Analysis Of Diversity Of Insecticidal Genes And Functional Proteins In Four Strains Of Bacillus Thuringiensis

Bacillus thuringiensis (Bt) was first reported in the early 20th century. Now it has become one of the most important microbial pesticides, offering a great contribution to integrated pest management. The first Bt commercial preparation came out in 1938. Nowadays it has achieved more than 90% market share in microbial pesticide industry. Bacillus thuringiensis can produce a variety of toxins to harmful insects, nematodes and mammals. Some of them can even be beneficial to the treatment of human disease. In this paper, four Bt strains (CPB008, CPB012, CPB016, CPB111) were previously obtained from Colorado potato beetle (Leptinotarsa decemlineata) collected from Xin-Jiang Autonomous Region by our laboratory research group. They were proved to have the ability to degrade chitin, behave well in killing Colorado potato beetle as well. However, their insecticidal genes and proteins were not known. Therefore, the objectives of the present study were to analyse diversity of insecticidal genes and proteins, and to test controlling effect of the bacteria on Bactrocera dorsalis (Diptera).1. Identification of insecticidal gene typesThe cry1-cry10 genes were identified with PCR-RFLP technique. A universal primer pair was designed based on the conservative regions of the cry1-cry10 genes and was used to amplify these genes with the PCR technique. The amplified products were then double-catalyzed with selected restriction enzymes so that the polymorphism of the insecticidal genes was detected. The results showed that both CPB008 and CPB012 contained cry 1, cry2 genes. Double-slicing cry 1 by Xba Ⅰ /Pst Ⅰ enzymes resulted in 1117 bp,801 bp,518 bp and 322 bp segments, while splicing cry2 by Hinc Ⅱ /Msp Ⅰ enzymes produced 958 bp,791 bp,297 bp 273 bp and 143 bp segments.The cry1-cry10 genes were not dected in the strains CPB016 and CPB111.Specific primers were designed and selected for identifying sub-groups of functional genes (cry2A, cry2Aa. etc.) in groups cryl-cry10 via PCR amplification. These genes were sequenced and compared with other genes in database in NCBI GenBank. CPB008 and CPB012 were shown to contain crylAa, crylAc, crylla, cry2Aa and cry2Ab and vip3A genes, but genes in the cry11-cry40 and vip groups were not detected. No insecticidal genes were found in CPB016 and CPB111.2. Morphology of crystal proteins and SDS-PAGESequential observation of different bacterial morphology showed that the crystal proteins were enriched 48h after culturing on LB plates. CPB008 produced diamond, irregular and spherical protein crystals; CPB012 produced quadrate, irregular and spherical protein crystals, but CPB016 and CPB111 produced only spherical crystals.The crystal proteins were extracted from the bacterial cells after cultured on LB plates for 24,48,72 and 96 hours, respectively. Then SDS-PAGE chromatographic fingerprint analysis was done to detect different insecticidal proteins. Based on the fingerprints CPB008 and CPB012 produced proeins of 135-140 kDa,80 kDa and 70 kDa, but only proteins of 27-35 kDa was found in the CPB016 and CPB111 strains.3. Test of killing Bactrocera dorsalisBioassays showed that both CPB008 and CPB012 infected caused death of 2-instar larvae of Bactrocera dorsalis (Diptera). The infected larvae stopped feeding soon after inoculation with CPB008 and CPB012. Their bodies became pigmented and then turned black and rottened completely. The cumulated lethality after 7 days (LR7d) of treatment was 70.86%(CPB008),76.72%(CPB012), but no death was found in CPB016, CPB111 and the no inoculation treatments. The half lethal time (LT50) was 3.1d (CPB008), and 2.4d (CPB012). The half lethal concentration (LC50) was 9.96×107cells/mL (CPB008) and 2.03×10’cells/mL (CPB012).Combining previous results of bioassays on Leptinotarsa decemlineata (Cleoptera) and on Bombyx mori (Lepidoptera) CPB008 and CPB012 were highly lethal to L. decemlineata, Bombyx mori and B. dorsalis. This lethal effect could be associated with the cry and vip genes identified in CPB008 and CPB012. CPB016 and CPB111 were demonstrated to have the ability of killing B. mori and L. decemlineata, but they were showed not significantly toxic to B. dorsalis. This result was also in agreement with the functional gene types revealt in the two strains.In conclution, the strains CPB008 and CPB012 of Bacillus thuringiensis from Colorado potato bettle contained cryl, cry2 and vip3A genes. These genes might regulate the synthesis of the 135 kDa,80 kDa and 70 kDa proteins which help B. thuringiensis kill the larvae of B. mori, L. decemlineata and B. dorsalis. The two strains had an important value of application development and promotion.