteins without C-terminus of Cry1.However,the C-terminus of Cry1 could not make Cry2Aa and Cry3Aa form the bipyramidal(Cry1), cuboidal(Cry2Aa),flat rectangular(Cry3Aa) crystal protein,only irregular inclusion bodies,but did not affect the toxicities of recombinant protein.This result demonstrate that the overexpression system can be used to increase the yields of other B.thuringiensis proteins which cannot form crystals or other low expression level proteins and could not affect the activity of recombinants.The expression strategy would facilitate the development of a suitable formulation for the application of this class of insecticidal proteins in the field,and offers an additional method for potentially improving the efficacy of insecticides based on B.thuringiensis.4.The C-terminus of Cry1C and Cry1Ac increase the solubility of protein of Cry7Ba1After exchanging the C-terminal halves of Cry7Ba1 and Cry1,we found that the limited solubility of Cry7Ba1 was due to its C-terminal half,and that recombinant Cry7Ba1 possessing the C-terminal half of Cry1 showed good solubility and toxicity to P. xylostella but could not form the bypimid crystal the same as that of the parent wild type strain Cry1C,Cry1Ac and Cry7Ba1.Our research opens a new way to release hidden biological function from "non-insecticidal" B.thuringiensis strains resulting from limited solubility by simply replacing their C-terminal halves.