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Biochemical and Multiplex PCR Analysis of Toxic Crystal Proteins to Determine Genes in Bacillus thuringiensis Mutants

Authors: Fatma N. Talkhan, H. H. Abo-Assy, K. A. Soliman, Marwa M. Azzam, A. Z. E. Abdelsalam, A. S. Abdel-Razek


The Egyptian Bacillus thuringiensis isolate (M5) produce crystal proteins that is toxic against insects was irradiated with UV light to induce mutants. Upon testing 10 of the resulting mutants for their toxicity against cotton leafworm larvae, the three mutants 62, 64 and 85 proved to be the most toxic ones. Upon testing these mutants along with their parental isolate by SDS-PAGE analysis of spores-crystals proteins as well as vegetative cells proteins, new induced bands appeared in the three mutants by UV radiation and also they showed disappearance of some other bands as compared with the wild type isolate. Multiplex PCR technique, with five sets of specific primers, was used to detect the three types of cryI genes cryIAa, cryIAb and cryIAc. Results showed that these three genes exist, as distinctive bands, in the wild type isolate (M5) as well as in mutants 62 and 85, while the mutant 64 had two distinctive bands of cryIAb and cryIAc genes, and a faint band of cryI Aa gene. Finally, these results revealed that mutant 62 is considered as the promising mutant since it is UV resistant, highly toxic against Spodoptera littoralis and active against a wide range of Lepidopteran insects.

Keywords: Biological Control, Bacillus thuringiensis, cry1 genes, multiplex PC, SDS- PAGE analysis

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[1] Chattopdhyay, A.; N. B. Bhatnagar and R. Bhatnagar "Bacterial insecticidal toxins" Criti. Rev. Microbiol. 30:33-54, 2004.
[2] Beegle, C. C. and T. Yamamoto "History of Bacillus thuringiensis Berliner research and development" Can. Entomol. 124:587-616, 1992.
[3] Feitelson, J. S. "The Bacillus thuringiensis family tree. In: Advanced Engineered Pesticides. Kim L. (ed.). Marcel Dekker, New York, 1993, pp. 63-71.
[4] Laemmli, U. K. "Cleavage of structural proteins during the assembly of the head bacteriophage T4". Nature. 277:680- 685. 1970.
[5] Donovan, W. P.; J. M. Gonzalez; M. P. Gilbert and C. Dankocsik" Isolation and characterization of EG2158, a new strain of Bacillus thuringiensis toxic to coleopteran larvae, and nucleotide sequence of the toxic gene" Mol. Gen. Genet. 214:366-372, 1988.
[6] Gill, S. H.; L. Kiehne; J. L. and T. Yamamoto "Cloning of a novel cryIC-type gene from a strain of Bacillus thuringiensis subsp. gallaiae" Appl. Environ. Microbiol. 59:1131-1137, 1995.
[7] Liang, H. ; Y. Liu ; J. Zhu ; P. Guan ; S.Li ; ; A. Zheng ; H. Liu and P. Li " Characterization of cry2-type genes of Bacillus thuringiensis strains from soil isolated of Sihuan Basin, China" Brazillian J. Microbiol. 42: 140-146, 2011.
[8] Kati, H; K. Sezen and Z. Demirabag "Characterization of a highly pathogenic Bacillus thuringiensis strain isolated from common cockchafer, Melolontha melolontha" Folia Microbiol. 52:147-152, 2007.
[9] Carlabrese, D. M. and K. W. Nickerson “A comparison of protein crystal subunit sizes in Bacillus thuringiensis” Can. J. Microbiol. 26:1006-1010, 1980.
[10] Jarrett, P. "Potency factors in the delta-endotoxin of Bacillus thuringiensis var. aizawi and the significance of plasmids in their control" J. Appli. Bacteriol. 58: 437-448, 1985.
[11] Baum, J. A.; C. Chu; M. Rupar; G. R. Brown; W. P. Donovan; J. E. Huesing; O. Ilagan; T. M. Malvar and M. Pleau "Binary toxins from Bacillus thuringiensis active against the western corn rootworm, Diabrotica virgifera virgifera LeConte" Appli. Environ. Microbiol. 70:4889-4898, 2004.
[12] Milne, R.; D. Murphy and P. G. Fast "Bacillus thuringiensis- endotoxin: An improved technique for the separation of crystals from spores" J. Invert. Pathol. 29:230-231, 1977.
[13] Lynch, M. J. and P. Baumann" Immunological comparisons of the crystal protein from strains of Bacillus thuringiensis" J. Invert. Pathol. 46:47-57. 1985.
[14] Bourque, S.; J. R. Valero; J. Mercier; M. C. Lavoie and C. Levesquer "Multiplex polymerase chain reaction for detection and differentiation of the microbial insecticide Bacillus thunrngiensis" Appli. Environ. Microbiol. 59: 523- 527, 1993.
[15] Ceron, J.; L. Cvarrubias; R. Quintero; A. Ortiz; M. Ortiz; E. Aranda; L. Lina and A. Bravo "PCR analysis of the cryI insecticidal crystal family genes from Bacillus thuringiensis" Appli. Environ. Microbiol. 60:353- 356, 1994.
[16] Ben-Dov, E.; A. Zaritsky; E. Dahan; Z. Barak; R. Siani; R.Manasherob; A. Khamraev; E. Trttskaya; A. Dubitsky; N.Berezina and Y. Margalith " Extended screening by PCR for seven cry-group genes from fieldcollected strains of Bacillus thuringiensis. Appli. Environ. Microbial" 63:4883-4890, 1994.
[17] Von-Tersch, M. A. and T. M. Gonzalez "Bacillus thuringiensis cry ET1 toxin gene and protein toxic to Lepidopteran insects, patent No: 5356623 supplemented for registration in (10-8-1994), 1994.
[18] Hames, B. D. "One-dimensional polyacrylamide gel electrophoresis. In: Gel electrophoresis of proteins. A practical approach. Hames B.D. and Rickwood D. (eds.). Oxford University Press, New York, 1995, pp. 1- 147.
[19] Saxena, D.; E. Ben-Dov; R. Manasherob; Z. Barak; S. Boussiba and A. Zaritsky "A UV tolerant mutant of Bacillus thuringiensis subsp. Kurstaki producing melanin" Curr. Microbiol. 44:25-30, 2002.
[20] Ruan, L.; Z. Yu; B. Fang; W. He; Y. Wang and P. Shen "Melanin pigment formation and increased UV resistance in Bacillus thuringiensis following high temperature induction" System. Appl. Microbiol. 27:286-289, 2004.
[21] Kumar, S. ; U. Tuteja ; K. Sarika ; D. Kumar Singh and O. Kumar "Rapid Multiplex PCR assay for the simultaneous detection of the Brucella Genus, B. abotus, B. melitensis, and B. suis." J. Microbiol. Biotechnol. 21(1):89-92, 2011.