Construction Of Engineering Strains HD1Ca15 And HD1Da6 Of Bacillus Thuringiensis And Analysis Of Biological Activities

Bacillus thuringiensis(Bt)is the most widely used and effective microbial insecticide in the world.Bt has the characteristics of high insecticidal specificity and high virulence,it is safety for persons and animals.The insecticidal crystal protein has insecticidal specificity and virulence,which produced during the sporulation process.In recent years,due to the continuous expansion of pest populations on agriculture,coupled with the unreasonable use of chemical pesticides in production and pesticides,the level of pest resistance has risen rapidly,and the prevention and control effect has decreased,and it has also affected the quality and safety of agricultural products.Therefore,finding suitable biocontrol bacteria has become a hot research topic.In order to screen insecticidal protein with broad-spectrum and high virulence,the engineering bacteria of Bt HD1Ca15 and Bt HDlDa6 were constructed in this study,Cry1Cal5 and Cry1Da6 proteins were expressed and the insecticidal activity was analyzed.This research has laid a solid foundation for the reduced application of chemical pesticides,and provided a basis for reduce the resistance of agricultural pests.The main results are as follows:1.The full-length gene sequences of cry1Ca and crylDa were obtained from Bt GS57 strain,and primers were designed according to the sequence.Recombinant plasmids pETcry1Ca15 and pETcry1Da6 were constructed and transformed into E.coli BL21(DE3),after IPTG induction expressed 135 kDa and 118 kDa protein respectively;To obtain the recombinant shuttle vectors pScrylCa15 and pScry1Da6,the cry1Ca15 and cry1Da6 genes were linked to the shuttle vector pSXY422b,then electric shock into Bt HD73(cry-),so engineering bacteria Bt HD1Ca15 and HD1Da6 were constructed.The crystal protein was extracted by isoelectric precipitation method and detected by SDS-PAGE.2.The insecticidal activities of Bt HD1Cal 5,Bt HDlDa6,Bt Cry1Ca15 and Cry1Da6 proteins against to newly-hatched and 3rd instar larvae were tested.The results are as follows:the LC50 of the Bt HD1Ca15 strain against to newly-hatched larvae of Spodoptera exigua,Spodoptera litura and Helicoverpa armigera were 0.089 mg/mL,0.128 mg/mL and 1.300 mg/mL,respectively,and the LC50 against to 3rd instar larvae were 0.770 mg/mL,1.270 mg/mL and 11.730 mg/mL.The LC50 of Bt HD1Da6 strain to newly-hatched and 3rd instar larvae of S.exigua were 0.097 mg/mL and 0.960 mg/mL,and the LC50 against to newly-hatched and 3rd instar larvae of S.litura were 0.170 mg/mL and 1.580 mg/mL,but it had no toxic effect to H.armigera.Bt Cry1Ca15 protein had strong insecticidal activity against to newly-hatched and 3rd instar larvae of S.exigua,the LC50 were 0.018 mg/mL and 0.157 mg/m L,respectively.The LC50 of Bt Cry1Ca15 protein to newly-hatched and 3rd instar larvae of S.litura were 0.034 mg/mL and 0.384 mg/mL,but lower virulence to H.armigera,LC50 were 0.272 mg/mL and 2.443 mg/mL.The LC50 of Bt Cry1Da6 protein to newly-hatched larvae of S.exigua and S.litura were 0.023 mg/mL and 0.057 mg/mL,while the Bt Cry1Da6 protein were 0.218 and 0.494 mg/mL,but had no toxic effect to newly-hatched and 3rd instar larvae of H.armigera.Therefore,Bt HD1Ca15,Bt HD1Da6,Bt Cry1Ca15 and Cry1Da6 proteins had the highest insecticidal activity against S.exigua,followed by S.litura,which was less toxic to H.armigera.3.The synergy test results showed that when Bt Cry1Ca15 and Cry1Da6.proteins were mixed at the ratio of 1:1,the LC50 to newly-hatched larvae S.exigua,S.litura and H.armigera were 0.011 mg/mL,0.028 mg/mL and 0.250 mg/mL,respectively,and the coefficient CTC was greater than 120,which indicatied that there was a synergistic effect between Bt Cry1Ca15 and Cry1Da6 proteins.4.The control effect of Bt HD1Ca15 and HD1Da6 strains on 3rd instar larvae of S.exigua was tested in the greenhouse.The results showed that after spraying Bt HD1Ca15 and HDlDa6 bacterial suspension for 5 days,the insect population reduction rate of S.exigua were 80.00%and 77.67%,the control effect reached 77.22%and 73.89%,respectively,there was no significant difference from the control group treated with 5%Chlorantraniliprole.