Study On The Function Of C-terminal Region Of The Cry1 Protoxins From Bacillus Thuringiensis

Bacillus thuringiensis(Bt)has become the most widely used microbial pesticide currently,and its insecticidal activities are mainly due to the insecticidal crystal proteins.Some studies have shown that C-terminal region of the Cry1 protoxins plays an important role in the insecticidal crystal formation and stability.It is important to study on the function of C-terminal region of the Cry1 protoxins for improving the yield of insecticidal crystal protein,enhancing its solubility and insecticidal virulence.Researches show that solubility of Cry1 Ac was obviously higher than Cry2Aa in alkaline environment of the insect midgut.In order to enhance Cry2Aa dissolving capacity,this study used N-terminal half of Cry2Aa protoxin and C-terminal half of Cry1 Ac protoxin to construct a fusion protein.Analysis of the fusion protein expression and insecticidal virulence was then performed.The cry1 Ac and cry2Aa genes were amplified from strain Bt4.0718 respectively.Then Red/ET homologous recombination technology was used to construct the fusion gene using N-terminal half of Cry2Aa protoxin and C-terminal half of Cryl Ac protoxin region encoding sequence.The expression vector containing the fusion gene was transformed into the acrystalliferous Bt strain XBU001.The recombinant strains were observed by phase contrast microscope and scanning electron microscope.The expressed products were analyzed by SDS-PAGE and identified by mass spectrometry.Furthermore,alkaline solubility,trypsin-activated characteristics and virulence to Helicoverpa armigera of the fusion protein were measured and analyzed.The results showed that the recombinant strain could produce amorphous crystalline inclusion and expressed 130 kDa fusion protein.Under the environment of the pH 10.5 the inclusion of fusion protein and Cry1 Ac crystal can be dissolved out of the original 80%protoxins,while only 10%protoxin could be dissolved from Cry2Aa crystal.Furthermore,the fusion protein can be activated normally by trypsin.In addition,when bioassays were carried out with different ratio combinations of CrylAc/Cry2Aa,obvious enhancement effect to Helicoverpa armigera was exhibited,while CrylAc/Cry2Aa-lAc had obvious synergistic activity.Thus,we concluded that the C-terminal region of Cry 1 Ac protoxin can enhance the solubility of Cry2Aa crystal and its virulence in the insect gut.The 130~140 kDa Cryl protoxins contained abundant cysteine(Cys)residues in the non-toxic C-terminal half.The researchers generally assumed that these Cys residues contributed to crystal formation.In the early research,we have successfully conducted serine scanning mutagenesis on Cryl Ac protoxin of HD73.In order to study the function of Cys residues in the non-toxic region in crystal formation,all mutant strains were observed by scanning electron microscopy.Results showed that all mutant strains could still form a typical diamond crystals.Solubility and trypsin stability analysis indicated that C-terminal mutant of Cryl Ac protein could be dissolved at pH7.5,while wild CrylAc protein was only soluble at pH9.5,indicating that the mutation can improve the solubility of Cryl Ac protein.All Cys residues mutant of CrylAc more sensitive to trypsin digestion than wild CrylAc,easily degrade into toxic fragment in a short time.Furthermore,the influence of wild CrylAc and all Cys residues mutant of CrylAc crystal protein on cell viability and cell proliferation of CF203/2.5 cell were conducted.Two kinds of toxins in different concentration gradient treated CF203/2.5 cells for 48h,observation with inverted microscope showed two toxins are toxic to cells and a lot of cell debris can be found.At the same time,the effect of protein on the proliferation of CF203/2.5 cells was detected by MTT test.Results show that with two protein concentration increased,the cytotoxicity was enhanced gradually.At 54 ?g/mL of wild CrylAc and all Cys residues mutant of CrylAc protein,mortality rate respectively is 69%and 82%,indicating that the mutation toxin had a obvious improvement.In summary,we conclude that the C-terminal region of CrylAc protoxin has an important role in the formation of crystals,but it does not necessarily depend on the Cys residues,which is different from the previous reports.The study help to elucidate the function of non-toxic C-terminal region of Cryl,and provide a theoretical basis for constructing insecticidal engineered bacteria of Bacillus thuringiensis.