Cloning And Expression Of Cry1Ib2 Gene From Bacillus Thuringiensis PP61 And Bioactivity Of Cry1Ib2 Protein Against Papilio Xuthus Linnaeus

For the purpose of obtaining new Cry proteins expressed the Bt PP61 strain which was isolated from Bauhinia purpurea as effective activity against citrus swallowtail butterfly,a series of physiological and biochemical reactions was conducted on PP61 and it suggested that the strain PP61 was a member of kurstaki.PCR-RFLP and PCR technologies were used to analyse the cry genes of PP61.It showed that the strain haboured at least four different cry genes including cry1Ba,cry1Cb,cry1Ib and cry2Ab.There hasn't been other reports except Shin cloned the cry1Ib1 in 1995.Thus we amplified the cry1Ib gene from Bt PP61 with 2160 bp in length,encoding a protein of 719 residues with a calculated molecular weight of 81.39 kDa and a predicted pI value of 6.425.The nucleotide sequence of cry1Ib of PP61 had been deposited in GenBank at the accession number AY945956 which was designated cry1Ib2 gene as a novel gene by Btδ-Endotoxin Nomenclature Committee.It showed that the deduced Cry1Ib2 had high similarity with Cry1I by Blast software. According to the signal peptide analysis,there were no signal peptides in front end of Cry1Ib2 protein,suggesting that Cry1Ib2 was an intracellular protein.Conserved Domain Searches revealed that Cry1Ib2 was mainly composed of three domains,structure domainâ… located at the N-end of toxin molecular,and structure domainâ…¢at the C-end and structure domainâ…¡between them.The structure domainâ… participated in the performation of membrane and the spore.Both domainâ…¡and domainâ…¢involved in the binding with the membrane receptor protein.There were 25%ofα-helix,28%ofβ-sheet,18%ofβ-turn and 29%of the coil in the secondary structure of the protein.In order to examine the expression of the cry1Ib2 gene in E.coli,the BamHI-SalI fragment corresponding to the ORF of cry1Ib2 was inserted into the expression vector pET29a(+).The recombinant plasmid,designated Pet29a-cry1Ib2,was transferred into E.coli BL21(DE3). Successful transformation was confirmed both by PCR amplification and nucleotide sequencing. The high expression of cry1Ib2 gene was induced by IPTG.SDS-PAGE showed that the molecular weight of the expressed fusion protein(Cry1Ib2 plus additional(His)₆ tag carrier protein)was about 81 kDa,which was corresponded with the ExPASy calculation.The expressessed products from cry1Ib2 gene were partially soluble.The expressed Cry1Ib2 protein was toxic against the 5^rdlarvae of Papilio xuthus.All insects fed with Cry1Ib2 protein exhibited obvious growth inhibition compared to the control and cloning of this new cry gene provided a new member for cry genetic family,and offered new genetic materials for constructing the new engineered strains as well as transgenic plants.In practice,we could use this new Cry1Ib2 protein for control of the Papilio xuthus pest.Furthermore,possibilities of using this gene on transgenic plants controlling pests which damage fruit trees were discussed.