Primary Location Of The Cluster Coding For Thuringiensin And Study On The Synergism Of Thuringiensin To Insecticidal Crystal Proteins

Thuringiensin is a non-specific heat-stable insecticidal toxin produced by someBacillus thuringiensis strains. This exotoxin is heat-stable and its chemical structure issimilar to that of nucleotides, its melecular weight is 701. The mechanism of insecticidalaction is to inhibit the production of DNA-dependent RNA polymerase by competitionwith ATP, so it has a broad-spectrum of effectivity against many pests. The toxicity ofthuringiensin is much less than that of most chemical insecticides; therefore it shows greatpotential to become a very useful insecticide for a wide range of pests.In the first part of this paper, in order to investigate the influence of the plasmids ofBacillus thuringiensis on thuringiensin production, the plasmid map, composition of ICPsand thuringiensin production of strain CT-43 and its mutants CT-43-1c, CT-43-5, CT-43-7,CT-43-55, CT-43-62, were analyzed. Moreover, the plasmids of strain CT-43-1c werecured by elevating growth temperature, and then the non-thuringiensin mutant F-15 wasconstructed. The differences of plasmids map and thuringiensin yield among the threestrains were identified by PFGE and HPLC, respectively, and the biologicalcharacterization of mutant F-15 was primarily studied. Comparing with strain CT-43 andCT-43-1c, the mutant F-15 respectively lost three and two large plasmid; and the resultsof PCR, SDS-PAGE and HPLC from mutant F-15 shown that the mutant F-15 becameunable to synthesize thuringiensin and 140kDa Insecticidal Crystal Protein coded bycrylB gene. At the same time, the differences between three strains indicated that theproduction of thuringiensin in Bacillus thuringiensis was directly associated with theplasmid harboring a cry1 B gene, so these results lay a solid theory foundation and offer aappropriate recipient for the cloning of thuringiensin gene cluster.In the second part, the synergism of thuringiensin to Cry1 Aa, Cry1 Ac, Cry1 B,Cry1 C were estimated by diet infection method using Helicoverpa armigera andSpodoptera exigua as the bioassay targets. The synergia was 66%, 58%, 85%, and 76%for Helicoverpa armigera when neonate larvae were fed with artificial diet containing themixture of ICPs and thuringiensin at a rate of 14.1:1; 1.2:1; 27.0:1; 6.8:1(W/W) separately;and the LT₅₀ decrease of 12.81 and 9.16 hours for Cry1 Aa and Cry1 Ac, separately. While,The synergia was 281%, 47%, 147% and 26% for Spodoptera exigua when first-instarlarvae were fed with artificial diet containing the mixture of ICPs and thuringiensin at arate of 246.2:1; 15.3:1; 246.2:1; 6.1:1 (W/W) separately, and the LT₅₀ were decreased by11.52 hours for Cry1 C.The results indicated that thuringiensin has remarkable synergismto ICP who has high toxicity to target. In the other hands, adding a little thuringiensin, thetoxicity could be increased significantly when the ICPs non-toxicity to targets.