Construction Of Recombinant Plasmids Carrying Cry2Ab And Its Expression In Bacillus Subtilis

cry2Ab is a kind of delta-endotoxins gene isolated from entomopathogenicbacterium Bacillus thuringiensis, which encoded an insecticidal crystal proteinCry2Ab specifically against Lepidoptera insects. B. thurigiensis and its delta-endotoxins have been widely exploited and applied as biological pesticide due to theirhigh efficiency, environmental safety and no residues. While Bacillus subtilis hasbeen enrolled as Generally Recognized As Safe （abbr. GRAS） bacterium by AmericanFood and Drug Administration （abbr. FDA）, and commonly used as biologicalpesticides for major crops suffered by plant pathogens or as a host for secretoryexpression in fermentation industries.In this study, the full length sequences of cry2Ab8gene （GenBank: DQ361266.1）from B. thuringiensis WB2and a strong promoter element PyxiE （which was firstpublished by Zhang Ai-Ling on BBRC） from B. subtilis168were amplified by PCRmethod using Pfu DNA polymerase with two pairs of specific primers （Cry2A-F/Rand PyxiE-F/R）, respectively. After the purification of each PCR products, the twofragments were employed as templates to generate PyxiE-cry2Ab fusion by SOE-PCRmethod with using primer pairs （PyxiE-F and Cry2Ab-R）. The PyxiE-cry2Ab fusionproducts with3’ A-overhangs by PCR-amplification using Taq DNA polymerase wascloned with pMD18-T cloning vector and transformed into Escherichia coli DH5αcompetent cell. Commercial sequencing was carried out to confirm the PyxiE-cry2Abfusion fragment. Then the PCR-amplication PyxiE-cry2Ab fusion products werecloned into pDG1662vector and pAD123vector with BamHI/EcoRI or KpnI/XbaIdouble digestion, resulting in the integrated pDG-PyxiE-cry2Ab plasmid and the E.coli-B. subtilis shuttle pAD-PyxiE-cry2Ab plasmid, respectively. The recombinantplasmid pAD-PyxiE-cry2Ab was transformed into B. subtilis ATCC6633byelectroporation methods. The obtained recombinant was validated by PCR method,designated as B. subtilis ATCC-PPC.Continuous passage culture （inoculated to fresh Luria-Bertani medium per12h）combined with colony PCR-diction was used to analyze the recombinant pAD-PyxiE-cry2Ab plasmid stability. The results showed that the recombinantplasmid exhibited good genetic stability （0.00%loss of recombinant plasmid） beforeits7^thpassage culture. However, after this, its genetic stability presented a decreasingtrend. A loss of42.22%for the recombinant plasmid appeared on the15thpassageculture. For bacterial growth curve （cultured in LB medium at30℃,120rpm）, nogrowth differences between the recombinant strain and control B. subtilis ATCC6633were observed during their exponential phase in the first10h. On the stationery phase,the OD600for the recombinant appeared to be slightly lower than that for the controlstrain. In addition, the recombinant tended to enter the death phase earlier. Byapplying multiple regression analysis to the growth data, the imitative univariateequations of higher degree were obtained, describing the relationship between the celldensity and growth time for the control B. subtilis ATCC6633strain and recombinantstrain. y1=-0.0171x13+0.2396x12-0.5028x1+0.502（R2=0.9793） and y2=-0.0161x23+0.2238x22-0.4276x2+0.4071（R2=0.9835）. It revealed that the growth rate of therecombinant was lowered by the introduction of recombinant plasmid. Finally,SDS-PAGE analysis indicated that there was a clear protein band with molecularweight of70kDa on expected position. The result suggested that the cry2Ab genewere expressed in both E. coli and B. subtilis ATCC6633host cells.